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1.  Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics 
Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses.
Cells respond in real time to the absolute number of TGF-β molecules in their environment.A single pulse of TGF-β stimulation results in transient SMAD activation whereas repeated short pulses of stimulation result in sustained SMAD activation.Ligand-induced short-term TGF-β/SMAD signaling activation is graded while long-term signaling response is switch-like or ultrasensitive.TGF-β ligand depletion is a major cause of conversion from graded short-term responses to ultrasensitive long-term responses.
The transforming growth factor-β (TGF-β) pathway is a prominent signaling pathway that regulates diverse aspects of cellular homeostasis, including proliferation, differentiation, migration, and death (Massague, 1998). Remarkably, the pleiotropic biological effects of TGF-β are mediated by a relatively simple signaling module (Clarke and Liu, 2008). An interesting question is how such an apparently straightforward and simple cascade can generate a wide array of biological responses depending on the cellular context.
Members of the TGF-β superfamily are frequently used as morphogens in early embryo development (Green, 2002). The best-studied examples include Dpp in Drosophila and Activin in Xenopus (Gurdon and Bourillot, 2001; Lander, 2007). In the developmental context, cells can respond to a graded ligand concentration and produce discrete biological responses (e.g., transcription of certain genes, proliferation, or differentiation; Green, 2002). To convert continuous morphogen stimulation into discrete responses, mechanisms must exist to provide a threshold for the cellular response. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remains poorly understood.
We have previously reported that ligand molecules per cell is the input variable to which the cells respond, and ligand number per cell is the best predictor of signaling responses (Zi and Klipp, 2007a; Clarke et al, 2009). Here, we developed an improved mathematical model to predict TGF-β signaling responses by calibrating the model with various experimental data sets from different TGF-β stimulations. Using a combined experimental and mathematical modeling approach, we showed that TGF-β pulse stimulation results in transient activation of the pathway while repeated short pulses at short time intervals lead to a sustained activation similar to persistent ligand exposure.
We next investigate the system response to variable doses of TGF-β in HaCaT cells. Our mathematical model predicts that the short-term Smad2 phosphorylation (after 45 min of TGF-β stimulation) is a graded response, while long-term Smad2 activation (after 24 h of TGF-β stimulation) is a switch-like response (Figure 5A and B). As shown in Figure 5A–D, both short- and long-term Smad2 phosphorylation can be saturated but doses of TGF-β that cause maximum response are quite different. Additionally, the shapes of response curves were different. The short-term Smad2 activation was a graded (Michaelis–Menten-like) response with a very low apparent Hill coefficient of about 0.8 (Figure 5A and C) while the long-term Smad2 activation (P-Smad2 at 24 h) yielded a switch-like response with an apparent Hill coefficient of about 4.5 (Figure 5B and D). Thus, the Smad2 response is initially graded and sharpens over time to become ultrasensitive. To address whether TGF-β-inducible gene expression responses are graded or switch-like in the short and long term, we measured mRNA levels of Smad7, an early responsive gene of TGF-β and protein levels of p21 and PAI-1 whose inductions are delayed and late, respectively. The experimental data show that Smad7 induction exhibits a graded response with corresponding Hill coefficients of about 1.3 (Figure 5E), which is consistent with the graded P-Smad2 response at 45 min (Figure 5A and C). PAI-1 induction in response to variable doses of TGF-β for 24 h is highly ultrasensitive with an apparent Hill coefficient of ∼5.3. Compared with Smad7 and PAI-1, p21 induction is only modest ultrasensitive (nHill≈2) (Figure 5G). These results suggest short-term gene induction by TGF-β appears to be graded while long-term targets are more switch-like. Finally, we measured the growth inhibitory response of HaCaT cells to variable doses of TGF-β. The level of BrdU incorporation is also ultrasensitive with an apparent Hill coefficient of about 4.3 (Figure 5H). Therefore, the long-term TGF-β growth inhibitory response also shows a switch-like behavior. Finally, we show that TGF-β depletion affects long-term Smad phosphorylation and switch-like response of TGF-β signaling system. These findings shed new light on how continuous ligand doses are translated into discontinuous cell fate decisions in biological systems.
In summary, we have shown that the dose and time course of TGF-β stimulation have profound effects on Smad signaling dynamics. The rate of ligand depletion controls the duration of Smad2 phosphorylation. Cells can respond to a short pulse of TGF-β stimulation, and periodic short ligand exposures are sufficient to generate long-term signaling responses. Short-term TGF-β stimulation causes only transient pathway activation and can be terminated by ligand depletion. TGF-β-induced Smad2 phosphorylation is graded in the short-term but ultrasensitive (switch-like) in the long-term (Figure 7). Additionally, cell growth arrest in response to TGF-β shows switch-like rather than graded behavior. Our modeling and experimental analyses suggest that ligand depletion is likely to be involved in sharpening a graded response into a switch-like response.
Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.
PMCID: PMC3130555  PMID: 21613981
mathematical model; Smad; TGF-β; ultrasensitivity
2.  Genome-wide transcriptional plasticity underlies cellular adaptation to novel challenge 
By recruiting the essential HIS3 gene to the GAL regulatory system and switching to a repressing glucose medium, we confronted yeast cells with a novel challenge they had not encountered before along their history in evolution.Adaptation to this challenge involved a global transcriptional response of a sizeable fraction of the genome, which relaxed on the time scale of the population adaptation, of order of 10 generations.For a large fraction of the responding genes there is no simple biological interpretation, connecting them to the specific cellular demands imposed by the novel challenge.Strikingly, repeating the experiment did not reproduce similar transcription patterns neither in the transient phase nor in the adapted state in glucose.These results suggest that physiological selection operates on the new metabolic configurations generated by the non-specific large scale transcriptional response to eventually stabilize an adaptive state.
Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. Some common perturbations, such as changes in nutrient composition, elicit well-characterized transcriptional responses that can be understood by simple engineering-like design principles as satisfying specific demands imposed by the perturbation. However, cells also have the ability to adapt to novel and unforeseen challenges. This ability is central in realizing the evolvability potential of cells as they respond to dramatic genetic or environmental changes along evolution. Little is known about the mechanisms underlying such adaptations to novel challenges; in particular, the role of the transcriptional regulatory network in such adaptations has not been characterized. Genome-wide measurements have revealed that, in many cases, perturbations lead to a global transcriptional response involving a sizeable fraction of the genome (Gasch et al, 2000; Jelinsky et al, 2000; Causton et al, 2001; Ideker et al, 2001; Lai et al, 2005). Such global behavior suggests that general collective properties of the genetic network, rather than specific pre-designed pathways, determine an important part of the transcriptional response. It is not known however what fraction of genes within such massive transcriptional responses is essential to the specific cellular demands. It is also unknown whether the non-pre-designed part of the response can have a functional role in adaptation to novel challenges.
To study these questions, we confronted yeast cells with a novel challenge they had not encountered before along their history in evolution. A strain of the yeast Saccharomyces cerevisiae was engineered to recruit the gene HIS3, an essential enzyme from the histidine biosynthesis pathway (Hinnebusch, 1992), to the GAL regulatory system, responsible for galactose utilization (Stolovicki et al, 2006). The GAL system is known to be strongly repressed when the cells are exposed to glucose. Therefore, upon switching to a medium containing glucose and lacking histidine, the GAL system and with it HIS3 are highly repressed immediately following the switch and the cells encounter a severe challenge. We have recently shown that a cell population carrying this rewired genome can adapt to grow competitively in a chemostat in a medium containing pure glucose (Stolovicki et al, 2006). This adaptation occurred on a timescale of ∼10 generations; applying a stronger environmental pressure in the form of a competitive inhibitor to HIS3 (3AT) resulted in a similar adaptation albeit with a longer timescale. Figure 1 shows the dynamics of the population's cell density (blue lines, measured by OD) following a medium switch from galactose to glucose in the chemostat without (A) and with (B) 3AT. The experiments revealed that adaptation occurs on physiological timescales (much shorter than required by spontaneous random mutations), but the mechanisms underlying this adaptation have remained unclear (Stolovicki et al, 2006).
Yeast cells had not encountered recruitment of HIS3 to the GAL system along their evolutionary history, and their genome could not possibly have been selected to specifically address glucose repression of HIS3. This experiment, therefore, provides a unique opportunity to characterize the spontaneous transcriptional response during adaptation to a novel challenge and to assess the functional role of the regulatory system in this adaptation. We used DNA microarrays to measure the genome-wide expression levels at time points along the adaptation process, with and without 3AT. These measurements revealed that a sizeable fraction of the genome responded by induction or repression to the switch into glucose. Superimposed on the OD traces, Figure 1 shows the results of a clustering analysis of the expression of genes as measured by the arrays along time in the experiments. This analysis revealed two dominant clusters, each containing hundreds of genes in each experiment, which responded to the medium switch to glucose by a strong transient induction or repression followed by relaxation to steady state on the timescale of the adaptation process, ∼ 10 generations. The two clusters in each experiment show similar but opposite dynamics.
A detailed analysis of the gene content in the two clusters revealed that only a small portion of the response was induced by a change in carbon source (15% overlap between the corresponding clusters in the two experiments, with and without 3AT). Moreover, it revealed a very low overlap with the universal stress response observed for a wide range of environmental stresses (Gasch et al, 2000; Causton et al, 2001) and with the typical response to amino-acid starvation (Natarajan et al, 2001). Additionally, all known specific responses to stress in the literature are characterized by transient induction or repression with relaxation to steady state within a generation time (Gasch et al, 2000; Koerkamp et al, 2002; Wu et al, 2004), whereas in our experiments relaxation of the transcriptional response occurs over many generations. Taken together, these results show that the transcriptional response observed here is neither a metabolic response to the change in carbon source nor is it a standard response to stress or amino-acid starvation. This raises the possibility that it is a spontaneous collective response that is largely composed of genes that do not have a specific function. This possibility was tested directly by repeating the experiment with different populations and comparing their responses. This procedure revealed reproducible adaptation dynamics and steady states in terms of population density, but showed significantly different transcriptional transient responses and steady states for the two repeated experiments. Thus, a significant portion of the genes that changed their expression during the adaptation process do not have a well-defined and reproducible function in the challenging environment.
