Dysregulated production of interleukin-10 (IL-10) and IL-12 by peripheral blood lymphocytes from human immunodeficiency virus-infected individuals is associated with altered proliferative responses to recall antigens.

The loss of immune function following infection with human immunodeficiency virus (HIV) may result from altered production of immunoregulatory cytokines such as interleukin-10 (IL-10) and IL-12. In this study, we analyzed IL-10 and IL-12 production by mitogen-stimulated peripheral blood mononuclear cells (PBMC) from HIV+ individuals and correlated their levels with proliferative responses to the recall antigens HIV p25 and influenza virus. We report two distinct groups of HIV+ patients. One group produced small amounts of IL-10, had PBMC that proliferated in response to recall antigens, and demonstrated enhanced recall antigen-induced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Conversely, the second group produced high levels of IL-10, had PBMC that failed to proliferate to recall antigens, and did not demonstrate enhanced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Mitogen-stimulated PBMC from both groups produced significantly lower levels of IL-12 than did those from HIV- controls. Analysis of the source of the IL-10-producing cell subset in PBMC demonstrated that in HIV+ individuals, IL-10 is produced by monocytes, while in HIV- controls, it is produced by both T cells and monocytes. Taken together, our results suggest that monocytes from HIV+ individuals secrete decreased amounts of IL-12, a Th1-type cytokine, which may lead to the development of Th2-type responses characterized by high IL-10 secretion and immune dysfunction.     

Systems Medicine: from molecular features and models to the clinic in COPD

Background and hypothesis

Chronic Obstructive Pulmonary Disease (COPD) patients are characterized by heterogeneous clinical manifestations and patterns of disease progression. Two major factors that can be used to identify COPD subtypes are muscle dysfunction/wasting and co-morbidity patterns. We hypothesized that COPD heterogeneity is in part the result of complex interactions between several genes and pathways. We explored the possibility of using a Systems Medicine approach to identify such pathways, as well as to generate predictive computational models that may be used in clinic practice.

Objective and method

Our overarching goal is to generate clinically applicable predictive models that characterize COPD heterogeneity through a Systems Medicine approach. To this end we have developed a general framework, consisting of three steps/objectives: (1) feature identification, (2) model generation and statistical validation, and (3) application and validation of the predictive models in the clinical scenario. We used muscle dysfunction and co-morbidity as test cases for this framework.

Results

In the study of muscle wasting we identified relevant features (genes) by a network analysis and generated predictive models that integrate mechanistic and probabilistic models. This allowed us to characterize muscle wasting as a general de-regulation of pathway interactions. In the co-morbidity analysis we identified relevant features (genes/pathways) by the integration of gene-disease and disease-disease associations. We further present a detailed characterization of co-morbidities in COPD patients that was implemented into a predictive model. In both use cases we were able to achieve predictive modeling but we also identified several key challenges, the most pressing being the validation and implementation into actual clinical practice.

Conclusions

The results confirm the potential of the Systems Medicine approach to study complex diseases and generate clinically relevant predictive models. Our study also highlights important obstacles and bottlenecks for such approaches (e.g. data availability and normalization of frameworks among others) and suggests specific proposals to overcome them.

     

Evaluation of self-reported skin problems among workers exposed to toluene diisocyanate (TDI) at a foam manufacturing facility

Toluene diisocyanate, or TDI (CAS 584-84-9) is a well-known asthmagen and respiratory irritant. TDI is also known for its ability to irritate the skin and mucous membranes. To further investigate the dermal effects of TDI, NIOSH investigators conducted a cross-sectional study at a flexible foam manufacturing plant. A total of 114 workers participated in the study. Participants completed a medical questionnaire, provided blood for antibody testing to TDI and other allergens, and a subset of participants reporting skin symptoms underwent skin patch testing to a standard diisocyanate panel. Production line workers were more than twice as likely to report skin problems as those working in nonproduction areas (PRR = 2.66; 95% CI = 1.14-16.32; P = 0.02). Age, gender and duration of employment at the plant were comparable among participants working in production and nonproduction areas. Of the 100 participants who provided blood samples for antibody testing, specific IgG antibody to TDI was detected in two individuals, and none of the samples demonstrated specific IgE antibody to TDI. Of the 26 workers who underwent skin patch testing, none developed reactions to the diisocyanate allergens. These results suggest that the skin symptoms among study participants represent an irritant rather than an immunologic reaction to TDI, or to an unidentified allergen present in the foam.     

