Leukotriene E4 (LTE4), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y12 is required for LTE4 mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y12 to cysteinyl leukotrienes. Models of human CysLT1, CysLT2 and P2Y12 overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT1 and CysLT2 but failed to induce any calcium response in cells expressing P2Y12 or P2Y12+ Gα16. In contrast, selective P2Y12 agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y12+ Gα16. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y12 when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y12 to analyse potential signalling and cell activation through P2Y12 receptor or receptor heterodimers but no specific LTE4 responses were observed. These results show that LTE4 as well as other cysLTs do not activate intracellular signalling acting through P2Y12 and suggest that another LTE4 specific receptor has yet to be identified.
In low-resource areas, the most common method of tuberculosis (TB) diagnosis is visual identification of rod-shaped TB bacilli in microscopic images of sputum smears. We present an algorithm for automated TB detection using images from digital microscopes such as CellScope , a novel, portable device capable of brightfield and fluorescence microscopy. Automated processing on such platforms could save lives by bringing healthcare to rural areas with limited access to laboratory-based diagnostics. Our algorithm applies morphological operations and template matching with a Gaussian kernel to identify candidate TB-objects. We characterize these objects using Hu moments, geometric and photometric features, and histograms of oriented gradients and then perform support vector machine classification. We test our algorithm on a large set of CellScope images (594 images corresponding to 290 patients) from sputum smears collected at clinics in Uganda. Our object-level classification performance is highly accurate, with Average Precision of 89.2% ± 2.1%. For slide-level classification, our algorithm performs at the level of human readers, demonstrating the potential for making a significant impact on global healthcare.
Visual loss in infectious posterior uveitis or panuveitis can occur if proper therapy is delayed because of diagnostic uncertainty. Some disorders, such as acute retinal necrosis and bacterial endophthalmitis, can be rapidly progressive, and therefore require prompt and accurate diagnosis to guide initial therapy. Other more slowly evolving infections, such as toxoplasmic chorioretinitis or fungal endophthalmitis, can be worsened by empiric use of corticosteroids without specific antimicrobial coverage. Key ocular diagnostic features are helpful but highly variable with overlap with both non-infectious uveitis and neoplastic masquerades, even for key signs such as hypopyon. Close examination of the fundus with attention to color, location, size, border, and opacity of lesions and associated arteriolitis or frosted branch angiitis is helpful in the diagnosis of chorioretinitis. Ultrasonography is an important tool in the evaluation of eyes with suspected endophthalmitis, especially those with intracapsular infection or focal infected deposits. Testing of intraocular fluid can be extremely useful but suffers from inaccessibility, poor sensitivity, and test selections dependent on a presumptive diagnosis, which may be wrong. The dilemma for clinician is to make the correct diagnosis of a rare, blinding, variegated disease quickly enough to intercede with specific therapy or to apply empiric therapy in a sufficiently skilled manner to avert disaster and confirm the diagnosis by response to treatment. When non-infectious uveitis is in the differential, empiric corticosteroids must sometimes be used, at great risk, if clinical examination, ancillary testing, and any available intraocular diagnostic tests have failed to confirm a diagnosis.
chorioretinitis; endophthalmitis; acute retinal necrosis; cytomegalovirus retinitis; syphilis; PCR
Pneumocystis jirovecii is an important opportunistic infection in HIV-infected patients. In the developed world, P. jirovecii epidemiology is marked by frequent colonization in immunosuppressed patients, but data on the prevalence of colonization is very limited in sub-Saharan Africa, where the majority of persons living with HIV reside. Our objective was to describe the epidemiology of P. jirovecii colonization among HIV-positive patients in a cross-sectional, hospital-based study of patients admitted with suspected pneumonia in Kampala, Uganda. P. jirovecii was detectable in bronchoalveolar lavage fluid from 7 of 124 (6%) consecutive patients with non-Pneumocystis pneumonia. Colonization was not associated with patient demographic or clinical information. This prevalence is substantially lower than in published studies in the developed world, and suggests that there is a limited reservoir of organisms for clinical infections in this Ugandan population. These findings may partially explain the low incidence of Pneumocystis pneumonia in Uganda and other sub-Saharan African countries.