The application of a stronger environmental pressure in the form of 3AT had a dramatic effect on the global characteristics of the transcriptional response: it induced a markedly higher correlation among the hundreds of responding genes. Figure 3A compares the array data in color code for the two experiments. It is seen that the emergent pattern of transcription exhibits a higher degree of order by the introduction of high external pressure. Observation of the transcriptional patterns for specific metabolic pathways illustrates the different contributions to the correlated dynamics (Figure 3B–D). A general energetic module such as glycolysis exhibited similar patterns of induction and relaxation in experiments with and without 3AT (Figure 3B). However, in general, we found that more than one-third of the known metabolic modules (30 out of 88 modules described in KEGG) exhibited high expression correlation among their genes when the environmental pressure was high but not when it was low. As an example, Figure 3C shows the histidine biosynthesis pathway and Figure 3D the purine pathway. Note the highly ordered trajectories in the lower panels (with 3AT) compared to the disordered ones in the upper panels (no 3AT). This order extends also between genes belonging to different and even distant metabolic modules. It indicates that a global transcriptional regulatory mechanism is in operation, rather than a local specific one. Surprisingly, genes belonging to the same metabolic pathway exhibited simultaneous positively and negatively correlated dynamics. Thus, an important conclusion of this work is that the global transcriptional response to a novel challenge cannot be explained by a simple cellular or metabolic logic. This is to be expected if the response had not been specifically selected in evolution and was not pre-designed for the challenge.
Our data clearly reveal that the massive transcriptional response underlies the adaptation process to a novel challenge. The novelty of the challenge presented to the cells excludes the possibility that this response has been specifically selected toward this challenge. Thus, transcriptional regulation has dynamic properties resulting in a general massive nonspecific response to a novel perturbation. Such a response in turn allows for metabolic rearrangements, which by feeding back on transcription lead to adaptation of the cells to the unforeseen situation. The drastic change in the expression state of the cell opens multiple new metabolic pathways. Physiological selection works then on these multiple metabolic pathways to stabilize an adaptive state that causes relaxation of the perturbed expression pattern. This scenario, involving the creation of a library of possibilities and physiological selection over this library, is compatible with our understanding of a broad class of biological systems, placing the cellular metabolic/regulatory networks on the same footing as the neural or the immune systems (Gerhart and Kirschner, 1997).
Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. An open question is to what extent transcriptional response to perturbations has been specifically selected along evolution. To test the possibility that transcriptional reprogramming does not need to be ‘pre-designed' to lead to an adaptive metabolic state on physiological timescales, we confronted yeast cells with a novel challenge they had not previously encountered. We rewired the genome by recruiting an essential gene, HIS3, from the histidine biosynthesis pathway to a foreign regulatory system, the GAL network responsible for galactose utilization. Switching medium to glucose in a chemostat caused repression of the essential gene and presented the cells with a severe challenge to which they adapted over approximately 10 generations. Using genome-wide expression arrays, we show here that a global transcriptional reprogramming (>1200 genes) underlies the adaptation. A large fraction of the responding genes is nonreproducible in repeated experiments. These results show that a nonspecific transcriptional response reflecting the natural plasticity of the regulatory network supports adaptation of cells to novel challenges.
PMCID: PMC1865588  PMID: 17453047
adaptation; cellular metabolism; expression arrays; plasticity; transcriptional response
3.  Achievement and Maintenance of Sustained Response during TADS Continuation and Maintenance Therapy 
Archives of general psychiatry  2008;65(4):447-455.
The Treatment for Adolescents with Depression Study evaluated fluoxetine (FLX), cognitive-behavioral therapy (CBT), and FLX/CBT combination (COMB), versus pill placebo (PBO) in 439 adolescents with major depressive disorder. Treatment consisted of three stages: (I) acute (12 weeks), (II) continuation (6 weeks), and (III) maintenance (18 weeks).
Examine rates of achieving and maintaining sustained response during continuation and maintenance treatments.
Design and Setting
Randomized controlled trial conducted in 13 US sites. Response was determined by blinded independent evaluators (IEs).
Clinical Trial Registry NCT00006286
242 FLX, CBT, and COMB patients in their assigned treatment at the end of Stage I.
Stage II treatment varied based on Stage I response. Stage III consisted of 3 CBT and/or pharmacotherapy sessions, and, if applicable, continued medication.
Main Outcome Measures
Sustained response was defined as two consecutive Clinical Global Impression-Improvement (CGI-I) ratings of 1 or 2 (“full response”). Patients achieving sustained response were classified on subsequent non-response status.
Among 95 patients (39.3%) who had not achieved sustained response by Week 12 (29.1% COMB, 32.5% FLX, 57.9% CBT), sustained response rates during Stages II/III were 80.0% COMB, 61.5% FLX, and 77.3% CBT (difference ns). Among the remaining 147 patients (60.7%) who achieved sustained response by Week 12, CBT patients were more likely than FLX patients to maintain sustained response through Week 36 (96.9% vs. 74.1%; p = 0.007; 88.5% of COMB patients maintained sustained response through Week 36). Total rates of sustained response by Week 36 were 88.4% COMB, 82.5% FLX, and 75.0% CBT.
Most depressed adolescents who had not achieved sustained response during acute treatment did achieve that level of improvement during continuation and maintenance therapies. The possibility that CBT may help the subset of depressed adolescents who achieve early sustained response maintain their response warrants further investigation.
The Treatment for Adolescents with Depression Study evaluated fluoxetine (FLX), cognitive-behavior therapy (CBT), and FLX/CBT combination (COMB) compared to pill placebo in 439 adolescents with major depressive disorder. Treatment consisted of acute, continuation, and maintenance stages. The present study examined rates of achieving and maintaining sustained response during continuation and maintenance therapy for the 242 active treatment patients who were in their assigned condition at the end of acute therapy. Sustained response was defined as two consecutive Clinical Global Impression-Improvement ratings by blinded assessors of 1 or 2 (“full response”). Among the 95 patients who had not achieved sustained response during acute therapy, sustained response rates during continuation and maintenance treatment were 80.0% COMB, 61.5% FLX, and 77.3% CBT (difference ns). Among the remaining 147 patients who had achieved sustained response during acute treatment, CBT patients were more likely than FLX patients to maintain sustained response through maintenance therapy (96.9% vs. 74.1%; p = 0.007; 88.5% of COMB patients maintained sustained response through Week 36). Most depressed adolescents achieved sustained response by the completion of treatment. The possibility that CBT may help the subset of depressed adolescents who achieve early sustained response maintain their response warrants further investigation.
PMCID: PMC2587406  PMID: 18391133
4.  IFN-γ production in response to in vitro stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1*0401 and HLA-DQ8 
Arthritis Research  1999;2(1):75-84.
IFN-γ was measured in supernatants after in vitro stimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA.
Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1] predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2,3,4] and that show that CII can induce experimental arthritis [5].
It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6,7,8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze IFN-γ responses as an alternative to proliferative responses.
To study cellular immune reactivity to CII in patients with RA and in healthy control individuals and to correlate this reactivity to HLA class II genotypes and to the presence of antibodies to CII in serum.
Forty-five patients who met the 1987 American College of Rheumatology classification criteria for RA [10] and 25 healthy control individuals of similar age and sex were included. Twenty-six of these patients who had low levels of anti-CII in serum were randomly chosen, whereas 19 patients with high anti-CII levels were identified by enzyme-linked immunosorbent assay (ELISA)-screening of 400 RA sera.
Heparinized blood was density gradient separated and PBMCs were cultured at 1 × 106/ml in RPMI-10% fetal calf serum with or without antigenic stimulation: native or denatured CII (100 μ g/ml), killed influenza virus (Vaxigrip, Pasteur Mérieux, Lyon, France; diluted 1 : 1000) or purified protein derivative (PPD; 10 μ g/ml). CII was heat-denatured in 56°C for 30 min.
Cell supernatants were collected after 7days and IFN-γ contents were analyzed using ELISA. HLA-DR and HLA-DQ genotyping was performed utilizing a polymerase chain reaction-based technique with sequence-specific oligonucleotide probe hybridization. Nonparametric statistical analyses were utilized throughout the study.
PBMCs from both RA patients and healthy control individuals responded with inteferon-γ production to the same degree to stimulation with native and denatured CII (Fig. 1a), giving median stimulation indexes with native CII of 4.6 for RA patients and 5.4 for healthy control individuals, and with denatured CII of 2.9 for RA patients and 2.6 for healthy control individuals. RA patients with elevated levels of anti-CII had a weaker IFN-γ response to both native and denatured CII than did healthy control individuals (P = 0.02 and 0.04, respectively).
Stimulation with the standard recall antigens PPD and killed influenza virus yielded a median stimulation index with PPD of 10.0 for RA patients and 51.3 for healthy control individuals and with influenza of 12.3 for RA patients and 25.7 for healthy, control individuals. The RA patients displayed markedly lower responsiveness to both PPD and killed influenza virus than did healthy control individuals (Fig. 1b). IFN-γ responses to all antigens were abrogated when coincubating with antibodies blocking MHC class II.
The low response to PPD and killed influenza virus in RA patients relative to that of healthy control individuals reflects a general downregulation of antigen-induced responsiveness of T cells from RA patients [6,7,8]. That no difference between the RA group and the control group was recorded in CII-induced IFN-γ production therefore indicates that there may be an underlying increased responsiveness to CII in RA patients, which is obscured by the general downregulation of T-cell responsiveness in these patients. In order to address this possibility, we calculated the fraction between individual values for the CII-induced IFN-γ production and the PPD-induced and killed influenza virus-induced IFN-γ production, and compared these fractions. A highly significant difference between the RA and healthy control groups was apparent after stimulation with both native CII and denatured CII when expressing the response as a fraction of that with PPD (Fig. 2a). Similar data were obtained using killed influenza virus-stimulated IFN-γ values as the denominator (Fig. 2b).
