HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B.

The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

Binding of type 3 reovirus by a domain of the sigma 1 protein important for hemagglutination leads to infection of murine erythroleukemia cells.

The recognition of cellular receptors by the mammalian reoviruses is an important determinant of cell and tissue tropism exhibited by reovirus strains of different serotypes. To extend our knowledge of the role of reovirus-receptor interactions in reovirus tropism, we determined whether type 1 and type 3 reovirus strains can infect cells derived from erythrocyte precursors. We found that reovirus type 3 Dearing (T3D), but not type 1 Lang, can grow in murine erythroleukemia (MEL) cells. This difference in growth was investigated by using reassortant viruses and we found that the capacity of T3D to infect MEL cells is determined by the viral cell-attachment protein, sigma 1. In experiments using murine monoclonal antibodies (mAbs) that bind to different sigma 1 regions, we show that T3D binding to MEL cells is inhibited by a mAb that identifies a domain important for hemagglutination (HA). We also determined that type 3 strains that can infect murine L cells but do not produce HA do not infect MEL cells. These results suggest that type 3 reovirus binds to and infects erythrocyte precursor cells via a sigma 1 domain important for HA. Moreover, this study suggests that different domains of some viral cell-attachment proteins are used to initiate productive infections of different types of cells.

Restricted heterogeneity of T cell receptor transcripts in rheumatoid synovium.

RA is characterized by massive proliferation of synovial tissue, accompanying infiltration of the tissue with CD4+ T lymphocytes, and a genetic linkage to the MHC antigen HLA-DR4. Since T cells are restricted by class II MHC molecules such as DR4, this suggests a direct role for these CD4+ cells in pathogenesis. To investigate T cell receptor (TCR) usage in RA, we used oligonucleotide primers specific for each of the major alpha and beta TCR subfamilies to amplify cDNA derived from whole synovium or synovial tissue T cell lines in a family-specific manner. Detection of amplified DNA was facilitated by utilizing oligonucleotide probes derived from the constant regions of the TCRs. The TCR repertoire present in the synovial T cell lines was quite heterogeneous, with an average of 15 alpha chains and 15.8 beta chains detected. When synovial tissue was analyzed, the predominant TCR subfamilies detected tended to be more restricted, with an average of 4.6 alpha chains and 8.6 beta chains detected. This compared with an average of six alpha chains and 12 beta chains in nonrheumatoid synovial samples. The average percentage of synovia positive per TCR beta family was significantly lower for RA versus non-RA specimens (46.1 vs 65.6%, P = 0.034). These findings indicate that while a polyclonal population of T cells is present in RA synovium, the predominant patterns of TCR transcript expression may be somewhat more restricted, suggesting that TCR-based therapy of RA is possible.

Vertical transmission of human immunodeficiency virus (HIV) infection. Reactivity of maternal sera with glycoprotein 120 and 41 peptides from HIV type 1.

The observation that approximately 70% of HIV-infected pregnant women do not transmit infection vertically suggests that antibody therapy may be effective in the prevention of transmission of HIV infection from mother to child. Currently, there is an incomplete understanding of the processes involved in vertical transmission of HIV infection. The elucidation of the serological basis of maternal immunity as it relates to protection from vertical transmission is the goal of this study. We have screened 20 maternal sera from HIV+ individuals of known vertical transmission status for reactivity with 31 peptides spanning the entire envelope glycoprotein of HIV-1. Of interest was reactivity to regions outside of the V3 loop of gp120. The findings have been examined in relationship to transmission status, as well as to in vitro anti-HIV-1 biological activity. Our results indicate that lack of vertical transmission is correlated with high viral neutralization activity, but not with antisyncytial activity nor with binding to the V3 peptides examined in this study. Also, the transmission group bound to fewer gp41 peptides when compared with the nontransmission group, suggesting that immune responses to gp41 may be important in preventing transmission. These findings may provide insights into the design of passive immunotherapies.

Expression of the neu gene-encoded protein (P185neu) in human non-small cell carcinomas of the lung.

The neu protooncogene is a recently described transforming gene originally isolated from ethylnitrosourea-induced rat neuroblastomas. We have examined the expression of the neu gene in human non-small cell lung carcinomas using immunoprecipitation and immunohistochemistry. The neu protein product (p185neu) was present in eight of 22 non-small cell carcinoma cell lines derived from human lung tumors. Expression of p185neu was found in all histological subtypes of non-small cell carcinomas including large cell carcinomas, squamous cell carcinomas, and adenocarcinomas. Extension of these data to biopsy specimens of human lung tumors demonstrated that normal ciliated bronchial epithelium of the peripheral airways expressed p185neu at low levels. Neoplastic cells in four of 12 adenocarcinomas and three of five squamous cell carcinomas also expressed p185neu at levels higher than the normal ciliated bronchial epithelium. Together these studies indicate that p185neu expression is a common feature of human lung tumors.