Pneumocystis jirovecii; colonization; pneumonia; AIDS
The etiology of colon cancer is a complex phenomenon that involves both genetic and environmental factors. However, only about 20% have a familial basis with the largest fraction being attributed to environmental causes that can lead to chronic inflammation. While the link between inflammation and colon cancer is well established, the temporal sequence of the inflammatory response in relation to tumorigenesis has not been characterized. We examined the timing and magnitude of the intestinal inflammatory cytokine response in relation to tumorigenesis in the ApcMin/+ mouse. ApcMin/+ mice and wildtype mice were sacrificed at one of 4 time-points: 8, 12, 16, and 20 wks of age. Intestinal tissue was analyzed for polyp burden (sections 1, 4 and 5) and mRNA expression and protein concentration of MCP-1, IL-1β, IL-6 and TNF-α (sections 2 and 3). The results show that polyp burden was increased at 12, 16 and 20 wks compared to 8 wks (P<0.05). Gene expression (mRNA) of MCP-1, IL-1β, IL-6 and TNF-α was increased in sections 2 and 3 starting at wk 12 (P<0.05), with further increases in MCP-1, IL-1β and IL-6 at 16 wks (P<0.05). Protein concentration for these cytokines followed a similar pattern in section 3. Similarly, circulating MCP-1 was increased at 12 wks (P<0.05) and then again at 20 wks (P<0.05). In general, overall polyp number and abundance of large polyps were significantly correlated with the inflammatory cytokine response providing further support for a relationship between polyp progression and these markers. These data confirm the association between intestinal cytokines and tumorigenesis in the ApcMin/+ mouse and provide new information on the timing and magnitude of this response in relation to polyp development. These findings may lead to the development of inflammatory mediators as important biomarkers for colon cancer progression. Further, these data may be relevant in the design of future investigations of therapeutic interventions to effectively target inflammatory processes in rodent models.
colon cancer; inflammation; mouse models; monocyte chemoattractant protein 1; pro-inflammatory cytokines
Little is known about the serologic responses to Pneumocystis jirovecii major surface glycoprotein (Msg) antigen in African cohorts, or the IgM responses to Msg in HIV-positive and HIV-negative persons with respiratory symptoms.
We conducted a prospective study of 550 patients, both HIV-positive (n = 467) and HIV-negative (n = 83), hospitalized with cough ≥2 weeks in Kampala, Uganda, to evaluate the association between HIV status, CD4 cell count, and other clinical predictors and antibody responses to P. jirovecii. We utilized ELISA to measure the IgM and IgG serologic responses to three overlapping recombinant fragments that span the P. jirovecii major surface glycoprotein: MsgA (amino terminus), MsgB (middle portion) and MsgC1 (carboxyl terminus), and to three variations of MsgC1 (MsgC3, MsgC8 and MsgC9).
HIV-positive patients demonstrated significantly lower IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 compared to HIV-negative patients. We found the same pattern of low IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 among HIV-positive patients with a CD4 cell count <200 cells/µl compared to those with a CD4 cell count ≥200 cells/µl. HIV-positive patients on PCP prophylaxis had significantly lower IgM responses to MsgC3 and MsgC9, and lower IgG responses to MsgA, MsgC1, MsgC3, and MsgC8. In contrast, cigarette smoking was associated with increased IgM antibody responses to MsgC1 and MsgC3 but was not associated with IgG responses. We evaluated IgM and IgG as predictors of mortality. Lower IgM responses to MsgC3 and MsgC8 were both associated with increased in-hospital mortality.
HIV infection and degree of immunosuppression are associated with reduced IgM responses to Msg. In addition, low IgM responses to MsgC3 and MsgC8 are associated with increased mortality.
Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.
The clinical impact of Xpert MTB/RIF for tuberculosis (TB) diagnosis in high HIV-prevalence settings is unknown.
To determine the diagnostic accuracy and impact of Xpert MTB/RIF among high-risk TB suspects.
We prospectively enrolled consecutive, hospitalized, Ugandan TB suspects in two phases: baseline phase in which Xpert MTB/RIF results were not reported to clinicians and an implementation phase in which results were reported. We determined the diagnostic accuracy of Xpert MTB/RIF in reference to culture (solid and liquid) and compared patient outcomes by study phase.