Two distinct pathways of immuno-modulation improve potency of p53 immunization in rejecting established tumors

The p53 gene product is overexpressed by almost 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated rejection of established p53-overexpressing tumors without stimulating autoimmunity by immunization with modified vaccinia Ankara-expressing murine p53 (MVAp53). Tumor rejection was enhanced through antibody-mediated CTL-associated antigen 4 (CTLA-4) blockade. We examined the role of synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODN) in enhancing MVAp53-mediated tumor rejection. CpG ODN with MVAp53 resulted in tumor rejection in BALB/c mice bearing poorly immunogenic 11A-1 murine mammary carcinomas or Meth A sarcomas and C57Bl/6 mice bearing MC-38 colon carcinomas. The effect was similar to that seen in tumor-bearing mice treated with MVAp53 along with CTLA-4 blockade. Monoclonal antibody depletion experiments demonstrated that the adjuvant effects of CpG ODN and CTLA-4 blockades were CD8 dependent. CpG ODN were partially natural killer cell dependent and ineffective in Toll-like Receptor 9(-/-) and interleukin 6(-/-) mice, whereas CTLA-4 blockade was partially CD4 dependent and functional in Toll-like Receptor 9(-/-) and interleukin 6(-/-) mice. In addition, when administered with MVAp53, both adjuvants enhanced p53-specific cytotoxicity and demonstrated an additive effect when combined. The combination of CpG ODN and CTLA-4 blockade worked synergistically to reject palpable 11A-1 and MC-38 tumors. These experiments demonstrate the potential for augmenting MVAp53-mediated antitumor immunity using CpG ODN and CTLA-4 blockade. This cell-free immunotherapy approach is a candidate for evaluation in cancer patients.     

In vivo electroporation and non-protein based screening assays to identify antibodies against native protein conformations

In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved.     

High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay

Viruses are extremely heterogeneous entities; the size and the nature of their genetic information, as well as the strategies employed to amplify and propagate their genomes, are highly variable. However, as obligatory intracellular parasites, replication of all viruses relies on the host cell. Having co-evolved with their host for several million years, viruses have developed very sophisticated strategies to hijack cellular factors that promote virus uptake, replication, and spread. Identification of host cell factors (HCFs) required for these processes is a major challenge for researchers, but it enables the identification of new, highly selective targets for anti viral therapeutics. To this end, the establishment of platforms enabling genome-wide high-throughput RNA interference (HT-RNAi) screens has led to the identification of several key factors involved in the viral life cycle. A number of genome-wide HT-RNAi screens have been performed for major human pathogens. These studies enable first inter-viral comparisons related to HCF requirements. Although several cellular functions appear to be uniformly required for the life cycle of most viruses tested (such as the proteasome and the Golgi-mediated secretory pathways), some factors, like the lipid kinase Phosphatidylinositol 4-kinase IIIα in the case of hepatitis C virus, are selectively required for individual viruses. However, despite the amount of data available, we are still far away from a comprehensive understanding of the interplay between viruses and host factors. Major limitations towards this goal are the low sensitivity and specificity of such screens, resulting in limited overlap between different screens performed with the same virus. This review focuses on how statistical and bioinformatic analysis methods applied to HT-RNAi screens can help overcoming these issues thus increasing the reliability and impact of such studies.     

Incidence and risk factors of retinopathy of prematurity and utility of the national screening criteria in a tertiary center in Iran

AIM: To determine the incidence and risk factors of retinopathy of prematurity (ROP) and the sensitivity of current screening criteria in a tertiary eye center in Tehran, Iran. METHODS: In a cross-sectional observational study, neonates weighing ≤2000 grams at birth or born <34wk gestational age (GA) and all other infants at risk of ROP admitted to the neonatal intensive care unit (NICU) or referred to our ROP clinic were investigated. The incidence of ROP and severe ROP (i.e. patients needing treatment) were determined. The associations between risk factors and the development and severity of ROP were assessed. We also examined the sensitivity of the current national screening guideline in Iran. RESULTS: Among 207 infants, the incidence of ROP and severe ROP was 33.3% and 11.1%, respectively. Mean GA and birth weight (BW) were significantly lower in ROP vs non-ROP infants (29±2wk vs 33±3wk, P<0.001; 1274±489 g vs 1916±550 g, P<0.001, respectively). Univariate analysis displayed significant association between ROP incidence and GA, BW, NICU admission period, blood transfusion, surfactant usage, sepsis, intraventricular hemorrhage and patent ductus arteriosus (P<0.05 for all). BW [relative risk (RR): 0.857 (0.711-0.873), P<0.001], GA [RR: 0.788 (0.711-0.873), P<0.001] and blood transfusion [RR: 1.888 (0.995-3.583), P=0.052] were independent ROP risk factors. The sensitivity of country-specific screening guidelines was 95.7% and 100% for overall and severe ROP detection, respectively. CONCLUSION: ROP incidence is relatively high in Iran. Identifying ROP risk factors results in more accurate screening and reduces the risk of irreversible vision loss. The ROP screening criteria utilized in Iran are efficient at the present time.     

CTLA4 blockade increases Th17 cells in patients with metastatic melanoma

Background  

Th17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature.