Analysis of multivariate longitudinal data becomes complicated when the outcomes
are of high dimension and informative right censoring is prevailing. Here, we propose a
likelihood based approach for high dimensional outcomes wherein we jointly model the
censoring process along with the slopes of the multivariate outcomes in the same
likelihood function. We utilized pseudo likelihood function to generate parameter
estimates for the population slopes and Empirical Bayes estimates for the individual
slopes. The proposed approach was applied to jointly model longitudinal measures of blood
urea nitrogen, plasma creatinine, and estimated glomerular filtration rate which are key
markers of kidney function in a cohort of renal transplant patients followed from kidney
transplant to kidney failure. Feasibility of the proposed joint model for high dimensional
multivariate outcomes was successfully demonstrated and its performance was compared to
that of a pairwise bivariate model. Our simulation study results suggested that there was
a significant reduction in bias and mean squared errors associated with the joint model
compared to the pairwise bivariate model.
Informative right censoring; Joint modeling; Likelihood based approach; Multivariate longitudinal outcomes; Random effect; Slope estimation
Although bradykinin (BK) and insulin like growth factor-1 (IGF-1) have been shown to modulate the functional and structural integrity of the arterial wall, the cellular mechanisms through which this regulation occurs is still undefined. The present study examined the role of second messenger molecules generated by BK and IGF-1 that could ultimately result in proliferative or anti-proliferative signals in vascular smooth muscle cells (VSMC).
Activation of BK or IGF-1 receptors stimulated the synthesis and release of prostacyclin (PGI2) leading to increased production of cAMP in VSMC. Inhibition of p42/p44mapk or src kinases prevented the increase in PGI2 and cAMP observed in response to BK or IGF-1, indicating a role for these kinases in the regulation of cPLA2 activity in the VSMC. Inhibition of PKC failed to alter production of PGI2 in response to BK, but further increased both p42/p44mapk activation and the synthesis of PGI2 produced in response to IGF-1. In addition, both BK and IGF-1 significantly induced the expression of c-fos mRNA levels in VSMC, and this effect of BK was accentuated in the presence a cPLA2 inhibitor. Finally, inhibition of cPLA2 activity and/or cyclooxygenase activity enhanced the expression of collagen I mRNA levels in response to BK and IGF-1 stimulation.
These findings indicate that the effect of BK or IGF-1 to stimulate VSMC growth is an integrated response to the activation of multiple signaling pathways. Thus, the excessive cell growth that occurs in certain forms of vascular disease could reflect dysfunction in one or more of these pathways.
The evidence for genetic susceptibility in the pathogenesis of diabetic nephropathy is well recognised, but the genes involved remain to be identified. It is hypothesised that mutations within the gene encoding connective tissue growth factor (CTGF/CCN2) will increase the propensity of diabetic subjects to develop nephropathy.
Methods and results
Genomic screening was performed for single nucleotide polymorphisms (SNPs) within the CTGF gene in 862 subjects from the DCCT/EDIC cohort of type 1 diabetes. A novel SNP was identified in the promoter region that changes a C-G at the position −20. The frequency of GG genotype in microalbuminuric patients (albumin excretion rate (AER) >40 mg/24 h) is significantly greater than diabetics with AER <40 mg/24 h, p<0.0001. The relative risk (RR) to develop microalbuminuria in diabetic subjects with the polymorphism is 3X higher than diabetic subjects without the polymorphism (RR 3.142, 95% CI 1.9238 to 5.1249; p<0.05). Kaplan–Meier survival curves demonstrated that the GG genotype group developed microalbuminuria and macroalbuminuria at a more rapid rate than the GC or CC genotypes. Functional studies demonstrated that the basal activity of the substituted allele/promoter (−20 GG allele) was significantly greater than that of the wild type promoter (−20 CC genotype). This higher level of basal activity of substituted allele CTGF/CCN2 promoter was abrogated upon suppression of Smad1 levels, indicating that SNP region in the CTGF/CCN2 promoter plays a vital role in the gene expression.
These findings provide the first evidence that variants within the promoter region of the CTGF/CCN2 gene predisposes diabetic subjects to develop albuminuria and demonstrate that Samd1 controls the expression of CTGF/CCN2 promoter through this region.
Diabetic nephropathy (DN), the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B2-receptors (B2R) in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B2R knockout (B2R−/−) mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER) were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B2R−/−D null mice had a significantly decreased AER levels compared to wild type B2R+/+D mice (P = 0.0005). Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B2R+/+C, B2R+/+D, B2R−/−C and B2R−/−D) highlighted the role of several altered pathological pathways in response to disruption of B2R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.
Aim. To assess severity of malocclusion in Lebanese elementary school children and the relationship between components of malocclusion and sociodemographic and behavioral factors. Methods. Dental screening was performed on 655 school children aged 6–11 from 2 public (PB) and 5 private (PV) schools in Beirut. A calibrated examiner recorded occlusion, overjet, overbite, posterior crossbite, midline diastema, and crowding. Another examiner determined the DMFT (Decayed/Missing/Filled Teeth) score. A questionnaire filled by the parents provided data on sociodemographic and behavioral factors. Multinomial, binomial, and multiple linear regressions tested the association of these factors with occlusal indices. Results. Malocclusion was more severe in PB students. Age and sucking habit were associated with various components of malocclusion. Crowding was more prevalent among males and significantly associated with the DMFT score. Income and educational level were significantly higher (P < 0.05) in PV pupils and deleterious habits were more frequent in PB children. Conclusions. Children of lower socioeconomic background had more severe malocclusions and poorer general dental health. Compared to Western and WHO norms, the findings prompt health policy suggestions to improve dental care of particularly public school children through regular screenings in schools, prevention methods when applicable, and cost effective practices through public and private enabling agencies.
Biosensors research is a fast growing field in which tens of thousands of papers have been published over the years, and the industry is now worth billions of dollars. The biosensor products have found their applications in numerous industries including food and beverages, agricultural, environmental, medical diagnostics, and pharmaceutical industries and many more. Even though numerous biosensors have been developed for detection of proteins, peptides, enzymes, and numerous other biomolecules for diverse applications, their applications in tissue engineering have remained limited. In recent years, there has been a growing interest in application of novel biosensors in cell culture and tissue engineering, for example, real-time detection of small molecules such as glucose, lactose, and H2O2 as well as serum proteins of large molecular size, such as albumin and alpha-fetoprotein, and inflammatory cytokines, such as IFN-g and TNF-α. In this review, we provide an overview of the recent advancements in biosensors for tissue engineering applications.
Therapies designed to decrease SOD1 are currently in clinical trial for patients with superoxide dismutase (SOD1)-linked Familial Amyotrophic Lateral Sclerosis (ALS),
To determine whether SOD1 protein in cerebral spinal fluid (CSF) may be a pharmacodynamic marker and whether SOD1 protein in CSF is a disease marker for ALS.
Antisense oligonucleotides targeting human SOD1 (hSOD1) were administered to SOD1G93A rats. hSOD1 protein levels were measured in rat brain and CSF. In human CSF, the following proteins were measured: SOD1, tau, p-tau, VILIP-1, and YKL-40. was measured in human CSF.
SOD1G93A ALS model rats. ALS subject CSF (N=93), healthy controls (N=880 and neurological disease controls (NDC, N=89), including subjects with Dementia of the Alzheimer’s Type (DAT) (55), multiple sclerosis (19), and peripheral neuropathy (15).
Antisense oligonucleotide-treated SOD1G93A rats had decreased hSOD1 mRNA (69%+/−4%) and protein levels (48%+/ −14%) in brain. Importantly, rat CSF showed a similar 42+/−14% decrease in hSOD1. In human CSF, SOD1 varied 7.1+/−5.7 % on repeat measurements separated by months. SOD1 CSF levels were higher in ALS (172+/−8ng/ml, p<0.05) and NDC (172+/−6 ng/ml, p<0.05) compared with healthy controls (134+/−4ng/ml). Elevated CSF SOD1 did not correlate with disease characteristics in ALS or DAT subjects, but did correlate with tau, p-tau, VILIP-1 and YKL-40 in DAT subjects and controls.
CSF SOD1 may be an excellent pharmacodynamic marker for SOD1-lowering therapies since antisense oligonucleotide therapy lowers protein levels in both rat brain and rat CSF and since SOD1 CSF in humans is stable upon repeat measurements.
Diabetic nephropathy (DN), a major complication of diabetes, is characterized by hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. Hypertrophy is a major factor inducing proximal tubular epithelial cells injury. However, the mechanisms leading to tubular injury is not well defined. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. In conclusion, we show that hyperglycemia in diabetes has a significant effect on the expression of Arachidonic Acid (AA)-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites.
Patients with anorexia may have elevated autistic traits. In this study, we tested test whether patients with anorexia nervosa (anorexia) have an elevated score on a dimensional measure of autistic traits, the Autism Spectrum Quotient (AQ), as well as on trait measures relevant to the autism spectrum: the Empathy Quotient (EQ), and the Systemizing Quotient (SQ).