477 patients were included (baseline phase 287, implementation phase 190). Xpert MTB/RIF had high sensitivity (187/237, 79%, 95% CI: 73–84%) and specificity (190/199, 96%, 95% CI: 92–98%) for culture-positive TB overall, but sensitivity was lower (34/81, 42%, 95% CI: 31–54%) among smear-negative TB cases. Xpert MTB/RIF reduced median days-to-TB detection for all TB cases (1 [IQR 0–26] vs. 0 [IQR 0–1], p<0.001), and for smear-negative TB (35 [IQR 22–55] vs. 22 [IQR 0–33], p = 0.001). However, median days-to-TB treatment was similar for all TB cases (1 [IQR 0–5] vs. 0 [IQR 0–2], p = 0.06) and for smear-negative TB (7 [IQR 3–53] vs. 6 [IQR 1–61], p = 0.78). Two-month mortality was also similar between study phases among 252 TB cases (17% vs. 14%, difference +3%, 95% CI: −21% to +27%, p = 0.80), and among 87 smear-negative TB cases (28% vs. 22%, difference +6%, 95% CI: −34 to +46%, p = 0.77).
Xpert MTB/RIF facilitated more accurate and earlier TB diagnosis, leading to a higher proportion of TB suspects with a confirmed TB diagnosis prior to hospital discharge in a high HIV/low MDR TB prevalence setting. However, our study did not detect a decrease in two-month mortality following implementation of Xpert MTB/RIF possibly because of insufficient powering, differences in empiric TB treatment rates, and disease severity between study phases.
Increasing evidence suggests that epigenetic dysfunction may account for the alteration of gene transcription present in neuropsychiatric disorders such as schizophrenia (SZ), bipolar disorder (BP) and autism. Here, we studied the expression of the ten-eleven translocation (TET) gene family and activation-induced deaminase/apolipoprotein B mRNA-editing enzymes (AID/APOBEC) in the inferior parietal lobule (IPL) (BA39-40) and the cerebellum of psychotic (PSY) patients, depressed (DEP) patients and nonpsychiatric (CTR) subjects obtained from the Stanley Foundation Neuropathology Consortium Medical Research Institute. These two sets of enzymes have a critical role in the active DNA demethylation pathway. The results show that TET1, but not TET2 and TET3, mRNA and protein expression was increased (two- to threefold) in the IPL of the PSY patients compared with the CTR subjects. TET1 mRNA showed no change in the cerebellum. Consistent with the increase of TET1, the level of 5-hydroxymethylcytosine (5hmC) was elevated in the IPL of PSY patients but not in the other groups. Moreover, higher 5hmC levels were detected at the glutamic acid decarboxylase67 (GAD67) promoter only in the PSY group. This increase was inversely related to the decrease of GAD67 mRNA expression. Of 11 DNA deaminases measured, APOBEC3A mRNA was significantly decreased in the PSY and DEP patients, while APOBEC3C was decreased only in PSY patients. The other APOBEC mRNA studied failed to change. Increased TET1 and decreased APOBEC3A and APOBEC3C found in this study highlight the possible role of altered DNA demethylation mechanisms in the pathophysiology of psychosis.
post-mortem brain; TET; AID/APOBECs; 5hmC; epigenetic
Skeletal muscle interleukin-6 (IL-6) expression is induced by continuous contraction, overload-induced hypertrophy and during muscle regeneration. The loss of IL-6 can alter skeletal muscle’s growth and extracellular matrix remodeling response to overload-induced hypertrophy. IGF-1 gene expression and related signaling through Akt/mTOR is a critical regulator of muscle mass. The significance of IL-6 expression during the recovery from muscle atrophy is unclear. This study’s purpose was to determine the effect of IL-6 loss on mouse gastrocnemius (GAS) muscle mass during recovery from hindlimb suspension (HS)-induced atrophy.
Female C57BL/6 (WT) and IL-6 knockout (IL-6 KO) mice at 10 wks of age were assigned to control, HS, or HS followed by normal cage ambulation groups.
GAS muscle atrophy was induced by 10 days of HS. HS induced a 20% loss of GAS mass in both WT and IL-6 KOmice. HS+7 days of recovery restored WT GAS mass to cage control values. GAS mass from IL-6 KO mice did not return to cage control values until HS+14 days of recovery. Both IGF-1 mRNA expression and Akt/mTOR signaling were increased in WT muscle after 1 day of recovery. In IL-6 KO muscle, IGF-1 mRNA expression was decreased and Akt/mTOR signaling was not induced after one day of recovery. MyoD and myogenin mRNA expression were both induced in WT muscle after 1 day of recovery, but not in IL-6 KO muscle.
