Omar Bagasra

The immunobiology of interferon-gamma inducible protein 10 kD (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily.

Interferon-gamma inducible protein 10 kD (IP-10) is a highly inducible, primary response gene that belongs to the C-X-C chemokine superfamily. Despite the original cloning of IP-10 in 1985, its biological functions are still unclear although accumulating reports indicate that it is a pleiotropic molecule capable of eliciting potent biological effects, including stimulation of monocytes, natural killer and T-cell migration, regulation of T-cell and bone marrow progenitor maturation, modulation of adhesion molecule expression as well as inhibition of angiogenesis. More interest is now likely to be focused on IP-10 due to the recent cloning of an IP-10 receptor. This paper aims to highlight our current knowledge of IP-10 and its homologues as well as defining its likely involvement in regulating fibroproliferation following inflammatory lung injury.

Detection of HIV-1 proviral DNA in sperm from HIV-1-infected men.

ObjectiveSexual transmission is a major mode of the spread of HIV-1, although the cellular and molecular mechanisms are poorly defined. In this study, we sought to assess the cellular reservoirs of HIV-1 proviral DNA in the semen of HIV-1-infected men. Design and methodsAn in situ polymerase chain reaction (IS-PCR), which amplifies specific genes within intact cells, was used to evaluate levels of HIV-1 provirus in seminal cells from HIV-1-infected men in various stages of clinical disease. ResultsInitial studies demonstated HIV-1 provirus in relatively low numbers (1:100 to 1:6000) of both the seminal mononuclear cells and sperm from certain HIV-1-infected men. To extend these findings, 94 seminal samples from HIV-1-infected men were evaluated. HIV-1 proviral DNA was detected in seminal cells of a significant percentage of HIV-1-infected men (45%) at all stages of clinical immunodeficiency. Both seminal mononuclear cells and sperm (35 and 33% of samples studied, respectively) harbored HIV-1 proviral sequences. HIV-1-harboring sperm are shown to stain positively for HIV-1 in the mid-pieces of these cells, with rarer staining of the sperm heads. ConclusionsHIV-1 proviral DNA can be demonstrated by IS-PCR in seminal mononuclear cells and sperm from certain HIV-1-infected men. The role played by proviral DNA in these cells in the sexual transmission of this retroviral agent will require further study.

In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal gangliaof latently infected mice

The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected by an in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with LAT probes. Sensitivity assays were shown to detect a single copy cellular gene in 48% of neuronal cell bodies. The results suggest that in situ PCR is an effective method to locate and detect HSV-1 within latently infected neurons. Moreover, the number of neurons found to be harboring HSV-1, by the method of in situ PCR, which does not depend upon virus gene expression, is within threefold of the number detected by in situ hybridization for LAT. Therefore, this report describes the first detection of HSV-1 DNA in latently infected murine trigeminal ganglia by the method of indirect in situ PCR, and compares the findings to the number of neurons expressing LAT, as assessed by conventional in situ hybridization.

Expression of cytokine mRNA in human melanoma tissues.

We have reported that patients with metastatic melanoma treated with an autologous, dinitrophenol-modified vaccine develop inflammatory responses at tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration, which is sometimes associated with tumor cell destruction. We tested biopsy specimens of eight subcutaneous metastases that had developed inflammation following vaccine treatment for expression of mRNA for interferon γ(IFNγ), interleukin-4 (IL-4), tumor necrosis factor α (TNFα), and IL-10. Post-vaccine, inflamed biopsies contained mRNA for IFNγ (5/8), IL-4 (4/8) or both (3/8), and for TNFα (4/7). In contrast, IFNγ mRNA was detected in only 1/17 and TNFα mRNA in 2/16 control specimens (pre-treatment lymph node metastases or non-inflamed subcutaneous metastases). mRNA for IL-10, a cytokine with anti-inflammatory properties, was detected in 24/25 melanoma metastases and was independent of lymphoid content; in situ the reverse transcriptase/polymerase chain reaction confirmed that melanoma cells were the major source. These findings may provide a new parameter by which to measure the effects of cancer immunotherapy.

Polymerase chain reaction in situ: intracellular amplification and detection of HIV-1 proviral DNA and other specific genes.

The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology and cell biology. We report here a unique, highly sensitive and specific technique which can be utilized to detect a single copy of human immunodeficiency virus type I (HIV-1) provirus and other genes, at the single cell level, by in situ amplification of a portion of a gene sequence. In this method, a polymerase chain reaction (PCR) can be carried out in situ, in fixed cells, on specially designed glass slides. After amplification one can detect the amplified signals by the in situ hybridization method, utilizing either biotinylated probes or 32P-labelled probes. The early molecular events in the retroviral life-cycle of HIV-1, in specific target cells, are demonstrated utilizing in situ PCR. The techniques utilized in this procedure and various potential uses of this methodology are described.

Cellular latency of human immunodeficiency virus type 1

The infection of humans by human immunodeficiency virus type 1 is characterized by a prolonged stage of clinical quiescence. This clinically asymptomatic period may be based, in part, on the development of cell populations within the body that maintain human immunodeficiency virus type 1 in a state of latency. Recent advances in the understanding of the molecular mechanisms involved in various forms of cellular latency of human immunodeficiency virus type 1 have begun to shed light on the variable period of asymptomatic infection. The elucidation of cellular retroviral latency, in vivo, will also be critical to the design of novel therapeutic approaches with which to combat human immunodeficiency virus type 1 infections.

