It has been known since 1986 that CD8 T lymphocytes from certain HIV-1-infected individuals who are immunologically stable secrete a soluble factor, termed CAF, that suppresses HIV-1 replication. However, the identity of CAF remained elusive despite an extensive search. By means of a protein-chip technology, we identified a cluster of proteins that were secreted when CD8 T cells from long-term nonprogressors with HIV-1 infection were stimulated. These proteins were identified as alpha-defensin 1, 2, and 3 on the basis of specific antibody recognition and amino acid sequencing. CAF activity was eliminated or neutralized by an antibody specific for human alpha-defensins. Synthetic and purified preparations of alpha-defensins also inhibited the replication of HIV-1 isolates in vitro. Taken together, our results indicate that alpha-defensin 1, 2, and 3 collectively account for much of the anti-HIV-1 activity of CAF that is not attributable to beta-chemokines.

Our objective was to identify metabolic pathways affected by ALS using non-targeted metabolomics in plasma, comparing samples from healthy volunteers to those from ALS patients. This discovery could become the basis for the identification of therapeutic targets and diagnostic biomarkers of ALS. Two distinct cross-sectional studies were conducted. Plasma was collected from 62 (Study 1) and 99 (Study 2) participants meeting El Escorial criteria for possible, probable, or definite ALS; 69 (Study 1) and 48 (Study 2) healthy controls samples were collected. Global metabolic profiling was used to detect and evaluate biochemical signatures of ALS. Twenty-three metabolites were significantly altered in plasma from ALS patients in both studies. These metabolites include biochemicals in pathways associated with neuronal change, hypermetabolism, oxidative damage, and mitochondrial dysfunction, all of which are proposed disease mechanisms in ALS. The data also suggest possible hepatic dysfunction associated with ALS. In conclusion, the data presented here provide insight into the pathophysiology of ALS while suggesting promising areas of focus for future studies. The metabolomics approach can generate novel hypotheses regarding ALS disease mechanisms with the potential to identify therapeutic targets and novel diagnostic biomarkers.

Insulin resistance and hepatic lipid accumulation constitute the metabolic underpinning of nonalcoholic steatohepatitis (NASH). We tested the hypothesis that saroglitazar, a PPAR α/γ agonist would improve nASH in the diet-induced animal model of nAfLD. Mice received chow diet and normal water (CDNW) or high fat western diet and ad lib sugar water (WDSW). After 12 weeks, WDSW fed mice were randomized to receive (1) WDSW alone, (2) WDSW + vehicle, (3) WDSW + pioglitazone or (4) WDSW + saroglitazar for an additional 12 weeks. Compared to mice on WDSW and vehicle controls, mice receiving WDSW + saroglitazar had lower weight, lower HOMA-IR, triglycerides, total cholesterol, and ALT. Saroglitazar improved steatosis, lobular inflammation, hepatocellular ballooning and fibrosis stage. NASH resolved in all mice receiving saroglitazar. These effects were at par with or superior to pioglitazone. Molecular analyses confirmed target engagement and reduced oxidative stress, unfolded protein response and fibrogenic signaling. Transcriptomic analysis further confirmed increased PPARtarget expression and an anti-inflammatory effect with saroglitazar. Lipidomic analyses demonstrated that saroglitazar also reduced triglycerides, diglycerides, sphingomyelins and ceramides. These preclinical data provide a strong rationale for developing saroglitazar for the treatment of nASH in humans. Nonalcoholic fatty liver disease (NAFLD) encompasses a continuum of liver disease ranging from fatty liver (NAFL) to steatohepatitis (NASH), fibrosis and cirrhosis 1-3. This rising prevalence of NASH is accompanied with an alarming increase in the number of patients with cirrhosis and hepatocellular carcinoma (HCC) necessitating liver transplantation 4,5. Dynamic models of disease progression predict a doubling of the burden of end-stage liver disease from the NAFLD epidemic by 2030 if left unmanaged 6. Despite progress in understanding the clinical drivers of disease progression and pathogenesis of NAFLD and an exponential increase in clinical trials investigating the therapeutic potential and identifying therapeutic targets, there are immediate unmet medical needs and challenges and the disease still remains without any approved drugs 7,8. A key consideration in therapeutic development for NASH is the identification of a rational therapeutic target. NASH often develops in the context of excess adiposity and systemic insulin resistance 9. The current paradigm for the pathogenesis of NASH starts with increased delivery of lipids such as free fatty acids (FFA), carbohydrates along with inflammatory cytokines and gut-microbiome-derived products e.g. endotoxin 10 .

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.