Muscle IL-6 expression appears important for the initial growth response during the recovery from disuse.
inflammation; myogenesis; muscle regeneration; hypertrophy; IGF-1; mTOR; Stat3
Contamination by bacterial or fungal organisms reduces the effectiveness of mycobacterial culture for diagnosis of pulmonary tuberculosis (TB). We evaluated the effect of an anti-microbial and an anti-fungal oral rinse prior to expectoration on culture-contamination rates.
We enrolled a consecutive random sample of adults with cough for ≥2 weeks and suspected TB admitted to Mulago Hospital (Kampala, Uganda) between October 2008 and June 2009. We randomly assigned patients to oral rinse (60 seconds with chlorhexidine followed by 60 seconds with nystatin) vs. no oral rinse prior to initial sputum collection. Uganda National Tuberculosis Reference Laboratory technicians blinded to the method of sputum collection (with or without oral rinse) processed all sputum specimens for smear microscopy (direct Ziehl-Neelsen) and mycobacterial culture (Lowenstein-Jensen media).
Of 220 patients enrolled, 177 (80%) were HIV-seropositive (median CD4-count 37 cells/uL, IQR 13–171 cells/uL). Baseline characteristics were similar between patients in the oral-rinse (N = 110) and no oral-rinse (N = 110) groups. The proportion of contaminated cultures was significantly lower in the oral-rinse group compared to the no oral-rinse group (4% vs. 15%, risk difference −11%, 95% CI −18 to −3%, p = 0.005). Oral rinse significantly reduced the proportion of contaminated cultures among HIV-infected patients (3% vs. 18%, risk difference −14%, 95% CI −23 to −6%, p = 0.002) but not HIV-uninfected (6% vs. 4%, risk difference 2%, 95% CI −12 to +15%, p = 0.81) patients. However, the proportion of smear-positive specimens (25% vs. 35%, p = 0.10) and culture-positive specimens (48% vs. 56%, p = 0.24) were lower in the oral-rinse compared to the no oral-rinse group, although the differences were not statistically significant.
Oral rinse prior to sputum expectoration is a promising strategy to reduce mycobacterial culture contamination in areas with high HIV prevalence, if strategies can be devised to reduce the adverse impact of oral rinse on smear- and culture-positivity.
Background and objectives
The frequency, aetiologies, and outcomes of normal chest radiographs (CXRs) among HIV-seropositive patients with suspected pulmonary tuberculosis (TB) have been infrequently described.
Consecutive HIV-seropositive adults hospitalized for cough of ≥ 2 weeks duration at Mulago Hospital (Kampala, Uganda), between September 2007 and July 2008, were enrolled. Baseline CXRs were obtained on admission. Patients with sputum smears that were negative for acid-fast bacilli (AFB) were referred for bronchoscopy with bronchoalveolar lavage (BAL). BAL fluid was examined for mycobacteria, Pneumocystis jirovecii, and other fungi. Patients were followed for two months after enrolment.
Of the 334 patients, 54 (16%) had normal CXRs. These patients were younger (median age 30 vs. 34 years, P=0.002), had lower counts of CD4+ T lymphocytes (median 13 vs. 57 cells/μL, P<0.001), and were less likely to be smear positive for AFB (17% vs. 39%, P=0.002) than those with abnormal CXRs. Pulmonary TB was the most frequent diagnosis (44%) among those with normal CXRs, followed by unknown diagnoses, pulmonary aspergillosis, and pulmonary cryptococcosis. The frequency of normal CXRs was 12% among pulmonary TB patients. There was a trend towards increased two-month mortality among patients with normal CXRs compared to those with abnormal CXRs (40% vs. 29%, P=0.15).
Normal CXR findings were common among HIV-seropositive patients with suspected TB, especially those who were young, those with low CD4+ T cell counts, and those with sputum smears that were negative for AFB. Mortality was high among those with normal CXRs. Normal CXR findings should not preclude further diagnostic evaluation in this population.
clinical epidemiology; critical care medicine; immunodeficiency; radiology and other imaging; tuberculosis
Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2 and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging (BLI). Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2 invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.