Linkage of tyrosine kinase activity with transforming ability of the p185neu oncoprotein.

The neu oncogene encodes a 185 kd glycoprotein (p185neu) with intrinsic tyrosine kinase activity. Sequencing data has demonstrated that oncogenic p185neu differs from c-neu by a single point mutation within the transmembrane region of the glycoprotein. This mutation results in the substitution of a glutamic acid residue for a nonpolar valine residue at amino acid position 664 of the rat neu gene product. Recent studies have demonstrated that this mutation results in specific aggregation of the p185neu oncoprotein mimicking ligand induced dimerization events. The cellular consequences of the aggregated phenotype may include the enzymatic activation of p185neu. We demonstrate that the oncoprotein p185neu possesses higher intrinsic tyrosine kinase activity and that this increase in enzymatic activity is apparent within the plasma membrane, manifesting itself through the increased tyrosine phosphorylation of substrates. Furthermore, the neu oncoprotein itself is also phosphorylated on tyrosine to a higher extent than the proto-oncoprotein. These results strongly link enzymatic activation of p185neu to cellular transformation events. To test directly the effect of p185neu tyrosine kinase activity on cellular transformation we constructed mutant p185neu devoid of ATP binding ability. This mutant protein is expressed at high levels, but is unable to induce the transforming phenotype. The point mutation within the transmembrane region of p185neu mimics aspects of ligand induced activation events including increases in the specific tyrosine kinase activity of the molecule leading to cellular transformation.

Expression pattern of the neu (NGL) gene-encoded growth factor receptor protein (p185neu) in normal and transformed epithelial tissues of the digestive tract.

The neu gene (also called NGL, erbB-2, and HER-2) encodes a 185-190 kDa transmembrane glycoprotein, p185neu, which has tyrosine-specific kinase activity and is homologous to but distinct from the epidermal growth factor receptor. The normal expression of neu mRNA and protein has been demonstrated in epithelial tissues of adult animals. Also, activation of the neu oncogene has been implicated in a variety of human adenocarcinomas. In the present study, we examined the expression of the p185neu protein in normal and transformed digestive tract tissues and in a panel of digestive tract-derived cell lines. By immunohistochemistry, strong reactivity was observed in the mucosal epithelium of the stomach, small intestine, and colon of both rodents and humans. In the small intestine, there was prominent p185neu expression by mucosal epithelium of the villus, with little or no staining in the crypts. Prominent expression was observed in the liver parenchyma, the endocrine and exocrine portions of the pancreas, and in the salivary gland. Immunoreactive p185neu was also demonstrated in fetal human intestinal epithelium. Tissue sections of selected benign and malignant colonic neoplasms were also examined. Immunoreactivity was consistently greater in adenomatous polyps than in adjacent normal colonic epithelium or areas showing malignant degeneration. By radioimmunoprecipitation, there was decreased expression in cell lines derived from more anaplastic colonic tumors. The p185neu protein is expressed widely in normal and transformed epithelial tissues of the digestive tract of the adult rat and human. This finding suggests that p185neu, a putative growth factor receptor, may play a role in the regulation of normal growth and function or in the malignant transformation of these cells.

HIV-1 viral protein R (Vpr) regulates viral replication and cellular proliferation in T cells and monocytoid cells in vitro.

Among the putative accessory genes of HIV-1, the 96-amino-acid virion-associated vpr gene product has been described to have several novel biological activities. These include cytoplasmic-to-nuclear translocation, which empowers HIV to infect and replicate in non-dividing cells and to increase viral replication, particularly in macrophages. Along with these viral effects, we found that HIV-1 Vpr induces dramatic biological changes in the target cells of HIV infection, including induction of changes in transcriptional patterns, morphological changes, and complete inhibition of proliferation, which collectively was termed differentiation. These changes occur in the absence of other viral gene products, suggesting that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T cell lines has been extended by several groups to demonstrate that the inhibition of proliferation is through G2 cell cycle arrest, further supporting the idea that Vpr acts directly on cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, which is involved in the regulation of the state of the cell, in cytoplasmic-to-nuclear translocation, and in modulation of host cell transcription. It is important to note that certain anti-glucocorticoid compounds modulate Vpr activity in vitro. These results support the idea that the host cell contains specific receptor molecule(s) through which Vpr mediates its activity. Consequently, Vpr represents a unique target for anti-HIV drug development and has significance for HIV-1 disease progression.