When comparing the compensated IFN-γ response to denatured CII stimulation between RA patients with different HLA genotypes, highly significant differences were evident, with HLA-DRB1*0401 patients having greater CII responsiveness than patients who lacked this genotype (Fig. 3a). HLA-DQ8 positive patients also displayed a high responsiveness to CII as compared with HLA-DQ8 negative RA patients (Fig. 3b). These associations between the relative T-cell reactivity to denatured CII and HLA class II genotypes were not seen in healthy control individuals. Similar results were achieved using influenza as denominator (P = 0.02 for HLA-DRB1*0401 and P = 0.01 for HLA-DQ8).
No reports have previously systematically taken the general T-cell hyporesponsiveness in RA into account when investigating specific T-cell responses in this disease. In order to address this issue we used the T-cell responses to PPD and killed influenza virus as reference antigens. This was made on the assumption that exposure to these antigens is similar in age-matched and sex-matched groups of RA patients and healthy control individuals. The concept of a general hyporesponsiveness in RA T cells has been documented in several previous reports, in which both nominal antigens [6,7,8] and mitogens [11,12,13] have been used. The fact that a similar functional downregulation in RA PBMCs was obtained with both PPD and killed influenza virus as reference antigens strengthens the validity of our approach.
We identified an association between the IFN-γ response to CII and HLA-DRB1*0401 and HLA-DQ8 in the RA patient group, which is of obvious interest because both these MHC class II alleles have been associated with high responsiveness to CII in transgenic mice that express these human MHC class II molecules [14,15]. There was no association between high anti-CII levels and shared epitope (HLA-DRB1*0401 or HLA-DRB1*0404).
CII, a major autoantigen candidate in RA, can elicit an IFN-γ response in vitro that is associated with HLA-DRB1*0401 and HLA-DQ8 in RA patients. This study, with a partly new methodological approach to a classical problem in RA, has provided some additional support to the notion that CII may be a target autoantigen of importance for a substantial group of RA patients. Continued efforts to identify mechanisms behind the general hyporesponsiveness to antigens in RA, as well as the mechanisms behind the potential partial anergy to CII, may provide us with better opportunities to study the specificity and pathophysiological relevance of anti-CII reactivity in RA.
PMCID: PMC17806  PMID: 11219392
collagen type II; human leucocyte antigen-DR; IFN-γ; rheumatoid arthritis; T cell
5.  Exquisite Sensitivity of TP53 Mutant and Basal Breast Cancers to a Dose-Dense Epirubicin−Cyclophosphamide Regimen 
PLoS Medicine  2007;4(3):e90.
In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.
Methods and Findings
In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m2 epirubicin and 1,200 mg/m2 cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.
This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin–cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.
Hugues de The and colleagues report thatTP53 status is a predictive factor for responsiveness in breast cancers to a dose-dense epirubicin-cyclophosphamide chemotherapy regimen, and suggests that this regimen might be well suited for patientsTP53 mutant tumors.
Editors' Summary
One woman in eight will develop breast cancer during her life. As with other cancers, breast cancer arises when cells accumulate genetic changes (mutations) that allow them to grow uncontrollably and to move around the body. These altered cells are called malignant cells. The normal human breast contains several types of cell, any of which can become malignant. In addition, there is more than one route to malignancy—different sets of genes can be mutated. As a result, breast cancer is a heterogeneous disease that cannot be cured with a single type of treatment. Ideally, oncologists would like to know before they start treating a patient which therapeutic approach is going to be successful for that individual. Recently, researchers have begun to identify molecular changes that might eventually allow oncologists to make such rational treatment decisions. For example, laboratory studies in cell lines or animals indicate that the status of a gene called TP53 determines the chemotherapy agents (drugs that preferentially kill rapidly dividing cancer cells) to which cells respond. p53, the protein encoded by TP53, is a tumor suppressor. That is, in normal cells it prevents unregulated growth by controlling the expression of proteins involved in cell division and cell death. Consequently, p53 is often inactivated during cancer development.
Why Was This Study Done?
Although laboratory studies have linked TP53 status to chemotherapy responses, little is known about this relationship in human breast cancers. The clinical studies that have investigated whether TP53 status affects chemotherapy responses have generally found that patients whose tumors contain mutant TP53 have a poorer response to therapy and/or a shorter survival time than those whose tumors contain normal TP53. In this study, the researchers have asked whether TP53 status affects tumor responses to a dose-intense chemotherapy regimen (frequent, high doses of drugs) given to women with advanced noninflammatory breast cancer before surgery. This type of treatment is called neoadjuvant chemotherapy and is used to shrink tumors before surgery.
What Did the Researchers Do and Find?
The researchers collected breast tumor samples from 80 women before starting six fortnightly cycles of chemotherapy with epirubicin and cyclophosphamide. After this, each woman had her affected breast removed and examined to see whether the chemotherapy had killed the tumor cells. The researchers determined which original tumor samples contained mutated TP53 and used a technique called microarray expression profiling to document gene expression patterns in them. Overall, 28 tumors contained mutated TP53. Strikingly, all 15 tumors that responded completely to neoadjuvant chemotherapy (no tumor cells detectable in the breast tissue after chemotherapy) contained mutated TP53. Nine of these responsive tumors were basal-cell–like breast tumors, a particularly aggressive type of breast cancer; only one basal-cell–like, TP53-mutated tumor did not respond to chemotherapy. Patients whose tumors were unresponsive to the neoadjuvant chemotherapy but contained mutated TP53 tended to die sooner than those whose tumors contained normal TP53 or those with chemotherapy-responsive TP53-mutated tumors. Finally, expression profiling identified changes in the expression of many p53-regulated genes, but did not identify an expression profile in the TP53-mutated tumors unique to those that responded to chemotherapy.
What Do These Findings Mean?
These findings indicate that noninflammatory breast tumors containing mutant TP53—in particular, basal-cell–like tumors—are very sensitive to dose-dense epirubicin and cyclophosphamide chemotherapy. Intensive regimens of this type have rarely been used in previous studies, which might explain the apparent contradiction between these results and the generally poor response to chemotherapy of TP53-mutated breast tumors. More tumors now need to be examined to confirm the association between complete response, TP53 status and basal-cell–like tumors. In addition, although complete tumor responses generally predict good overall survival, longer survival studies than those reported here are needed to show that the tumor response to this particular neoadjuvant chemotherapy regimen translates into improved overall survival. If the present results can be confirmed and extended, dose-dense neoadjuvant chemotherapy with epirubicin and cyclophosphamide could considerably improve the outlook for patients with aggressive TP53-mutant, basal-cell–like breast tumors.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides patient and physician information on breast cancer and general information on understanding cancer
Cancer Research UK offers patient information on cancer and breast cancer
The MedlinePlus encyclopedia has pages on breast cancer
Emory University's CancerQuest discusses the biology of cancer, including the role of tumor suppressor proteins
Wikipedia has pages on p53 (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1831731  PMID: 17388661
6.  Metabolomic and transcriptomic stress response of Escherichia coli 
GC-MS-based analysis of the metabolic response of Escherichia coli exposed to four different stress conditions reveals reduction of energy expensive pathways.Time-resolved response of E. coli to changing environmental conditions is more specific on the metabolite as compared with the transcript level.Cease of growth during stress response as compared with stationary phase response invokes similar transcript but dissimilar metabolite responses.Condition-dependent associations between metabolites and transcripts are revealed applying co-clustering and canonical correlation analysis.
The response of biological systems to environmental perturbations is characterized by a fast and appropriate adjusting of physiology on every level of the cellular and molecular network.
Stress response is usually represented by a combination of both specific responses, aimed at minimizing deleterious effects or repairing damage (e.g. protein chaperones under temperature stress) and general responses which, in part, comprise the downregulation of genes related to translation and ribosome biogenesis. This in turn is reflected by growth cessation or reduction observed under essentially all stress conditions and is an important strategy to adjust cellular physiology to the new condition.
E. coli has been intensively investigated in relation to stress responses. Thus far, however, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. To better understand system response to perturbation, we designed a time-resolved experiment to compare and integrate metabolic and transcript changes of E. coli using four stress conditions including non-lethal temperature shifts, oxidative stress, and carbon starvation relative to cultures grown under optimal conditions covering both states before and directly after stress application, resumption of growth after stress-induced lag phase, and finally the stationary phase.
Metabolic changes occurring after stress application were characterized by a reduction in metabolites of central metabolism (TCA cycle and glycolysis) as well as an increase in free amino acids. Whereas the latter is probably due to protein degradation and stalling of translation, the former supports and extends conclusions based on transcriptome data demonstrating a major decrease in energy-consuming processes as a general stress response. Further comparative analysis of the response on the metabolome and transcriptome, however, revealed in addition to these similarities major differences. Thus, the response on the metabolome displayed a significantly higher specificity towards the specific stress as compared with the transcriptome. Further, when comparing the metabolome of cells ceasing growth due to stress application with cells ceasing growth due to reaching stationary phase the metabolome response differed to a significant extent between both growth arrest phases, whereas the transcriptome response showed significant overlap again, suggesting that the response of E. coli on the metabolome level displays a higher level of significance as compared with the transcriptome level.
Subsequently, both data sets were jointly analyzed using co-clustering and canonical correlation approaches to identify coordinated changes on the transcriptome and the metabolite level indicative metabolite–transcript associations. A first outcome of this study was that no association was preserved during all conditions analyzed but rather condition-specific associations were observed. One set of associations found was between metabolites from the oxidative pentose phosphate pathway such as glc-6-P, 6-P-gluconic acid, ribose-5-P, and E-4-P and metabolites from the glycolytic pathway (3PGA and PEP in addition to glc-6-P with two genes encoding pathway enzymes, that is rpe encoding ribulose phosphate 3-epimerase and pps encoding PEP synthase.