Bioluminescence imaging; pancreatic ductal adenocarcinoma; ARF; CtBP2; xenograft model of metastasis
Many observational epidemiologic studies suggest an association between exercise and breast cancer risk. However, the lack of controlled experimental studies that examine this relationship and the mechanisms involved weaken the basis for inferring a causal relationship. Inflammation plays a role in breast cancer progression and exercise has been reported to reduce inflammation; however, the inflammatory effects of exercise in breast cancer have yet to be established. We examined the relationship between exercise training and systemic inflammation in relation to breast cancer progression in C3(1)SV40Tag mice. Female C3(1)SV40Tag mice were assigned to either exercise (Ex) or sedentary (Sed) treatment (n=12-14/group). Beginning at 4wks of age mice (Ex) were run on a treadmill for 60 min/d (20 m/min and 5% grade), 6 d/wk for a period of 20wks. Mice were examined weekly for palpable tumors, and tumor number and volume were recorded. At 24wks of age mice were sacrificed and a more direct measure of tumor number and volume, and spleen weight was recorded. Plasma was analyzed for MCP-1 and IL-6 concentration using ELISA. Ex reduced palpable tumor number at sacrifice (24wks) by approximately 70% (P<0.05). Tumor volume was also reduced in Ex at 21 - 23wks (P<0.05). This reduction in tumor progression by Ex was associated with a reduction in plasma concentration of MCP-1 and IL-6, and spleen weight (P<0.05). These data provide strong support for a beneficial effect of exercise training on tumor progression in the C3(1)SV40Tag mouse model of breast cancer that may be partly mediated by its anti-inflammatory potential.
physical activity; mammary tumorigenesis; mouse models; cytokines
Peripheral blood interferon-gamma release assays (IGRAs) have sub-optimal sensitivity and specificity for diagnosis of active pulmonary tuberculosis (TB). However, assessment of local immune responses has been reported to improve the accuracy of TB diagnosis.
We enrolled HIV-infected adults with cough ≥2 weeks’ duration admitted to Mulago Hospital in Kampala, Uganda and referred for bronchoscopy following two negative sputum acid-fast bacillus smears. We performed an ELISPOT-based IGRA (T-SPOT.TB®, Oxford Immunotec, Oxford, UK) using peripheral blood and bronchoalveolar lavage (BAL) fluid mononuclear cells, and determined the accuracy of IGRAs using mycobacterial culture results as a reference standard.
94 HIV-infected patients with paired peripheral blood and BAL IGRA results were included. The study population was young (median age 34 years [IQR 28–40 years]) and had advanced HIV/AIDS (median CD4+ T-lymphocyte count 60 cells/µl [IQR 22–200 cells/µl]). The proportion of indeterminate IGRA results was higher in BAL fluid than in peripheral blood specimens (34% vs. 14%, difference 20%, 95% CI 7–33%, p = 0.002). BAL IGRA had moderate sensitivity (73%, 95% CI 50–89%) but poor specificity (48%, 95% CI 32–64%) for TB diagnosis. Sensitivity was similar (75%, 95% CI 57–89%) and specificity was higher (78%, 95% CI 63–88%) when IGRA was performed on peripheral blood.
BAL IGRA performed poorly for the diagnosis of smear-negative TB in a high HIV/TB burden setting. Further studies are needed to examine reasons for the large proportion of indeterminate results and low specificity of BAL IGRA for active TB in high HIV/TB burden settings.
Numerous in vitro studies argue for quercetin’s chemopreventive potential in colon cancer; however experimental studies in rodents are limited. Macrophages play a role in tumorigenesis, but the effects of quercetin on macrophage infiltration in colon cancer is unknown. We examined the effects of quercetin on intestinal polyp multiplicity and macrophage number in ApcMin/+ mice. ApcMin/+ mice were assigned to placebo or quercetin (n=8/group) groups. Mice were given a placebo or quercetin (0.02%) diet from 4–20 wks of age after which intestines were analyzed for polyp number and size in the small intestine (sections 1–4) and colon (section 5) and for macrophage number in the small intestine (section 1 and 3). Spleen weight was determined as a marker of systemic inflammation. Quercetin decreased total intestinal polyps by 67% (p<0.05). Specifically, quercetin reduced intestinal polyps in categories >2 mm (69%) and 1–2 mm (79%) (P<0.05), and in sections 2 (75%), 3 (80%) and 4 (79%) (P<0.05). Quercetin also decreased macrophage number in sections 1 (57%) and 3 (81%), and spleen weight (P<0.05). These data suggest that quercetin can reduce polyp number and size distribution in the ApcMin/+ mouse and that these effects may be related to a reduction in macrophage infiltration.