Two groups were tested: (1) female adolescents with anorexia: n = 66, aged 12 to 18 years; and (2) female adolescents without anorexia: n =1,609, aged 12 to 18 years. Both groups were tested using the AQ, EQ, and SQ, via the parent-report adolescent versions for patients aged 12 to 15 years old, and the self-report adult versions for patients aged over 16 years.
As predicted, the patients with anorexia had a higher AQ and SQ. Their EQ score was reduced, but only for the parent-report version in the younger age group. Using EQ-SQ scores to calculate ‘cognitive types’, patients with anorexia were more likely to show the Type S profile (systemizing (S) better than empathy (E)), compared with typical females.
Females with anorexia have elevated autistic traits. Clinicians should consider if a focus on autistic traits might be helpful in the assessment and treatment of anorexia. Future research needs to establish if these results reflect traits or states associated with anorexia.
Autistic traits; Anorexia; Autism spectrum conditions; Broader autism phenotype
Epithelial ovarian cancer (EOC) is usually discovered after extensive metastasis have developed in the peritoneal cavity. The ovarian surface is exposed to peritoneal fluid pressures and shear forces due to the continuous peristaltic motions of the gastro-intestinal system, creating a mechanical micro-environment for the cells. An in vitro experimental model was developed to expose EOC cells to steady fluid flow induced wall shear stresses (WSS). The EOC cells were cultured from OVCAR-3 cell line on denuded amniotic membranes in special wells. Wall shear stresses of 0.5, 1.0 and 1.5 dyne/cm2 were applied on the surface of the cells under conditions that mimic the physiological environment, followed by fluorescent stains of actin and β-tubulin fibers. The cytoskeleton response to WSS included cell elongation, stress fibers formation and generation of microtubules. More cytoskeletal components were produced by the cells and arranged in a denser and more organized structure within the cytoplasm. This suggests that WSS may have a significant role in the mechanical regulation of EOC peritoneal spreading.
In mammalian pregnancy, maternal cardiovascular adaptations must match the requirements of the growing fetus(es), and respond to physiologic and pathologic conditions. Such adaptations are particularly demanding for mammals bearing large-litter pregnancies, with their inherent conflict between the interests of each individual fetus and the welfare of the entire progeny. The mouse is the most common animal model used to study development and genetics, as well as pregnancy-related diseases. Previous studies suggested that in mice, maternal blood flow to the placentas occurs via a single arterial uterine loop generated by arterial-arterial anastomosis of the uterine artery to the uterine branch of the ovarian artery, resulting in counter bi-directional blood flow. However, we provide here experimental evidence that each placenta is actually supplied by two distinct arterial inputs stemming from the uterine artery and from the uterine branch of the ovarian artery, with position-dependent contribution of flow from each source. Moreover, we report significant positional- and inter-fetal dependent alteration of placental perfusion, which were detected by in vivo MRI and fluorescence imaging. Maternal blood flow to the placentas was dependent on litter size and was attenuated for placentas located centrally along the uterine horn. Distinctive apposing, inter-fetal hemodynamic effects of either reduced or elevated maternal blood flow, were measured for placenta of normal fetuses that are positioned adjacent to either pathological, or to hypovascular Akt1-deficient placentas, respectively. The results reported here underscore the critical importance of confounding local and systemic in utero effects on phenotype presentation, in general and in the setting of genetically modified mice. The unique robustness and plasticity of the uterine vasculature architecture, as reported in this study, can explain the ability to accommodate varying litter sizes, sustain large-litter pregnancies and overcome pathologic challenges. Remarkably, the dual arterial supply is evolutionary conserved in mammals bearing a single offspring, including primates.
Our objective was to test the hypothesis that aberrantly modified forms of superoxide dismutase (SOD1) influence the disease course for sporadic amyotrophic lateral sclerosis (SALS). We probed for anti-SOD1 antibodies (IgM and IgG) against both the normal and aberrantly oxidized-SOD1 (SODox) antigens in sera from patients with SALS, subjects diagnosed with other neurological disorders and healthy individuals, and correlated the levels of these antibodies to disease duration and/or severity. Anti-SOD1 antibodies were detected in all cohorts; however, a subset of ~5–10% of SALS cases exhibited elevated levels of anti-SOD1 antibodies. Those SALS cases with relatively high levels of IgM antibodies against SODox exhibit a longer survival of 6.4 years, compared to subjects lacking these antibodies. By contrast, SALS subjects expressing higher levels of IgG antibodies reactive for the normal WT-SOD1 antigen exhibit a shorter survival of 4.1 years. Anti-SOD1 antibody levels did not correlate with disease severity in either the Alzheimer’s or Parkinson’s disease cohorts. In conclusion, the association of longer survival with elevated levels of anti-SODox antibodies suggests that these antibodies may be protective. By extension, these data implicate aberrantly modified forms of WT-SOD1 (e.g. oxidized SOD1) in SALS pathogenesis. In contrast, an immune response against the normal WT-SOD1 appears to be disadvantageous in SALS, possibly because the anti-oxidizing activity of normal WT-SOD1 is beneficial to SALS individuals.