A second example comprises metabolites of the TCA cycle such as pyruvic acid, 2-ketoglutaric acid, fumaric acid, malic acid, and succinic acid and the mqo gene encoding malate-quinone oxidoreductase (MQO). MQO catalyses the irreversible oxidation of malate to oxaloacetate that in turn regulates the activity of citrate synthase, which is a major rate determining enzyme of the TCA cycle. The strong association between mqo gene expression and multiple members of the TCA cycle as well as pyruvate suggest mqo expression to have a major function for the regulation of the TCA cycle, which need to be experimentally validated.
Multiple further associations identified show on the one hand the power of integrative systems oriented approaches for developing new hypothesis, on the other hand their condition-dependent behavior shows the extreme flexibility of the biological systems studied thus requesting a much more intense effort toward parallel analysis of biological systems under several environmental conditions.
Environmental fluctuations lead to a rapid adjustment of the physiology of Escherichia coli, necessitating changes on every level of the underlying cellular and molecular network. Thus far, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. Here, we incorporate the metabolite composition together with gene expression data to provide a more comprehensive insight on system level stress adjustments by describing detailed time-resolved E. coli response to five different perturbations (cold, heat, oxidative stress, lactose diauxie, and stationary phase). The metabolite response is more specific as compared with the general response observed on the transcript level and is reflected by much higher specificity during the early stress adaptation phase and when comparing the stationary phase response to other perturbations. Despite these differences, the response on both levels still follows the same dynamics and general strategy of energy conservation as reflected by rapid decrease of central carbon metabolism intermediates coinciding with downregulation of genes related to cell growth. Application of co-clustering and canonical correlation analysis on combined metabolite and transcript data identified a number of significant condition-dependent associations between metabolites and transcripts. The results confirm and extend existing models about co-regulation between gene expression and metabolites demonstrating the power of integrated systems oriented analysis.
PMCID: PMC2890322  PMID: 20461071
Escherichia coli; metabolomic; response to stress; time course; transcriptomic
7.  Effects of short- and long-term sympathectomy on vasoconstrictor responses of the rat mesenteric arterial bed. 
British Journal of Pharmacology  1996;119(7):1347-1354.
1. The effects of short- and long-term sympathectomy were evaluated on vasoconstrictor function of constantly perfused mesenteric arterial beds isolated from rats: the effects of short-term sympathectomy were assessed at 3 and 8 days after 6-hydroxydopamine (6-OHDA) treatment of adult rats; the effects of long-term sympathectomy were assessed in adult rats treated at youth with guanethidine. 2. The relative degree of residual sympathetic innervation of the mesenteric arterial preparations was assessed by responses to electrical field stimulation (EFS; 16 Hz, 1 ms, 90 V, 30 s). Control responses were 95.6 +/- 3.9 mmHg (n = 35). Responses after sympathectomy were: 3 days after 6-OHDA, 2.9 +/- 0.9 mmHg (n = 15) < 8 days after 6-OHDA, 14.1 +/- 2.1 mmHg (n = 14) < guanethidine, 21.1 +/- 4.1 mmHg (n = 16). 3. Three days after 6-OHDA treatment there was an increase in the sensitivities of response to vasopressin and endothelin, producing leftward shifts of the dose-response curves of 0.66 +/- 0.11 and 0.88 +/- 0.13 log units respectively (n = 7-11), and a small increase in sensitivity of responses to noradrenaline (NA) and ATP. The maximal response to 5-hydroxytryptamine (5-HT) was increased. In contrast, there was a decrease in maximal constriction to NA and to the alpha 1-adrenoceptor agonist methoxamine. The alpha 2-adrenoceptor agonist clonidine did not elicit vasoconstriction at basal tone. There was no difference in vasodilator responses to the beta-adrenoceptor agonist isoprenaline in preparations with tone raised with prostaglandin F2 alpha (PGF2 alpha; 0.1-0.3 microM). 4. Eight days after 6-OHDA sympathectomy there was no significant difference in sensitivities or maximal responses to ATP, vasopressin and endothelin, but a small increase in the sensitivity of responses to 5-HT. Maximal responses to NA and methoxamine were significantly lower than the controls, but sensitivities were similar. There was no significant difference in vasodilator responses to isoprenaline in PGF2 alpha-raised tone preparations. 5. After long-term guanethidine sympathectomy maximal responses to 5-HT and NA were significantly reduced. Responses to ATP, vasopressin and endothelin were unchanged. 6. In mesenteric arterial preparations from untreated rats, ouabain (0.1 mM), a blocker of the Na+/K+ pump, significantly augmented the sensitivity and maximal responses to EFS, NA, methoxamine and 5-HT. Responses to ATP, vasopressin and endothelin were unaffected. 7. It is concluded that in the rat mesenteric arterial bed, short-term sympathectomy, where only 3% of the sympathetic nerve-mediated response remained, results in non-uniform changes in sensitivity and maximal responses to different vasoconstrictors, which cannot be entirely explained by changes in the Na+/K+ pump. Most of these changes disappeared at 8 days after 6-OHDA treatment, when nerve-mediated responses had partially returned. After long-term guanethidine sympathectomy, there was little change in responses to vasoconstrictors, and nerve-mediated responses were reduced to 22%. Although the variable factors are complex, it appears that in general, changes in responses of smooth muscle to vasoconstrictor substances after sympathetic denervation only occur if there is near-complete loss of nerve-mediated responses.
PMCID: PMC1915809  PMID: 8968542
8.  Conjunctival FOXP3 Expression in Trachoma: Do Regulatory T Cells Have a Role in Human Ocular Chlamydia trachomatis Infection? 
PLoS Medicine  2006;3(8):e266.
Trachoma, caused by ocular infection with Chlamydia trachomatis, remains the leading infectious cause of blindness and in 2002 was responsible for 3.6% of total global blindness. Although transmission can be successfully interrupted using antibiotics and improvements in public and personal hygiene, the long-term success of the control programmes advocated by the World Health Organization are still uncertain. For the complete control and prevention of trachoma, a vaccine would be highly desirable. Currently there are no licensed vaccines for trachoma, and no human vaccine trials have been conducted since the 1960s. A barrier to new attempts to design and introduce a vaccine is the identification of immunologic correlates of protective immunity or immunopathology. We studied important correlates of the immune response in a trachoma-endemic population in order to improve our knowledge of this disease. This is essential for the successful development of a vaccine against both ocular and genital C. trachomatis infection.
Methods and Findings
We used quantitative real-time PCR for C. trachomatis 16S rRNA to identify conjunctival infection. The expression of IFN-γ, IDO, IL-10, and FOXP3 mRNA transcripts was measured. We evaluated the role of immune effector and regulatory responses in the control of chlamydial infection and in the resolution of clinical signs of trachoma in endemic communities in Gambia. All host transcripts examined were detectable even in normal conjunctiva. The levels of these transcripts were increased, compared to normal uninfected conjunctiva, when infection was detected, with or without clinical disease signs. Interestingly, when clinical disease signs were present in the absence of infection, the expression of a regulatory T cell transcription factor, FOXP3, remained elevated.
There is evidence of an increase in the magnitude of the local anti-chlamydial cytokine immune responses with age. This increase is coupled to a decline in the prevalence of infection and active trachoma, suggesting that effective adaptive immunity is acquired over a number of years. The anti-chlamydial and inflammatory immune response at the conjunctival surface, which may control chlamydial replication, is closely matched by counter inflammatory or regulatory IL-10 expression. Differences in the level of FOXP3 expression in the conjunctiva may indicate a role for regulatory T cells in the resolution of the conjunctival immune response, which is important in protection from immunopathology. However, the expression of cytokines that control chlamydial replication and those that regulate the conjunctival immune response is not simply juxtaposed; the interaction between the infection and the clinical disease process is therefore more complex.
The immune response in a trachoma-endemic population showed an increase in local anti-chlamydial cytokine responses with age, associated with a decline in the prevalence of infection and active trachoma.
Editors' Summary
Trachoma is the leading infectious cause of blindness worldwide. Six million people—most of whom live in crowded, unhygienic conditions with limited water supplies—are blind because of repeated eye infections with Chlamydia trachomatis. This bacterium passes easily from person to person on hands or clothing and is also spread by flies. Successive infections starting in childhood cause progressive scarring of the inside of the eyelid. Eventually, the eyelashes turn inwards and rub painfully over the front of the eye (the cornea). This causes corneal scarring, loss of corneal transparency, and, finally, irreversible loss of sight, usually in adulthood. C. trachomatis infections can be prevented by improving personal hygiene and by reducing fly breeding sites, and they can be treated with antibiotics. In addition, early scarring of the eyelid and turned-in eyelashes can be treated surgically.
Why Was This Study Done?
Through the above interventions, the World Health Organization hopes to eliminate trachoma by 2020, but a vaccine might also be necessary. To develop a vaccine, the human immune response to C. trachomatis needs to be better understood. As with other diseases, the immune response to C. trachomatis includes a pro-inflammatory side, which activates immune cells to attack the bacteria, and a regulatory side, which keeps the pro-inflammatory responses in check. The balance between these two sides is not perfect, however. Although the immune response deals with C. trachomatis infections efficiently, it also causes some of the tissue damage that leads to scarring and loss of sight. In this study, the researchers have investigated the human immune response to C. trachomatis to provide immunological information that might help vaccine development.
What Did the Researchers Do and Find?
The researchers examined school children living in Gambia, where trachoma is very common, for clinical signs of active trachoma (for example, red or swollen eyelids). To find out which children were infected with C. trachomatis, the researchers collected a few cells from the surface of their eyes and looked for a ribonucleic acid (RNA) molecule that is only made by C. trachomatis. The researchers also looked in these samples for human messenger RNA (mRNA) molecules that are made during pro-inflammatory and regulatory immune responses.
The children formed four groups based on infection with C. trachomatis and clinical signs. Some children—particularly the older ones—were uninfected and had no clinical signs. Others were infected but showed no clinical signs—these children were incubating the bacteria. Some were infected and had clinical disease; these children had the highest bacterial loads. Finally, children recovering from an infection carried no bacteria but still had some clinical signs.