Predicting drug-drug interactions involving multiple mechanisms using physiologically based pharmacokinetic modeling: a case study with ruxolitinib.

Physiologically based pharmacokinetic modeling was applied to characterize the potential drug-drug interactions for ruxolitinib. A ruxolitinib physiologically based pharmacokinetic model was constructed using all baseline PK data in healthy subjects, and verified by retrospective predictions of observed drug-drug interactions with rifampin (a potent CYP3A4 inducer), ketoconazole (a potent CYP3A4 reversible inhibitor) and erythromycin (a moderate time-dependent inhibitor of CYP3A4). The model prospectively predicts that 100-200 mg daily dose of fluconazole, a dual inhibitor of CYP3A4 and 2C9, would increase ruxolitinib plasma concentration area under the curve by ∼two-fold, and that as a perpetrator, ruxolitinib is highly unlikely to have any discernible effect on digoxin, a sensitive P-glycoprotein substrate. The analysis described here illustrates the capability of physiologically based pharmacokinetic modeling to predict drug-drug interactions involving several commonly encountered interaction mechanisms and makes the case for routine use of model-based prediction for clinical drug-drug interactions. A model verification checklist was explored to harmonize the methodology and use of physiologically based pharmacokinetic modeling.

Cloning and biological characterization of human single-chain Fv fragments that mediate neutralization of HIV-1.

OBJECTIVE: To develop recombinant single-chain Fv fragments against HIV-1 gp120. METHODS: A panel of human monoclonal antibody Fv fragments were generated against the HIV-1 gp120 by affinity selection from an antibody library expressed on the surface of filamentous phage. The library was prepared from peripheral blood lymphocytes of an asymptomatic HIV-1-infected mother with a high neutralization titer. This mother did not transmit HIV-1 to her offspring (non-transmitter). Heavy and light chains were initially amplified separately and combined by splicing by overlap extension to generate Fv fragments. RESULTS: Several clones expressing single-chain Fv fragments bind strongly to HIV-1 gp120 and several were found to neutralize cell-free HIV-1IIIB. Gross epitope mapping suggest that different clones bound to different functional regions on the envelope. The clones also exhibited sequence diversity. CONCLUSIONS: This strategy of cloning resulted in the development of functional human-derived antibody reagents with different anti-HIV-1 biological properties in vitro. These recombinant Fv fragments have potential utility as immune reagents, as well as in the design of potential immunotherapeutics. In addition, these antibody reagents may provide information on the relationship between humoral immunity and maternal-fetal (vertical) HIV-1 transmission.

PASSHIV-1 treatment of patients with HIV-1 infection. A preliminary report of a phase I trial of hyperimmune porcine immunoglobulin to HIV-1.

OBJECTIVE: To study the safety of intravenously administered porcine-derived hyperimmune immunoglobulin to HIV-1, PASSHIV-1, in humans. METHODS: Fourteen HIV-1-infected individuals were treated for 5-7 days with intravenous infusions of highly purified PASSHIV-1 (> 95% pure). Two of the 14 patients were retreated 3 months later with PASSHIV-1 for an additional 5 days to evaluate side-effects from retreatment with porcine immunoglobulins. RESULTS: Ten of the patients had no side-effects from PASSHIV-1 therapy. Three patients experienced transient urticarial eruptions, which responded to antihistamine administration and did not require discontinuation of therapy. One patient, who received concomitant administration of human gammaglobulin, experienced serum sickness (type 3 hypersensitivity reaction). All patients demonstrated a significant improvement in fatigue (100% response), weight (all those with previous weight loss gained weight), fever (100% response), polyneuropathy (100% response), bronchitis (100% response), candidiasis (100% response), diarrhea (100% response), and dermatitis (100% response). One out of the five patients with Kaposi's sarcoma demonstrated > 50% improvement. Mean CD4+ cell counts in the group rose from 143 +/- 263 to 234 +/- 323 x 10(6)/l 4-6 months following completion of therapy (P = 0.013, paired Student's t-test); CD4+ counts rose > twofold in six individuals. p24 antigen, present in four patients, was negative following therapy in all patients. Other laboratory parameters that responded to therapy included: platelet counts (71% response), leukopenia (57% response), elevated lactic dehydrogenase (100% response), and elevated alkaline phosphatase (100% response). PASSHIV-1 was well tolerated by HIV-1-infected individuals. CONCLUSION: This therapy appears to be efficacious in ameliorating some of the clinical aspects and symptoms of HIV-1 infection.

Identifying structure-function relationships in four-helix bundle cytokines: towards de novo mimetics design.