Viral Burden and Disease Progression in HIV-1-Infected Patients With Sickle Cell Anemia

The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV‐1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV‐1 virus, will the course of the infection be altered, resulting in a prolonged symptom‐free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV‐1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV‐1‐related disease. All SS patients with a diagnosis of HIV‐1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow‐up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV‐1 viral burden. The studies included the branched‐DNA signal amplification assay, quantitative DNA‐polymerase chain reaction (PCR), quantitative reverse transcription (RT)‐initiated–PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow‐up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV‐1–infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T‐lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long‐term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long‐term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV‐1–seropositive SS patients (44%) may be asymptomatic long‐term nonprogressors. In these patients, the CD4+ T‐lymphocyte counts remained high and their viral burdens were remarkably lower than in non‐SS HIV‐1–seropositive individuals. Whereas this study does not prove an “autosplenectomy” hypothesis, it suggests that in patients with both SS and HIV‐1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV‐1 infection may be one. Am. J. Hematol. 59:199–207, 1998.

Absence of the inducible form of nitric oxide synthase in the brains of patients with the acquired immunodeficiency syndrome.

A majority of human immunodeficiency virus type I (HIV-1)-infected-individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including acquired immune deficiency syndrome (AIDS)-dementia complex (ADC), encephalitis, and various other disorders of the CNS. A series of devastating clinical conditions in the CNS of certain HIV-1-infected-individuals may be caused by infection of cells in the brain parenchyma. ADC is characterized by cognitive dysfunction, motor difficulties, coordination abnormalities and other neurological signs and symptoms, which develop in many HIV-1-infected-individuals. The precise molecular mechanisms leading to AIDS dementia remain incompletely explained. Various mechanisms including cytokine dysregulation, toxic effects of viral proteins and release of certain toxic substances from macrophages, especially nitric oxide, have been implicated as pathogenic mediators in the development of ADC. We have examined post mortem CNS tissues collected from 22 patients, previously diagnosed with AIDS, to explore if nitric oxide is responsible for the observed pathology in ADC. As controls, we utilized tissues collected from the brains of patients who expired without AIDS or other CNS pathologies. In addition, we also utilized post-mortem brain tissues from eight patients who were diagnosed with multiple sclerosis (MS) and were found to express inducible nitric oxide synthase (iNOS) in our previous studies, as positive controls. Highly sensitive in situ reverse transcriptase-initiated polymerase chain reaction (RT-IS-PCR) studies demonstrated that iNOS mRNA was present in the CNS tissues from all the positive MS controls, but were absent in all 22 specimens from AIDS patients, as well as in the brain tissues from normal controls. We have also analyzed the tissues for the presence of the NO reaction product, nitrotyrosine, to evaluate the presence of a protein nitrosalation adduct. Nitrotyrosine was not demonstrable in any of the AIDS brains. These findings indicate that iNOS may not play a significant role in the neuropathogenesis of most cases of ADC.

A Herpes simplex virus type 1 mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics

The latency associated transcripts (LATs) are the only abundant viral gene products detected during latent herpes simplex virus (HSV) infection of peripheral nerves in animals and people. A LAT promoter has been identified and mutant viruses with lesions removing the promoter and surrounding region have been observed to reactivate slowly from trigeminal ganglia (TG) explanted from latently infected mice. Previous work has shown that most mutants with lesions limited to regions downstream of the LAT promoter reactivate normally. Therefore, to help map the boundaries of the slow reactivation phenotype, a mutant virus with lesions located immediately upstream of the LAT promoter was constructed and called 17 delta S/N. 17 delta S/N contains a 437 nucleotide (nt) deletion 332 nts upstream of the TATAA box of the LAT promoter. In productively infected cells, 17 delta S/N failed to synthesize detectable amounts of the 1.1 and 1.8 kb transcripts which are produced during wild-type infections and are specified by a region just upstream of the LAT promoter. However, 17 delta S/N did produce normal amounts of LAT in tissue culture as well as in neurons derived from latently infected cells, as ascertained by Northern blot and in situ hybridization analysis. Moreover, in latently infected mice, 17 delta S/N established and maintained infection in as many neurons as did wild type virus, as determined by in situ polymerase chain reaction (PCR) to detect viral DNA. Finally, the virus reactivated from TG derived from latently infected mice with kinetics indistinguishable from those of wild-type virus. Therefore, reactivation from latency, in this model system, does not appear to require function from the viral genomic region located immediately upstream of the LAT promoter.

A quantitative reverse transcriptase-polymerase chain reaction for HIV-l-specific RNA species

The ability to evaluate the patterns and levels of human immunodeficiency virus type I (HIV-1)-specific RNA in latently and productively-infected cell lines, and primary human cells, is critical to the understanding of HIV-1 expression in cell cultures and possibly in vivo. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing in vitro transcribed RNA standards, to evaluate the copy number per cell and per microgram of total cellular RNA of multiply-spliced, unspliced and total HIV-1-specific RNA species. The latently-infected monocytic and T-lymphocyte cell lines, U1 and ACH-2 respectively, are shown to express between 10(4) to 10(6) copies of total HIV-1-specific RNA per cell, based on the state of cellular stimulation. A dramatic increase of unspliced HIV-1-specific RNA in both the U1 cell line and the ACH-2 cell line is demonstrated by this quantitative RT-PCR, 24 h after stimulation with phorbol esters. These data suggest that a single integrated HIV-1 provirus can rapidly express large quantities of HIV-1-specific RNA. Quantitative RT-PCR, for HIV-1-specific transcripts, should prove extremely useful in evaluating retroviral load and pathogenesis in cell cultures and in vivo.