The researchers detected different types of immune response in each of these groups. Children incubating the bacteria had a strong pro-inflammatory response—their immune systems were trying to fight off infection. The pro-inflammatory response was even stronger in the infected children with clinical signs, but now the regulatory response had also increased, presumably to limit inflammation. In children in the recovery phase, only regulatory immune cells, which were making an mRNA from a gene called FOXP3, remained active.
What Do These Findings Mean?
The relative rarity of infections and active disease in older children together with indications of a more active immune response to infection indicates that protective immunity to C. trachomatis is acquired through repeated exposure to it. This bodes well for the development of a vaccine, which would speed up the acquisition of this natural immunity. Furthermore, the new information about immune responses at different stages of infection with C. trachomatis should help in vaccine design. The findings need to be confirmed by tracking immune responses in individual children during episodes of infection, but could then be used to help design vaccines that produce good protective immunity against C. trachomatis without causing too much collateral tissue damage. The current results suggest, for example, that regulatory immune cells are important in limiting the inflammatory response, so vaccine developers may need to ensure that their vaccines stimulate the production of this sort of cell as well as of the pro-inflammatory cells needed to clear the infection.
Additional Information.
Please access these Web sites via the online version of this summary at
• NHS Direct Online patient information on trachoma
• World Health Organization information on trachoma and its elimination
• US Centers for Disease Control and Prevention general information on trachoma
• MedlinePlus encyclopedia entry on trachoma
PMCID: PMC1526769  PMID: 16881731
9.  Allelic Variation on Murine Chromosome 11 Modifies Host Inflammatory Responses and Resistance to Bacillus anthracis 
PLoS Pathogens  2011;7(12):e1002469.
Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36–74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.
Author Summary
We show that genetic variation within an 8.3 Mb region on mouse chromosome 11 controls host response to anthrax lethal toxin (LT) and resistance to infection by the Sterne strain of Bacillus anthracis. Specifically, congenic C57BL/6 mice in which this region of chromosome 11 is derived from a genetically divergent CAST/Ei strain presented with a rapid and strong innate immune response to LT and displayed increased survival following infection with Sterne spores. CAST/Ei chromosome 11 encodes a dominant LT-responsive allele of Nlrp1b that may partially account for the severe response to LT. However, the strength of this response was attenuated in mice with only one copy of chromosome 11 derived from CAST/Ei indicating the existence of a recessive modifier of the inflammatory response to LT. In addition, congenic mice displayed a pronounced immune response using an experimental model of sepsis, indicating that one or more genes within the chromosome 11 region control host response to multiple inflammatory stimuli. Analyzing the influence of allelic variation on gene expression identified 25 genes as candidates for controlling these responses. In summary, we report a genetic model to study inflammatory responses beneficial to the host during anthrax.
PMCID: PMC3248472  PMID: 22241984
10.  A Central Regulatory System Largely Controls Transcriptional Activation and Repression Responses to Phosphate Starvation in Arabidopsis 
PLoS Genetics  2010;6(9):e1001102.
Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF) has an important role in the control of phosphate (Pi) starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1) mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS) in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like) control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide) corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress.
Author Summary
As sessile organisms, plants are often exposed to stress conditions, and have evolved adaptive responses to protect themselves from different types of stress. Some responses are stress type-specific whereas others are common to different stress types. Understanding how these responses are controlled is crucial for rational improvement of stress tolerance, a limiting factor in crop productivity. Here we examined the physiological and molecular responses to phosphate starvation and found that a single transcription factor family, represented by PHOSPHATE STARVATION RESPONSE REGULATOR 1 (PHR1), has a central role in the control of specific and shared phosphate starvation stress responses. In consonance with the importance of PHR1, we found that the PHR1-binding sequence, present in most PHR1 direct targets, is a crucial cis motif for Pi starvation responsiveness. An artificial promoter controlled by PHR1 recapitulates responsiveness to Pi starvation and to modulators of this response, qualifying PHR1 family members as central integrators in Pi starvation signalling. This central integrator system also controls most transcriptional repression responses to Pi starvation, indicating that they are an integral part of the adaptive response, and not a consequence of plant malfunction due to stress.
PMCID: PMC2936532  PMID: 20838596
11.  Competition for Antigen between Th1 and Th2 Responses Determines the Timing of the Immune Response Switch during Mycobaterium avium Subspecies paratuberulosis Infection in Ruminants 
PLoS Computational Biology  2014;10(1):e1003414.
Johne's disease (JD), a persistent and slow progressing infection of ruminants such as cows and sheep, is caused by slow replicating bacilli Mycobacterium avium subspecies paratuberculosis (MAP) infecting macrophages in the gut. Infected animals initially mount a cell-mediated CD4 T cell response against MAP which is characterized by the production of interferon (Th1 response). Over time, Th1 response diminishes in most animals and antibody response to MAP antigens becomes dominant (Th2 response). The switch from Th1 to Th2 response occurs concomitantly with disease progression and shedding of the bacteria in feces. Mechanisms controlling this Th1/Th2 switch remain poorly understood. Because Th1 and Th2 responses are known to cross-inhibit each other, it is unclear why initially strong Th1 response is lost over time. Using a novel mathematical model of the immune response to MAP infection we show that the ability of extracellular bacteria to persist outside of macrophages naturally leads to switch of the cellular response to antibody production. Several additional mechanisms may also contribute to the timing of the Th1/Th2 switch including the rate of proliferation of Th1/Th2 responses at the site of infection, efficiency at which immune responses cross-inhibit each other, and the rate at which Th1 response becomes exhausted over time. Our basic model reasonably well explains four different kinetic patterns of the Th1/Th2 responses in MAP-infected sheep by variability in the initial bacterial dose and the efficiency of the MAP-specific T cell responses. Taken together, our novel mathematical model identifies factors of bacterial and host origin that drive kinetics of the immune response to MAP and provides the basis for testing the impact of vaccination or early treatment on the duration of infection.
Author Summary
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminants such as sheep and cows. Due to early culling and reduction in milk production of affected animals, MAP inflicts high economic cost to diary farms. MAP infection has a long incubation period of several years, and during the asymptomatic stage a strong cellular (T helper 1) immune response is thought to control MAP replication. Over time, Th1 response is lost and ineffective antibody response driven by Th2 cells becomes predominant. We develop the first mathematical model of helper T cell response to MAP infection to understand impact of various mechanisms on the dynamics of the switch from Th1 to Th2 response. Our results suggest that in contrast to the generally held belief, Th1/Th2 switch may be driven by the accumulation of long-lived extracellular bacteria, and therefore, may be the consequence of the disease progression of MAP-infected animals and not its cause. Our model highlights limitations of our current understanding of regulation of helper T cell responses during MAP infection and identifies areas for future experimental research.
PMCID: PMC3886887  PMID: 24415928
12.  Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila 
PLoS Pathogens  2009;5(12):e1000694.
The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient RelishE20 flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that RelishE20 flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.
Author Summary
Innate immunity is the first line of antimicrobial defence for vertebrates and the only immune response present in invertebrates such as the fruitfly Drosophila, which provides a powerful model system to study innate immunity. Interestingly, local infections of epithelia like the gut and, in our study, the genital tract, result not only in a local immune response, but in an immune response of the whole body. The latter seems to protect Drosophila against the potential spread of local infections. We have investigated the immune response to bacteria placed on the genitalia, at the entrance to both the genital tract and hindgut. This could be a natural entry route of pathogens, possibly linked to sexually transmitted infections. We observe a strong immune response to Gram-negative bacteria, mediated by the immune responsive Imd signalling pathway. This response depends on peptidoglycan, a crucial component of the bacterial cell wall, as pure peptidoglycan placed on the genitalia is sufficient to trigger a whole body immune response. Finally, we present strong evidence that peptidoglycan fragments within the genital tract or hindgut can cross these epithelia, enter the body cavity and thus induce a system wide immune response to a local infection.
PMCID: PMC2787014  PMID: 20019799
13.  Behavioral and Immune Responses to Infection Require Gαq- RhoA Signaling in C. elegans 
PLoS Pathogens  2012;8(2):e1002530.
Following pathogen infection the hosts' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection.
Author Summary
Once infected by a pathogen the nervous and immune systems of many animals react with coordinated responses to the danger. A key question is what are the pathways by which responses to infection occur and to what extent are the same pathways involved in differing responses? Here we demonstrate that a Gαq-RhoA pathway is required for both behavioral and immune responses to infection in C. elegans. We show that Gαq-RhoA signaling is a late step in the response to infection and their site of action defines the cellular targets of signals generated internally in response to infection. One response is to move away from sites of pathogenic bacteria and Gαq-RhoA signaling acts in motorneurons to achieve this. A second response is an innate immune response where Gαq-RhoA signaling acts within cells close to sites of infection, the rectal epithelial cells, to cause major changes in their size and shape to mitigate the effects of infection. Our work demonstrates that ligands for Gq coupled GPCRs are likely to be required for response to infection. Identifying these ligands and the cells that release them will help define the mechanisms by which C. elegans recognizes pathogens and coordinates behavioral and immune responses to infection.
PMCID: PMC3280986  PMID: 22359503
14.  Bifactor Item Response Theory Model of Acute Stress Response 
PLoS ONE  2013;8(6):e65291.
Better understanding of acute stress responses is important for revision of DSM-5. However, the latent structure and relationship between different aspects of acute stress responses haven’t been clarified comprehensively. Bifactor item response model may help resolve this problem.
The purpose of this study is to develop a statistical model of acute stress responses, based on data from earthquake rescuers using Acute Stress Response Scale (ASRS). Through this model, we could better understand acute stress responses comprehensively, and provide preliminary information for computerized adaptive testing of stress responses.
Acute stress responses of earthquake rescuers were evaluated using ASRS, and state/trait anxiety were assessed using State-trait Anxiety Inventory (STAI). A hierarchical item response model (bifactor model) was used to analyze the data. Additionally, we tested this hierarchical model with model fit comparisons with one-dimensional and five-dimensional models. The correlations among acute stress responses and state/trait anxiety were compared, based on both the five-dimensional and bifactor models.