We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knockdown of Gcn5 by siRNA silencing decreased mRNA and protein levels of GCN5, HAT activity, and also attenuated ethanol induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥ 1.5 fold up- or down- regulated). Among these, 492 transcripts were up- and 399 were down- regulated compared to their respective controls. Using a more stringent threshold (≥ 2.5 fold) the array data from GCN5 silenced samples showed 57 genes differentially expressed (39 up-regulated and 18 down-regulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥ 1.5 fold change (35 gene up-regulated and 22 down-regulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly up-regulated by ethanol (3 fold), and GCN5 silencing down regulated it (1.5 fold). The quantitative RT-PCR profile corroborated the array findings. This report demonstrates for the first time that (a) GCN5 differentially affects expression of multiple genes, (b) ethanol induced histone H3-lysine 9 acetylation is mediated via GCN5 and (c) that GCN5 is involved in ethanol induced expression of the putative choline transporter SLC44A2.
Treatment with the chemotherapeutic agent oxaliplatin produces a robust painful neuropathy similar to various other neuropathic conditions which result in loss of nerve fibers innervating the skin. This loss of intraepidermal nerve fibers (IENFs) appears to play an important role in neuropathy, but has yet to be investigated in oxaliplatin-induced neuropathic pain. For this study, mechanical hyperalgesia and IENF density were measured in rats receiving oxaliplatin, given at a dosage of 2 mg/kg every other day for four injections. The immuomodulatory agent minocycline (25 mg/kg) was also administered and was given 24 hours prior to the first dose of oxaliplatin and continued throughout oxaliplatin treatment. Immunohistochemistry using the pan-neuronal marker PGP9.5 was used to investigate IENF densities in hind paw skin on Day 15 and Day 30. The results show that a robust mechanical sensitivity developed in oxaliplatin treated animals, as did a pronounced decrease in epidermal nerve fibers, and these outcomes were effectively prevented by minocycline treatment. This is the first study to show changes in IENF density in oxaliplatin treated animals, and confirms not only a relationship between IENF loss and hypersensitivity but also prevention of both with minocycline treatment.
neuropathy; oxaliplatin; intraepidermal nerve fiber; minocycline; chemotherapy
During the past 30 years, major advances have been made in our understanding of HIV/AIDS and Pneumocystis pneumonia (PCP), but significant gaps remain. Pneumocystis is classified as a fungus and is host-species specific, but an understanding of its reservoir, mode of transmission, and pathogenesis is incomplete. PCP remains a frequent AIDS-defining diagnosis and is a frequent opportunistic pneumonia in the United States and in Europe, but comparable epidemiologic data from other areas of the world that are burdened with HIV/AIDS are limited. Pneumocystis cannot be cultured, and bronchoscopy with bronchoalveolar lavage is the gold standard procedure to diagnose PCP, but noninvasive diagnostic tests and biomarkers show promise that must be validated. Trimethoprim-sulfamethoxazole is the recommended first-line treatment and prophylaxis regimen, but putative trimethoprim-sulfamethoxazole drug resistance is an emerging concern. The International HIV-associated Opportunistic Pneumonias (IHOP) study was established to address these knowledge gaps. This review describes recent advances in the pathogenesis, epidemiology, diagnosis, and management of HIV-associated PCP and ongoing areas of clinical and translational research that are part of the IHOP study and the Longitudinal Studies of HIV-associated Lung Infections and Complications (Lung HIV).