More than 10 million people are thought to be infected with Trypanosoma cruzi, primarily in the Americas. The clinical manifestations of Chagas' disease (CD) are variable, but most subjects remain asymptomatic for decades. Only 15 to 30% eventually develop terminal complications. All current diagnostic tests have limitations. New approaches are needed for blood bank screening as well as for improved diagnosis and prognosis. Sera from subjects with asymptomatic CD (n ‫؍‬ 131) were compared to those from uninfected controls (n ‫؍‬ 164) and subjects with other parasitic diseases (n ‫؍‬ 140), using protein array mass spectrometry. To identify biomarkers associated with CD, sera were fractionated by anion-exchange chromatography and bound to two commercial ProteinChip array chemistries: WCX2 and IMAC3. Multiple candidate biomarkers were found in CD sera (3 to 75.4 kDa). Algorithms employing 3 to 5 of these biomarkers achieved up to 100% sensitivity and 98% specificity for CD. The biomarkers most useful for diagnosis were identified and validated. These included MIP1 alpha, C3a anaphylatoxin, and unusually truncated forms of fibronectin, apolipoprotein A1 (ApoA1), and C3. An antipeptide antiserum against the 28.9-kDa C terminus of the fibronectin fragment achieved good specificity (90%) for CD in a Western blot format. We identified full-length ApoA1 (28.1 kDa), the major structural and functional protein component of high-density lipoprotein (HDL), as an important negative biomarker for CD, and relatively little full-length ApoA1 was detected in CD sera. This work provides proof of principle that both platform-dependent (i.e., mass spectrometry-based) and platform-independent (i.e., Western blot) tests can be generated using high-throughput mass profiling.

There is a need for continued drug development for nonalcoholic steatohepatitis (NASH). Bergamot is a plant whose fruit juice is enriched with flavonoids and phenolic compounds which improves dyslipidemia and markers of systemic inflammation in patients with Metabolic Syndrome. The aim of this study was to perform a preclinical "proof of concept" study of Bergamot polyphenolic formulation (BPF99) for the treatment of NASH. A disease reversal study was performed in the diet-induced animal model of NAFLD (DIAMOND). Groups of 8 weeks old mice were randomly assigned to receive chow diet, high fat diet with sugar in drinking water (Western diet-WD). Mice on WD were further randomized to continue on WD gavaged with vehicle or continue on WD with additional gavage of BPF99 (50 mg/ kg) after 16 weeks of diet. Mice were euthanized after 11 additional weeks. The primary endpoint was resolution of NASH. Secondary endpoints included changes in individual histological features, body weight, liver enzymes, dyslipidemia, markers of oxidative stress and molecular markers of disease activity and fibrosis. The results showed that BPF99 reduced ALT (mean 71.6 vs 44.6 IU/l, p < 0.01), triglycerides (38.8 vs 28.1 mg/dl, p < 0.05), LDL-C (39.2 vs 23.7 mg/dl, p < 0.001). It significantly improved nASH resolution (p < 0.001) and the SAF scores (p < 0.05) while the NAS improvement approached significance. BPF99 reduced markers of oxidative stress, along with reduced JNK and p38 MAP kinase activity. BPF99 did not reduce the number of mice with fibrosis but improved collagen proportional area (p < 0.04) and procollagen I and III expression. Collectively our results showed that BPF99 resolves NASH and ameliorates key histological and pathophysiological features of NASH along with improvement in ALT and dyslipidemia in the DIAMOND mice. Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and mortality for which there are no approved therapies 1. The clinical-histological spectrum of NAFLD extends from a nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) 2. NASH is a more aggressive phenotype of NAFLD and is

Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N-acetyl-L-cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L-buthionine-(S,R)-sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen-presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.

ObjectiveSerum α-hydroxybutyrate (α-HB) is elevated in insulin resistance and diabetes. We tested the hypothesis that the α-HB level predicts abnormal 1 h glucose levels and β-cell dysfunction inferred from plasma insulin kinetics during a 75 g oral glucose tolerance test (OGTT).Research design and methodsThis cross-sectional study included 217 patients at increased risk for diabetes. 75 g OGTTs were performed with multiple postload glucose and insulin measurements over a 30–120 min period. OGTT responses were analyzed by repeated measures analysis of variance (ANOVA). Multivariable logistic regression was used to predict 1 h glucose ≥155 mg/dL with α-HB added to traditional risk factors.ResultsMean±SD age was 51±15 years (44% male, 25% with impaired glucose tolerance). Fasting glucose and insulin levels, but not age or body mass index (BMI), were significantly higher in the second/third α-HB tertiles (>3.9 µg/mL) than in the first tertile. Patients in the second/third α-HB tertiles exhibited a higher glucose area under the receiver operating characteristics curve (AUC) and reduced initial slope of insulin response during OGTT. The AUC for predicting 1 h glucose ≥155 mg/dL was 0.82 for a base model that included age, gender, BMI, fasting glucose, glycated hemoglobin (HbA1c), and insulin, and increased to 0.86 with α-HB added (p=0.015), with a net reclassification index of 52% (p<0.0001).ConclusionsFasting serum α-HB levels predicted elevated 1 h glucose during OGTT, potentially due to impaired insulin secretion kinetics. This association persisted even in patients with an otherwise normal insulin–glucose homeostasis. Measuring serum α-HB could thus provide a rapid, inexpensive screening tool for detecting early subclinical hyperglycemia, β-cell dysfunction, and increased risk for diabetes.