Model fit comparisons showed bifactor model fit the data best. Item loadings on general and specific factors varied greatly between different aspects of stress responses. Many symptoms (40%) of physiological responses had positive loadings on general factor, and negative loadings on specific factor of physiological responses, while other stress responses had positive loadings on both general and specific factors. After extracting general factor of stress responses using bifactor analysis, significant positive correlations between physiological responses and state/trait anxiety (r = 0.185/0.112, p<0.01) changed into negative ones (r = −0.177/−0.38, p<0.01).
Our results demonstrated bifactor structure of acute stress responses, and positive and negative correlations between physiological responses and stress responses suggested physiological responses could have negative feedback on severity of stress responses. This finding has not been convincingly demonstrated in previous research.
PMCID: PMC3676469  PMID: 23762336
15.  Typhoid Fever in Young Children in Bangladesh: Clinical Findings, Antibiotic Susceptibility Pattern and Immune Responses 
PLoS Neglected Tropical Diseases  2015;9(4):e0003619.
Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Typhi) in endemic areas. However, immune responses and clinical findings in children are not well defined. Here, we describe clinical and immunological characteristics of young children with S. Typhi bacteremia, and antimicrobial susceptibility patterns of isolated strains.
As a marker of recent infection, we have previously characterized antibody-in-lymphocyte secretion (TPTest) during acute typhoid fever in adults. We similarly assessed membrane preparation (MP) IgA responses in young children at clinical presentation, and then 7-10 days and 21-28 days later. We also assessed plasma IgA, IgG and IgM responses and T cell proliferation responses to MP at these time points. We compared responses in young children (1-5 years) with those seen in older children (6-17 years), adults (18-59 years), and age-matched healthy controls.
Principal Findings
We found that, compared to age-matched controls patients in all age cohorts had significantly more MP-IgA responses in lymphocyte secretion at clinical presentation, and the values fell in all groups by late convalescence. Similarly, plasma IgA responses in patients were elevated at presentation compared to controls, with acute and convalescent IgA and IgG responses being highest in adults. T cell proliferative responses increased in all age cohorts by late convalescence. Clinical characteristics were similar in all age cohorts, although younger children were more likely to present with loss of appetite, less likely to complain of headache compared to older cohorts, and adults were more likely to have ingested antibiotics. Multi-drug resistant strains were present in approximately 15% of each age cohort, and 97% strains had resistance to nalidixic acid.
This study demonstrates that S. Typhi bacteremia is associated with comparable clinical courses, immunologic responses in various age cohorts, including in young children, and that TPTest can be used as marker of recent typhoid fever, even in young children.
Author Summary
The highest disease burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Typhi) is seen in children under five years of age in endemic areas. We investigated both mucosal and systemic immune responses in S. Typhi bacteremic young children (aged, 1 to 5 years) by measuring S. Typhi membrane preparation (MP) specific IgA response in lymphocyte culture secretion and plasma IgA, IgG and IgM responses using ELISA. We also measured T cell proliferation responses using 3H-thymidine incorporation assay. We compared the responses to S. Typhi bacteremic older children (6 to 17 years) and adults (18 to 59 years) and with age-matched healthy controls (HC). Younger children, older children and adults show comparable responses in lymphocyte secretions after onset of illness. Plasma antibody responses to MP vary between young children and other age groups. T cell proliferative responses increased in all age cohorts by late convalescence. Clinical characteristics were similar in all age cohorts. Emergence of MDR S. Typhi strains is seen in young children which does not impact on the clinical symptoms or the immune responses. The results of this study show that natural infections do induce immune response in young children as well as in adults.
PMCID: PMC4388457  PMID: 25849611
16.  Pharmacological analysis of salivary and blood flow responses to histamine of the submandibular gland of the dog. 
British Journal of Pharmacology  1980;68(4):651-661.
1 The submandibular gland in situ was perfused with blood through the glandular artery at constant pressure in anaesthetized dogs. Drugs were administered intra-arterially. 2 Histamine produced both salivation and an increase in blood flow, each response having an early and a late component. 3 Marked tachyphylaxis to histamine developed in both of the salivary responses but only in the late blood flow response to histamine. 4 The early and late salivary responses were abolished and the late blood flow response was diminished by infusion of tetrodotoxin in doses that abolished the salivary and blood flow responses to electrical stimulation of the chorda-lingual nerve. 5 The whole salivary response to histamine was abolished by infusion of (--)-hyoscyamine in doses that greatly antagonized the salivary and blood flow responses to acetylcholine, whereas the blood flow responses to histamine were scarcely modified. These doses of (--)-hyoscyamine abolished the salivary response to chorda-lingual nerve stimulation but left the blood flow response to it unaffected. 6 The salivary and blood flow responses to histamine were unaffected by infusion of hexamethonium in doses that almost abolished the salivary and blood flow responses to chorda-lingual nerve stimulation. 7 The whole salivary response to histamine was abolished and the late blood response to histamine was partially inhibited by the histamine H1-receptor antagonist, mepyramine, but not by the histamine H2-receptor antagonist, metiamide. 8 The early blood flow response to histamine was antagonized by both mepyramine and metiamide but mepyramine was far more effective than metiamide. 9 These results led to the following conclusions: (1) the whole salivary response and a part of the late blood flow response to histamine are due entirely to excitation of parasympathetic postganglionic neurones; (2) neuronal histamine receptors involved are exclusively of the H1-type; (3) histamine has no direct stimulant action on the glandular cells; (4) the early blood flow response and the remaining part of the late blood flow response to histamine result from the direct action on vascular smooth muscle in the glandular vascular bed; (5) vascular histamine receptors consist of H1- and H2-receptors.
PMCID: PMC2044235  PMID: 7378641
17.  Evolution of Stress-Regulated Gene Expression in Duplicate Genes of Arabidopsis thaliana 
PLoS Genetics  2009;5(7):e1000581.
Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time) is >∼0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.
Author Summary
Plants have developed a multitude of response mechanisms to survive stressful environments. Since the environment is highly variable, these stress response mechanisms are expected to undergo frequent innovation. Duplicate genes represent a potential source for such innovation. In this paper, we explored the evolutionary changes in stress responses at the transcriptional level among duplicated genes in the model plant Arabidopsis thaliana. We found that after gene duplication, ancestral stress responses tend to be retained by only one of the gene duplicates (partitioning). In addition, the pattern of partitioning of multiple stress responses is extremely asymmetric, where one duplicate tends to inherit most or all of the ancestral stress responses. We present evidence that the asymmetric loss of stress responses is correlated with the asymmetric loss of putative transcription factor binding sites. Interestingly, those duplicate genes inheriting few or no ancestral responses tend to have gained new stress responses, providing support for the model that gene duplicates are a source of innovation. Our findings provide important insight into the mechanisms of gene function evolution and lay the foundation for experimental studies to determine the significance of gain of stress responses in plant adaptation.
PMCID: PMC2709438  PMID: 19649161
18.  The Genetic Architecture of the Genome-Wide Transcriptional Response to ER Stress in the Mouse 
PLoS Genetics  2015;11(2):e1004924.
Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. The cellular response to ER stress involves complex transcriptional and translational changes, important to the survival of the cell. ER stress is a primary cause and a modifier of many human diseases. A first step to understanding how the ER stress response impacts human disease is to determine how the transcriptional response to ER stress varies among individuals. The genetic diversity of the eight mouse Collaborative Cross (CC) founder strains allowed us to determine how genetic variation impacts the ER stress transcriptional response. We used tunicamycin, a drug commonly used to induce ER stress, to elicit an ER stress response in mouse embryonic fibroblasts (MEFs) derived from the CC founder strains and measured their transcriptional responses. We identified hundreds of genes that differed in response to ER stress across these genetically diverse strains. Strikingly, inflammatory response genes differed most between strains; major canonical ER stress response genes showed relatively invariant responses across strains. To uncover the genetic architecture underlying these strain differences in ER stress response, we measured the transcriptional response to ER stress in MEFs derived from a subset of F1 crosses between the CC founder strains. We found a unique layer of regulatory variation that is only detectable under ER stress conditions. Over 80% of the regulatory variation under ER stress derives from cis-regulatory differences. This is the first study to characterize the genetic variation in ER stress transcriptional response in the laboratory mouse. Our findings indicate that the ER stress transcriptional response is highly variable among strains and arises from genetic variation in individual downstream response genes, rather than major signaling transcription factors. These results have important implications for understanding how genetic variation impacts the ER stress response, an important component of many human diseases.
Author Summary
Genetic variation among individuals can greatly impact the severity of disease outcomes. To understand the effects of different genetic backgrounds on disease presentation, we focused on ER stress, an important cellular stressor that impacts many human diseases. We examined how genetic variation affects ER stress response, at the RNA level, in eight laboratory mouse strains and their hybrid progeny. We find that each mouse strain responds in a unique way to ER stress, and we characterized the patterns of genetic variation that underlie the differences in ER stress response between the strains. We find that the strains show major differences in their inflammatory response to ER stress, a critical component to disease. The results of this study are important for understanding potential ways in which genetic variation in ER stress response could impact disease, and lays the groundwork for future studies in human patients.
PMCID: PMC4412289  PMID: 25651210
19.  Anion transport blockers inhibit DL-2-amino-4-phosphonobutyrate responses induced by quisqualate in the rat cerebral cortex. 
British Journal of Pharmacology  1993;109(2):449-458.