acquired immune deficiency syndrome; HIV; Pneumocystis; Pneumocystis pneumonia; dihydropteroate synthase
Genomic selection can increase genetic gain per generation through early selection. Genomic selection is expected to be particularly valuable for traits that are costly to phenotype and expressed late in the life cycle of long-lived species. Alternative approaches to genomic selection prediction models may perform differently for traits with distinct genetic properties. Here the performance of four different original methods of genomic selection that differ with respect to assumptions regarding distribution of marker effects, including (i) ridge regression–best linear unbiased prediction (RR–BLUP), (ii) Bayes A, (iii) Bayes Cπ, and (iv) Bayesian LASSO are presented. In addition, a modified RR–BLUP (RR–BLUP B) that utilizes a selected subset of markers was evaluated. The accuracy of these methods was compared across 17 traits with distinct heritabilities and genetic architectures, including growth, development, and disease-resistance properties, measured in a Pinus taeda (loblolly pine) training population of 951 individuals genotyped with 4853 SNPs. The predictive ability of the methods was evaluated using a 10-fold, cross-validation approach, and differed only marginally for most method/trait combinations. Interestingly, for fusiform rust disease-resistance traits, Bayes Cπ, Bayes A, and RR–BLUB B had higher predictive ability than RR–BLUP and Bayesian LASSO. Fusiform rust is controlled by few genes of large effect. A limitation of RR–BLUP is the assumption of equal contribution of all markers to the observed variation. However, RR-BLUP B performed equally well as the Bayesian approaches.The genotypic and phenotypic data used in this study are publically available for comparative analysis of genomic selection prediction models.
Rationale: Smear-positive tuberculosis (TB) case detection rates are far below targets in most low-income countries. The standard approach to smear microscopy involves sputum collection over multiple days and examination of sputum smears by light microscopy (LM), an insensitive and time-consuming technique.
Objective: To determine whether two alternative approaches can increase smear-positive case detection by increasing the efficiency (single-specimen microscopy) or sensitivity (light-emitting diode [LED] fluorescence microscopy [FM]) of TB suspect evaluation.
Methods: We enrolled patients with cough of 2 weeks or more admitted to Mulago Hospital in Kampala, Uganda and collected spot and early morning sputum specimens. We compared the diagnostic accuracy of four prespecified strategies based on the number of sputum specimens collected (one specimen versus two specimens) and the type of microscopy (LM versus LED FM) using mycobacterial culture as a reference standard.
Measurements and Main Results: Two hundred thirty-three of 464 (50%) patients had culture-positive TB. There was no difference in sensitivity between single-specimen and two-specimen strategies when smears were examined with LM (55 vs. 56%; difference, −1%; 95% confidence interval [CI], −5 to +2%) or LED FM (61 vs. 64%; difference, −3%; 95% CI, −7 to +1%). LED FM was more sensitive than LM with both the single-specimen (61 vs. 55%; difference, 6%; 95% CI, 2–10%) and two-specimen strategies (64 vs. 56%; difference, 8%; 95% CI, 3–12%). Findings were similar among the HIV-infected patient subset (n = 321 patients).
Conclusions: In low-income, high TB burden settings, single-specimen microscopy and LED FM, either alone or in combination, could considerably increase identification of smear-positive TB cases.
tuberculosis; diagnosis; smear-positive case detection
Loss of intraepidermal nerve fibers (IENFs) has been speculated to play a critical role in the development of various neuropathies. In this study, the density of IENFs were studied over time during the induction of Taxol-induced chemoneuropathy and compared with the changes in IENFs in animals co-treated with Taxol plus the protective agent minocycline. Rats were injected (i.p.) with 2mg/kg of Taxol every other day for 4 injections (Days 1, 3, 5, and 7). Minocycline (25 mg/kg) was given in a separate group of rats 24 hours prior to the first dose of Taxol and every day for the next 9 days (Days 0 through 9). Animals were tested for mechanical paw withdrawal thresholds prior to any drug administrations and again on Days 7, 14, and 30. Immunohistochemistry using the pan-neuronal marker PGP9.5 was performed on glabrous skin of the hind-paw foot pad to stain for IENFs also on Days 7, 14, and 30. The results show that Taxol-treated animals developed mechanical sensitivity and corresponding IENF loss. Animals receiving minocycline plus Taxol showed no hyperalgesia or loss of IENFs. This study confirms, for the first time, that a loss of IENFs occurs as a neuropathy develops, and further shows a protection against both IENF loss and hyperalgesia with minocycline treatment.