Sporadic amyotrophic lateral sclerosis; superoxide dismutase; immunotherapy; Parkinson’s disease; Alzheimer’s disease
Recent studies using mass spectrometry have discovered candidate biomarkers for amyotrophic lateral sclerosis (ALS). However these studies utilize small numbers of ALS and control subjects. Additional studies using larger subject cohorts are required to verify these candidate biomarkers.
Cerebrospinal fluid (CSF) from 100 patients with ALS, 100 disease control and 41 healthy control subjects were examined by mass spectrometry.
61 mass spectral peaks exhibited altered levels between ALS and controls. Mass peaks for cystatin C and transthyretin were reduced in ALS, whereas mass peaks for post-translational modified transthyretin and C-reactive protein (CRP) were increased. CRP levels were 5.84±1.01 ng/mL for controls and 11.24±1.52 ng/mL for ALS subjects as determined by ELISA.
This study verified prior mass spectrometry results for cystatin C and transthyretin in ALS. CRP levels were increased in the CSF of ALS patients, and cystatin C level correlated with survival in patients with limb-onset disease. Our biomarker panel predicted ALS with an overall accuracy of 82%.
amyotrophic lateral sclerosis; mass spectrometry; biomarkers; cerebrospinal fluid; cystatin C
Demographical factors such as race, vital status, gender and age could affect the final renal outcome of patients who undergo renal transplantation. These demographical factors could be assessed at a recipient and donor levels. Repeated measures for blood urea nitrogen (BUN) are typically recorded for each patient following renal transplantation, as a biomarker to assess renal progress. However, once a patient develops renal failure due to graft rejection, no measurement of BUN can be registered and the patient goes back to dialysis. This causes loss of follow-up and incomplete data on BUN measurements, a problem referred to as informative right censoring. If this problem is ignored inaccurate and biased estimates will be generated. In this study unbiased estimates for rate of change for BUN levels over time adjusted for informative right censoring and demographical factors were acquired using a sophisticated model of analysis. Our results demonstrated that BUN levels for Caucasians were decreasing at a greater rate than African-Americans (P<0.0001). When donors are deceased, African-American recipients showed an increase instead of a decrease in their BUN levels following transplantation. Moreover, African-Americans showed a decrease in their BUN levels when donors were African-Americans compared to when donors were Caucasians (P=0.03). Our results also showed that BUN levels were decreasing at a greater rate when donors and recipients were of different gender than when they were of same gender (P = 0.009). These results suggest that success of renal transplantation is impacted by donor/recipient demographical factors.
BUN rate of change; Renal transplant; Longitudinal data; Informative missing Values
Patients undergoing renal transplantation are prone to graft failure which causes lost of follow-up measures on their blood urea nitrogen and serum creatinine levels. These two outcomes are measured repeatedly over time to assess renal function following transplantation. Loss of follow-up on these bivariate measures results in informative right censoring, a common problem in longitudinal data that should be adjusted for so that valid estimates are obtained. In this study, we propose a bivariate model that jointly models these two longitudinal correlated outcomes and generates population and individual slopes adjusting for informative right censoring using a discrete survival approach. The proposed approach is applied to the clinical dataset of patients who had undergone renal transplantation. A simulation study validates the effectiveness of the approach.
Bivariate correlated outcomes; Discrete Survival distributions; Informative right censoring; Longitudinal data; Slope estimation
OBJECTIVE—Because endothelial cell dysfunction and inflammation are key contributors to the development of complications in type 1 diabetes, we studied risk factors related to endothelial dysfunction and inflammation (C-reactive protein and fibrinogen, soluble vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin, and fibrinolytic markers) in a subgroup of patients from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study cohort.
RESEARCH DESIGN AND METHODS—We determined which of these risk factors or clusters thereof are associated with the presence of and subsequent development of nephropathy and macrovascular complications (reflected by carotid intima-media thickness [IMT]).
RESULTS—After adjustment for conventional risk factors (age, sex, DCCT treatment group, diabetes duration, A1C, systolic blood pressure, waist-to-hip ratio, total and HDL cholesterol, and smoking status), fibrinogen remained strongly associated with progression of internal and common carotid IMT (P < 0.01) and soluble E-selectin had a strong association with nephropathy (P < 0.01).
CONCLUSIONS—The best predictor for IMT progression in the DCCT/EDIC cohort was plasma fibrinogen, and the levels of soluble E-selectin discriminate patients with albuminuria better than conventional risk factors.
Urinary kallikrein excretion (UKE) is decreased in rats with passive Heymann nephritis (PHN), but increases after converting enzyme inhibition (CEI). Although CEI potentiates bradykinin activity, neither the effect of CEI on kallikrein secretion nor the abnormal renal kallikrein metabolism in PHN has been examined previously. To determine the mechanism by which CEI increases UKE, normal rats and PHN received enalapril, 40 mg/kg per d orally for 4 d. UKE was 85% lower in PHN than in normals and increased in both groups after CEI, although UKE in PHN remained significantly less than in normals. Kallikrein mRNA was significantly lower in PHN compared to normals but not in PHN treated with CEI and did not change in normal rats. Renin mRNA was significantly lower in PHN, and was stimulated by CEI only in normals. Renal kallikrein and renin content were not different and were not altered by CEI. Both kallikrein and renin genes appear to be transcriptionally suppressed in rats with PHN and the depressed kallikrein mRNA levels can be reversed by CEI. The modest increase in UKE despite normalization of kallikrein mRNA after CEI suggests that there is also a posttranscriptional defect in synthesis and/or secretion of kallikrein.
This study examined the role of tissue kallikrein and kinins in renal vasodilation produced by infusion of amino acids (AA). In rats fed a 9% protein diet for 2 wk, intravenous infusion of a 10% AA solution over 60-90 min reduced total renal vascular resistance and increased glomerular filtration rate (GFR) by 25-40% and renal plasma flow (RPF) by 23-30% from baseline. This was associated with a two- to threefold increase in urinary kinin excretion rate. Acute treatment of rats with aprotinin, a kallikrein inhibitor, resulted in deposition of immunoreactive aprotinin in kallikrein-containing connecting tubule cells and inhibited renal kallikrein activity by 90%. A protinin pretreatment abolished the rise in urinary kinins and prevented significant increases in GFR and RPF in response to AA. In a second group of rats pretreated with a B2 kinin receptor antagonist, [DArg Hyp3, Thi5,8 D Phe7]bradykinin, AA infusion raised urinary kinins identically as in untreated controls, but GFR and RPF responses were absent. Aprotinin or the kinin antagonist produced no consistent change in renal function in rats that were not infused with AA.AA-induced increases in kinins were not associated with an increase in renal kallikrein activity. Notably, tissue active kallikrein level fell 50% in AA-infused rats. These studies provide evidence that kinins generated in the kidney participate in mediating renal vasodilation during acute infusion of AA.
A mother and daughter were the sole survivors of a house fire. Support, maintenance of the parent-child relationship, and monitoring and facilitating the grieving process in a way that was consistent with the developmental stages of the patients were the basis of management.
The effects of streptozotocin (STZ) diabetes and insulin on regulation of renal kallikrein were studied in the rat. 1 and 2 wk after STZ injection, diabetic rats had reduced renal levels and urinary excretion of active kallikrein. Tissue and urinary prokallikrein levels were unchanged, but the rate of renal prokallikrein synthesis relative to total protein synthesis was reduced 30-45% in diabetic rats. Treatment of diabetic rats with insulin prevented or reversed the fall in tissue level and excretion rate of active kallikrein and normalized prokallikrein synthesis rate. To further examine insulin's effects, nondiabetic rats were treated with escalating insulin doses to produce hyperinsulinemia. In these rats, renal active kallikrein increased. Although renal prokallikrein was not increased significantly by hyperinsulinemia, its synthesis was increased. As this was accompanied by proportionally increased total protein synthesis, relative kallikrein synthesis rate was not changed. Excretion of active kallikrein was unchanged, but prokallikrein excretion was markedly reduced. Therefore, increased tissue active kallikrein seen with hyperinsulinemia can be explained not only by increased synthesis but also by retention and increased activation of renal prokallikrein. These studies show that STZ diabetes produces an impairment in renal kallikrein synthesis and suggest that this disease state also impairs renal prokallikrein activation. The findings also suggest that insulin modulates renal kallikrein production, activation, and excretion.