Many cytokines (growth-factor proteins) are constructed from a common four-helix bundle structural framework. Rapid advances have been made in relating the structure and function of a growing number of four-helix bundle cytokines. This understanding opens the way to design de novo mimetics through such strategies as cytokine hybrids, structure-excerpted scaffolds and contact residue topology mimics. These may provide leads for agonists and antagonists of cell growth and differentiation.

Antibody geometry and form: three-dimensional relationships between anti-idiotypic antibodies and external antigens.

Anti-idiotypes have been developed to mimic a wide variety of antigens in studies of antibody-antigen interactions. Molecular, biochemical and structural analysis of antibody-anti-idiotype pairs presents an excellent model for study of complex protein-protein interactions. An understanding of the ways in which anti-idiotypes mimic antigens and the features determining binding characteristics will facilitate rational design of compounds with biological, enzymatic, pharmaceutical and chemical applications.

Immunization by direct DNA inoculation induces rejection of tumor cell challenge.

Direct DNA inoculation is the basis for a new technology that has been successfully used for in vivo induction of both humoral and cellular immune responses. However, the immunological parameters of this new approach remain to be evaluated in detail. We report here that direct DNA inoculation can induce protection from malignant tumor cell challenge through the generation of specific immune responses directed against antigens displayed on the tumor cells. The protected mice remain tumor-free for more than 1 year post-challenge. Memory responses upon tumor rechallenge were observed for both humoral and cellular immunity. Inoculated animals were able to reject otherwise lethal tumors several months following the original DNA inoculation protocol. These in vivo protective responses suggest that further analysis of this technology for vaccine development or immune therapeutic strategies is warranted.

Prevention in mental health: a controlled study.

The authors conducted a controlled study in which families who had experienced the sudden death of a family member were given crisis intervention services and compared at follow-up with two untreated control groups. Results did not support the hypothesis that such services decrease the risk of psychiatric illness, disturbed family functioning, or increased social cost to the families. The authors suggest that environmental and social systems factors and individual variables are powerful predictors of outcome in bereavement.

Hypophyseal-pituitary-adrenal axis in autoimmune and rheumatic diseases.

This article discusses the effects of sex steroids and anterior pituitary hormones on the immune system. Data from clinical and experimental studies on the effects of CRH, FSH, LH, and prolactin are reviewed. This is followed by a summary of results from our studies on the effects of FSH, LH, and prolactin on PBMC, CD4+ cells, and CD8+ cells in vitro.

T cell receptor analysis in rheumatoid arthritis: what have we learnt?

Many clues point to a role for T lymphocytes in the pathogenesis of rheumatoid arthritis (RA), although the importance of these cells and their position within the rheumatoid pathogenic scheme remain unknown. Encouraged by data from animal models of T-lymphocyte-mediated autoimmunity, a major focus of research into the role of T lymphocytes in RA has been the usage of T cell receptor V genes in rheumatoid synovitis. Despite many methodologic problems, involving choice of patients and controls, choice of specimens, and technical factors, several conclusions can be drawn from the published research. In particular, synovial T lymphocyte populations, as a whole, frequently show biased V gene usage and restricted clonality within those T lymphocyte subsets that utilize over-represented V gene families. Continued research into these synovial T lymphocyte subsets should provide important insights into the pathogenesis of RA, particularly if solutions to the identified methodologic problems are implemented.

The antigen-major histocompatibility complex-T cell receptor interaction. A structural analysis.

In summary, we wish to propose that regions on MHC molecules interact with complementary regions on processed peptide antigens, and that the resultant Ag-MHC complex forms a conformation with separate functional regions that are able to interact with similarly complementary areas on T cell receptors. It is the product of these interactions that determines whether a given peptide Ag is capable of binding the MHC molecule, and whether a given Ag-MHC complex is capable of stimulating a particular T cell. As more becomes known about the molecular aspects of MHC-restricted, Ag-specific T cell activation, it will become clear which amino acid residues on the Ag, MHC, and TCR are involved in these interactions.

Structural properties of a subset of nephritogenic anti-DNA antibodies.

Structural analysis of lupus autoantibodies is beginning to provide clues to the molecular basis for antigenic specificity and pathogenicity. The present analysis indicates that multiple light and heavy chains contain residues which can facilitate DNA binding, reaffirming the notion that there are multiple ways that different amino acids combine to form an antigen-binding pocket with affinity for dsDNA and ssDNA. Furthermore, this analysis suggests that these conformations and contact residues are intrinsic to germline sequences, although amino acid changes at critical locations (somatically introduced) modulate antigen binding, and appear to influence the capacity of individual immunoglobulin to form immune deposits. Analysis of additional individual immunoglobulins with closely related V-region sequences and differing pathogenic properties will be required to resolve the contribution of specific motifs to pathogenecity.