Intraepidermal nerve fibers; Taxol; Neuropathy; Minocycline; Skin biopsy; Chemotherapy
Curcumin's benefits on tumorigenesis are thought to be mediated by its antiinflammatory activity; however, these effects have not been well characterized in a mouse model of colon cancer. We examined the effects of curcumin on intestinal inflammation in the ApcMin/+ mouse. ApcMin/+ mice were given a placebo or curcumin (2%) diet from 4 to 18 weeks of age (n = 10/group). C57BL/6 mice were used as a wild-type control (n = 10/group). Intestines were analyzed for polyp burden (sections 1, 4, and 5) and for mRNA expression, and concentration of interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and chemokine ligand 2 (CCL2) (sections 2 and 3). Plasma was collected for concentration of CCL2. Curcumin decreased total intestinal polyps by 75% (P < 0.05) in all size categories [>2 mm (65%), 1–2 mm (72%), <1 mm (82%); P < 0.05]. mRNA expression of IL-1β, IL-6, tumor necrosis factor-α, and CCL2 was elevated (P < 0.05) and curcumin blunted this increase (P < 0.05). Protein concentration of IL-1β, IL-6 (section 3), and CCL2 was increased (P < 0.05) and curcumin reduced this response for IL-1β (section 2) and CCL2 (P < 0.05). Curcumin also offset the increase in plasma CCL2 (P < 0.05). The benefits of curcumin in colon cancer may be at least in part mediated by its antiinflammatory activity.
A flexible statistical framework is developed for the analysis of read counts from RNA-Seq gene expression studies. It provides the ability to analyse complex experiments involving multiple treatment conditions and blocking variables while still taking full account of biological variation. Biological variation between RNA samples is estimated separately from the technical variation associated with sequencing technologies. Novel empirical Bayes methods allow each gene to have its own specific variability, even when there are relatively few biological replicates from which to estimate such variability. The pipeline is implemented in the edgeR package of the Bioconductor project. A case study analysis of carcinoma data demonstrates the ability of generalized linear model methods (GLMs) to detect differential expression in a paired design, and even to detect tumour-specific expression changes. The case study demonstrates the need to allow for gene-specific variability, rather than assuming a common dispersion across genes or a fixed relationship between abundance and variability. Genewise dispersions de-prioritize genes with inconsistent results and allow the main analysis to focus on changes that are consistent between biological replicates. Parallel computational approaches are developed to make non-linear model fitting faster and more reliable, making the application of GLMs to genomic data more convenient and practical. Simulations demonstrate the ability of adjusted profile likelihood estimators to return accurate estimators of biological variability in complex situations. When variation is gene-specific, empirical Bayes estimators provide an advantageous compromise between the extremes of assuming common dispersion or separate genewise dispersion. The methods developed here can also be applied to count data arising from DNA-Seq applications, including ChIP-Seq for epigenetic marks and DNA methylation analyses.
Respiratory infections are a leading cause of death in Africa, especially among HIV-infected patients. Data on the etiology of fatal respiratory diseases are largely based on autopsy studies. We evaluated causes of pneumonia associated with early mortality among hospitalized HIV-infected patients in Kampala, Uganda.
Prospective cohort study of HIV-infected patients admitted to Mulago Hospital, Kampala, with at least 2 weeks of cough. Consecutively enrolled patients with negative Ziehl Neelsen sputum smears for acid-fast bacilli underwent bronchoscopy with bronchoalveolar lavage and examination for mycobacteria (smear, solid culture), Pneumocystis jirovecii (Giemsa stain), and fungi (KOH mount, India ink stain, Sabouraud culture). Early mortality was defined as death before the 2-month follow-up visit.
Follow-up data were available for 353 (87%) of 407 patients enrolled. Of participants with follow-up data, 112 (32%) died within 2 months. Among patients with early mortality, a diagnosis was confirmed in 74 (66%), including tuberculosis (TB) (56%), cryptococcal pneumonia (1%), Pneumocystis pneumonia (3%), pulmonary Kaposi sarcoma (4%), and pneumonia caused by 2 or more disease processes (3%).
Mortality in HIV-infected TB suspects is high, with TB associated with the largest proportion of deaths. A significant proportion of patients die without a confirmed diagnosis.
HIV; tuberculosis; mortality; hospital admission; Africa