1. Depolarizing responses to DL-2-amino-4-phosphonobutyrate (AP4) and related amino acids have been studied in the rat cerebral cortex slice following the application of quisqualate (Quis). 2. Before exposure to Quis, 500 microM DL-AP4 had little or no effect. However, following a single application of 40 microM Quis for 2 min, DL-AP4 produced depolarizing responses. With repeated applications of DL-AP4, there was a decline in response amplitude. A second application of Quis restored the depolarizing potency of DL-AP4 to a level above that for the first DL-AP4 response after the first Quis application. With a sequence of alternate applications of Quis and DL-AP4, the amplitude of DL-AP4 responses became maximal after the second Quis application. Responses to DL-AP4 could also be induced by the application of 1 microM Quis for 60 min, but were smaller in amplitude. 3. Responses to the normally inactive amino acids L-cysteine (Cys), L-cystathionine (CTN) and L-alpha-aminoadipate (AA) were also induced once Quis was applied. These responses were also maximized after a second application of Quis, except those to L-Cys, which failed to reach a plateau after three Quis applications. 4. The co-application of DL-AP4 with the first Quis application depressed the subsequent mean DL-AP4 response by 47%. Re-application of Quis restored the amplitude of DL-AP4 responses to levels comparable to control. L-alpha-AA also suppressed the induction of DL-AP4 responses, when co-applied with the first Quis exposure, reducing mean response amplitude by 98%. Unlike DL-AP4, however, the effect with L-alpha-AA persisted so that DL-AP4 responses were significantly suppressed compared to control, even after further applications of Quis. 5. The effects of the anion transport blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetoamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) on the induction process and the DL-AP4 responses themselves were examined. DIDS (100 microM) significantly inhibited the DL-AP4 responses, and to a lesser extent the induction of the responses by 40 microM Quis (2 min), while SITS (300 microM) only inhibited the DL-AP4 responses. However, the induction of responses by 1 microM Quis (60 min) was significantly affected by this concentration of SITS. 6. DIDS (100 microM) had no effect on responses to alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA), but selectively potentiated those to Quis.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2175710  PMID: 7689393
20.  Multiple sources of calcium for contraction of the human urinary bladder muscle. 
British Journal of Pharmacology  1989;98(3):1021-1031.
1. KCl, carbachol, neurokinin A and endothelin produced concentration-dependent contractions of mucosa-free muscle strips from the dome of the human urinary bladder. The maximal response to carbachol or neurokinin A exceeded that to KCl, while the maximal response to endothelin approached that to KCl. 2. Nifedipine (1 microM) abolished the response to KCl, reduced the response to carbachol or neurokinin A but had no effect on the response to endothelin. Bay K 8644 (1 microM) markedly potentiated the response to KCl but had little or no effect on the response produced by the other stimulants. 3. Superfusion of the strips with a nominally calcium (Ca)-free medium containing EDTA (1 mM) for 30 min markedly reduced the response to carbachol, neurokinin A and endothelin, although a small response was still evident at high concentrations. Likewise, after a prolonged (60 min) superfusion of the strips with a high K (80 mM) Ca-free medium plus EDTA (1 mM) these three agonists still produced a small contractile response. 4. The nifedipine (1 microM) resistant response to carbachol, neurokinin A or endothelin was markedly depressed by LaCl3 (1 mM). In contrast, the nifedipine-(1 microM) resistant response to carbachol was not modified by NiCl2 (0.1 mM) or omega-conotoxin (0.1 microM). 5. Caffeine produced divergent effects depending upon the temperature of incubation: a relaxation at 37 degrees C and a concentration-dependent (2.5-20 mM) contraction at 25 degrees C. The latter was markedly inhibited by procaine (3 mM) but unaffected by nifedipine (1 microM). 6. After a prolonged (60 min) superfusion with a high K, Ca-free medium containing EDTA the response to carbachol (100 microM) was abolished by previous exposure to procaine (3 mM). Conversely, the response to endothelin (1 microM) was unaffected by procaine. The response to endothelin in these experimental conditions was also resistant to LaCl3 (1 mM). 7. These findings indicate that multiple sources of Ca are mobilized for contraction of the human bladder muscle by different stimulants. Dihydropyridine- and voltage-sensitive Ca channels provide the major if not the sole source of Ca for the response to KCl, play some role in the response to muscarinic (carbachol) or NK-2 tachykinin receptor stimulation but are not involved in the response to endothelin. Carbachol, neurokinin A and endothelin all mobilize a Ca pool (either extracellular or located at membrane level) which is LaCl3-sensitive but nifedipine-resistant. Neither T- nor N-type channels appear to be involved in the response to carbachol.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC1854771  PMID: 2480167
21.  Evaluation and Validation of Neo-Adjuvant Response Index (NRI) and It’s Correlation with Various Predictive Biomarkers and RECIST in Locally Advanced Breast Cancer 
Response evaluation following neo-adjuvant chemotherapy in breast cancer is usually done without taking in to account the axillary response and the available tools like ‘response evaluation criteria in solid tumors’ (RECIST) have this limitation. These criteria rely solely on the response observed in the primary tumour. Neoadjuvant response index is one such attempt to have a comprehensive assessment of response both in the primary tumour and the axilla.
30 cases of locally advanced breast cancer (LABC) were assessed for response using ‘Neo-adjuvant Response Index’. The index always gives score between ‘0’ (no response or progressive disease) and ‘1’ (pathological complete response i.e. no invasive tumor in breast as well as axilla). This index includes axillary response as well and provides a spectrum of response rather than dividing patients into simply responders and non-responders .
Mean reading of index was found to be 0.2925 in this study. Three patients achieved an index of 1. This index correlates significantly with the existing scales for assessing response. Hormone negative tumors were found to be more chemo responsive with higher rates of pathological complete response (pCR) while ER/PR + Her2- tumors showed a very poor response to NACT.
Based on the observations of the present study it may be submitted that Neoadjuvant Response Index (NRI) is a reliable and simple tool that can serve as a comprehensive and accurate method of assessing response to neo-adjuvant chemotherapy as it takes in to consideration both the tumor and axillary response unlike the existing RECIST, binary system (responders are those with greater than 50 % reduction), RCB method and the available biomarkers. This study being first of it’s kind in Indian population, in spite of it’s limitations, could prove to be a launching ground for further reasearch and contribute substantially to the evidence base.
PMCID: PMC4235867  PMID: 25419059
Neo-adjuvant response index; NRI; Locally advanced breast cancer; RECIST; LABC
22.  Response Rates and Response Bias for 50 Surveys of Pediatricians 
Health Services Research  2005;40(1):213-226.
Research Objective
To track response rates across time for surveys of pediatricians, to explore whether response bias is present for these surveys, and to examine whether response bias increases with lower response rates.
Data Source/Study Setting
A total of 63,473 cases were gathered from 50 different surveys of pediatricians conducted by the American Academy of Pediatrics (AAP) since 1994. Thirty-one surveys targeted active U.S. members of the AAP, six targeted pediatric residents, and the remaining 13 targeted AAP-member and nonmember pediatric subspecialists. Information for the full target samples, including nonrespondents, was collected using administrative databases of the AAP and the American Board of Pediatrics.
Study Design
To assess bias for each survey, age, gender, location, and AAP membership type were compared for respondents and the full target sample. Correlational analyses were conducted to examine whether surveys with lower response rates had increasing levels of response bias.
Principal Findings
Response rates to the 50 surveys examined declined significantly across survey years (1994–2002). Response rates ranged from 52 to 81 percent with an average of 68 percent. Comparisons between respondents and the full target samples showed the respondent group to be younger, to have more females, and to have less specialty-fellow members. Response bias was not apparent for pediatricians' geographical location. The average response bias, however, was fairly small for all factors: age (0.45 years younger), gender (1.4 percentage points more females), and membership type (1.1 percentage points fewer specialty-fellow members). Gender response bias was found to be inversely associated with survey response rates (r=−0.38). Even for the surveys with the lowest response rates, amount of response bias never exceeded 5 percentage points for gender, 3 years for age, or 3 percent for membership type.
While response biases favoring women, young physicians, and nonspecialty-fellow members were found across the 52–81 percent response rates examined in this study, the amount of bias was minimal for these factors that could be tested. At least for surveys of pediatricians, more attention should be devoted by investigators to assessments of response bias rather than relying on response rates as a proxy of response bias.
PMCID: PMC1361134  PMID: 15663710
Response rate; response bias; physician survey
23.  A modular gradient-sensing network for chemotaxis in Escherichia coli revealed by responses to time-varying stimuli 
Combining in vivo FRET with time-varying stimuli, such as steps, ramps, and sinusoids allowed deduction of the molecular mechanisms underlying cellular signal processing.The bacterial chemotaxis pathway can be described as a two-module feedback circuit, the transfer functions of which we have characterized quantitatively by experiment. Model-driven experimental design allowed the use of a single FRET pair for measurements of both transfer functions of the pathway.The adaptation module's transfer function revealed that feedback near steady state is weak, consistent with high sensitivity to shallow gradients, but also strong steady-state fluctuations in pathway output.The measured response to oscillatory stimuli defines the frequency band over which the chemotaxis system can compute time derivatives.
In searching for better environments, bacteria sample their surroundings by random motility, and make temporal comparisons of experienced sensory cues to bias their movement toward favorable directions (Berg and Brown, 1972). Thus, the problem of sensing spatial gradients is reduced to time-derivative computations, carried out by a signaling pathway that is well characterized at the molecular level in Escherichia coli. Here, we study the physiology of this signal processing system in vivo by fluorescence resonance energy transfer (FRET) experiments in which live cells are stimulated by time-varying chemoeffector signals. By measuring FRET between the active response regulator of the pathway CheY-P and its phosphatase CheZ, each labeled with GFP variants, we obtain a readout that is directly proportional to pathway activity (Sourjik et al, 2007). We analyze the measured response functions in terms of mechanistic models of signaling, and discuss functional consequences of the observed quantitative characteristics.
Experiments are guided by a coarse-grained modular model (Tu et al, 2008) of the sensory network (Figure 1), in which we identify two important ‘transfer functions': one corresponding to the receptor–kinase complex, which responds to changes in input ligand concentration on a fast time scale, and another corresponding to the adaptation system, which provides negative feedback, opposing the effect of ligand on a slower time scale. For the receptor module, we calibrate an allosteric MWC-type model of the receptor–kinase complex by FRET measurements of the ‘open-loop' transfer function G([L],m) using step stimuli. This calibration provides a basis for using the same FRET readout (between CheY-P and CheZ) to further study properties of the adaptation module.
It is well known that adaptation in E. coli's chemotaxis system uses integral feedback, which guarantees exact restoration of the baseline activity after transient responses to step stimuli (Barkai and Leibler, 1997; Yi et al, 2000). However, the output of time-derivative computations during smoothly varying stimuli depends not only on the presence of integral feedback, but also on what is being integrated. As this integrand can in general be any function of the output, we represent it by a black-box function F(a) in our model, and set out to determine its shape by experiments with time-varying stimuli.
We first apply exponential ramp stimuli—waveforms in which the logarithm of the stimulus level varies linearly with time, at a fixed rate r. It was shown many years ago that during such a stimulus, the kinase output of the pathway changes to a new constant value, ac that is dependent on the applied ramp rate, r (Block et al, 1983). A plot of ac versus r (Figure 5A) can thus be considered as an output of time-derivative computations by the network, and could also be used to study the ‘gradient sensitivity' of bacteria traveling at constant speeds.
To obtain the feedback transfer function, F(a), we apply a simple coordinate transformation, identified using our model, to the same ramp-response data (Figure 5B). This function reveals how the temporal rate of change of the feedback signal m depends on the current output signal a. The shape of this function is analyzed using a biochemical reaction scheme, from which in vivo kinetic parameters of the feedback enzymes, CheR and CheB, are extracted. The fitted Michaelis constants for these enzymatic reactions are small compared with the steady-state abundance of their substrates, thus indicating that these enzymes operate close to saturation in vivo. The slope of the function near steady state can be used to assess the strength of feedback, and to compute the relaxation time of the system, τm. Relaxation is found to be slow (i.e. large τm), consistent with large fluctuations about the steady-state activity caused by the near-saturation kinetics of the feedback enzymes (Emonet and Cluzel, 2008).
Finally, exponential sine-wave stimuli are used to map out the system's frequency response (Figure 5C). The measured data points for both the amplitude and phase of the response are found to be in excellent agreement with model predictions based on parameters from the independently measured step and ramp responses. No curve fitting was required to obtain this agreement. Although the amplitude response as a function of frequency resembles a first-order high-pass filter with a well-defined cutoff frequency, νm, we point out that the chemotaxis pathway is actually a low-pass filter if the time derivative of the input is viewed as the input signal. In this latter perspective, νm defines an upper bound for the frequency band over which time-derivative computations can be carried out.
The two types of measurements yield complementary information regarding time-derivative computations by E. coli. The ramp-responses characterize the asymptotically constant output when a temporal gradient is held fixed over extended periods. Interestingly, the ramp responses do not depend on receptor cooperativity, but only on properties of the adaptation system, and thus can be used to reveal the in vivo adaptation kinetics, even outside the linear regime of the kinase response. The frequency response is highly relevant in considering spatial searches in the real world, in which experienced gradients are not held fixed in time. The characteristic cutoff frequency νm is found by working within the linear regime of the kinase response, and depends on parameters from both modules (it increases with both cooperativity in the receptor module, and the strength of feedback in the adaptation module).
Both ramp responses and sine-wave responses were measured at two different temperatures (22 and 32°C), and found to differ significantly. Both the slope of F(a) near steady state, from ramp experiments, and the characteristic cutoff frequency, from sine-wave experiments, were higher by a factor of ∼3 at 32°C. Fits of the enzymatic model to F(a) suggest that temperature affects the maximal velocity (Vmax) more strongly than the Michaelis constants (Km) for CheR and CheB.
Successful application of inter-molecular FRET in live cells using GFP variants always requires some degree of serendipity. Genetic fusions to these bulky fluorophores can impair the function of the original proteins, and even when fusions are functional, efficient FRET still requires the fused fluorophores to come within the small (<10 nm) Förster radius on interactions between the labeled proteins. Thus, when a successful FRET pair is identified, it is desirable to make the most of it. We have shown here that combined with careful temporal control of input stimuli, and appropriately calibrated models, a single FRET pair can be used to study the structure of multiple transfer functions within a signaling network.
The Escherichia coli chemotaxis-signaling pathway computes time derivatives of chemoeffector concentrations. This network features modules for signal reception/amplification and robust adaptation, with sensing of chemoeffector gradients determined by the way in which these modules are coupled in vivo. We characterized these modules and their coupling by using fluorescence resonance energy transfer to measure intracellular responses to time-varying stimuli. Receptor sensitivity was characterized by step stimuli, the gradient sensitivity by exponential ramp stimuli, and the frequency response by exponential sine-wave stimuli. Analysis of these data revealed the structure of the feedback transfer function linking the amplification and adaptation modules. Feedback near steady state was found to be weak, consistent with strong fluctuations and slow recovery from small perturbations. Gradient sensitivity and frequency response both depended strongly on temperature. We found that time derivatives can be computed by the chemotaxis system for input frequencies below 0.006 Hz at 22°C and below 0.018 Hz at 32°C. Our results show how dynamic input–output measurements, time honored in physiology, can serve as powerful tools in deciphering cell-signaling mechanisms.
PMCID: PMC2913400  PMID: 20571531
adaptation; feedback; fluorescence resonance energy transfer (FRET); frequency response; Monod–Wyman–Changeux (MWC) model
24.  SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens 
PLoS Pathogens  2013;9(8):e1003539.
Enteric bacterial pathogens such as enterohemorrhagic E. coli (EHEC) and Salmonella Typhimurium target the intestinal epithelial cells (IEC) lining the mammalian gastrointestinal tract. Despite expressing innate Toll-like receptors (TLRs), IEC are innately hypo-responsive to most bacterial products. This is thought to prevent maladaptive inflammatory responses against commensal bacteria, but it also limits antimicrobial responses by IEC to invading bacterial pathogens, potentially increasing host susceptibility to infection. One reason for the innate hypo-responsiveness of IEC is their expression of Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and TLR signaling. To address whether SIGIRR expression and the innate hypo-responsiveness of IEC impacts on enteric host defense, Sigirr deficient (−/−) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr −/− mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses but surprisingly, Sigirr −/− mice proved dramatically more susceptible to infection than wildtype mice. Through haematopoietic transplantation studies, it was determined that SIGIRR expression by non-haematopoietic cells (putative IEC) regulated these responses. Moreover, the exaggerated responses were found to be primarily dependent on IL-1R signaling. Whilst exploring the basis for their susceptibility, Sigirr −/− mice were found to be unusually susceptible to intestinal Salmonella Typhimurium colonization, developing enterocolitis without the typical requirement for antibiotic based removal of competing commensal microbes. Strikingly, the exaggerated antimicrobial responses seen in Sigirr −/− mice were found to cause a rapid and dramatic loss of commensal microbes from the infected intestine. This depletion appears to reduce the ability of the microbiota to compete for space and nutrients (colonization resistance) with the invading pathogens, leaving the intestine highly susceptible to pathogen colonization. Thus, SIGIRR expression by IEC reflects a strategy that sacrifices maximal innate responsiveness by IEC in order to promote commensal microbe based colonization resistance against bacterial pathogens.
Author Summary
Despite being in close contact with billions of commensal bacteria, the epithelial cells that line the intestine develop very weak innate inflammatory responses to bacterial products. The goal of this study was to explore why these cells respond so poorly, and how increasing their innate responsiveness would impact on host defense against invading bacterial pathogens. We show that a negative regulator of innate signaling called SIGIRR, limits the inflammatory responses of the intestine to bacteria. Following infection by the bacterial pathogen Citrobacter rodentium, the intestines of mice lacking SIGIRR showed exaggerated inflammatory, antimicrobial and proliferative responses. Through transplantation studies, we showed it was SIGIRR expression by intestinal epithelial cells that limits these responses, and that the exaggerated responses were driven by cytokine signaling through the interleukin-1 receptor. Despite their exaggerated responses, SIGIRR deficient mice proved extremely susceptible to infection by C. rodentium and other intestinal bacterial pathogens. We found the exaggerated inflammatory responses rapidly depleted intestinal commensal microbes, reducing their ability to outcompete invading pathogens for space and nutrients (colonization resistance). Our study thus clarifies that the hypo-responsiveness of epithelial cells plays an unexpected but critical role in host defense, by promoting commensal microbe based competition against enteric pathogens.
PMCID: PMC3738496  PMID: 23950714
25.  Vaccination with Melanoma Helper Peptides Induces Antibody Responses Associated with Improved Overall Survival 
A melanoma vaccine incorporating six peptides designed to induce helper T cell responses to melanoma antigens has induced Th1-dominant CD4+ T cell responses in most patients, and induced durable clinical responses or stable disease in 24% of evaluable patients. The present study tested whether this vaccine also induced antibody (Ab) responses to each peptide, and whether Ab responses were associated with T cell responses and with clinical outcome.
Patients and Methods
Serum samples were studied from 35 patients with stage III–IV melanomas vaccinated with 6 melanoma helper peptides (6MHP). IgG Ab responses were measured by ELISA. Associations with immune response and overall survival were assessed by log-rank test and Chi square analysis of Kaplan-Meier data.
Ab responses to 6MHP were detected by week 7 in 77% of patients, and increased to peak 6 weeks after the last vaccine and persisted to 6 months. Ab responses were induced most frequently to longer peptides. Of those with T cell responses, 82% had early Ab responses. Survival was improved for patients with early Ab response (p = 0.0011) or with early T cell response (p < 0.006), and was best for those with both Ab and T cell responses (p = 0.0002).
Vaccination with helper peptides induced both Ab responses and T cell responses, associated with favorable clinical outcome. Such immune responses may predict favorable clinical outcome to guide combination immunotherapy. Further studies are warranted to understand mechanisms of interaction of these Abs, T cell responses, and tumor control.
PMCID: PMC4558239  PMID: 25967144
Melanoma; Immune therapy; Vaccination; Antibody; Helper T cell; peptides; Cancer Vaccines; Human; Clinical Trial; Survival

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