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M S McGrath

Publications and source records attributed to M S McGrath.

At least 19 recordsLinked to original sources
PubMed1998

A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages.

Primary cultured human macrophages are difficult to transfect. We have developed a DEAE-dextran DNA transfection method that mediates the reproducible transfection of primary cultured adherent human macrophages. Three factors essential for successful transfection were identified: DEAE-dextran concentration, the quantity of DNA per transfection and the incubation time of the macrophages with the transfection medium. Maximum levels of luciferase expression were attained within 24 h and maintained for at least 56 h after transfection. While serum in the transfection medium attenuated transfection, the treatment of the macrophages with chloroquine, DMSO, or glycerol did not enhance transfection within this system. A CMV enhancer/promoter mediated substantially greater luciferase expression in the macrophages than either HIV or RSV LTRs. DEAE-dextran facilitated superior transfection compared to either cationic liposome and calcium phosphate methods, and was more practical compared to electroporation for multiple transfections. This transfection protocol provides a simple, inexpensive, reproducible method for the evaluation of gene expression in primary cultured adherent human macrophages.

Adult↗
PubMed1998

The immunology of AIDS-associated lymphomas.

Lymphomas that occur in patients with human immunodeficiency virus (HIV) infection are predominantly of B-cell origin and subsets show evidence for Epstein-Barr virus (EBV) infection or chromosomal translocations in the c-myc locus. The only subset of lymphoma clearly related to the immunodeficiency caused by HIV infection (similar to transplantation-associated lymphomas) is the EBV+ primary central nervous system lymphoma. The systemic AIDS-related lymphomas (ARLs) represent a complex set of disease processes histologically categorized as large cell or small non-cleaved (Burkitt's-like) lymphomas. Molecular analyses of the ARLs have demonstrated polyclonal lymphomas as likely early representatives of monoclonal immunoglobulin (Ig)-expressing B-cell lymphomas. Variable region analysis of lymphoma-associated Ig has shown evidence for extensive somatic mutation with little evidence for appropriate affinity maturation. These observations suggest that abnormal control of B-cell maturation in response to polyclonal antigenic stimulation may play a central role in the pathogenesis of ARL. The recent finding of clonal HIV integrated within macrophages in a subset of early lymphomas also provides evidence for abnormalities outside the B-cell compartment playing roles in this disease. Overall, ARLs generally appear to be outgrowths of antigen-driven B-cells with significant growth control influence provided by abnormal T-cell and antigen-presenting cell processes.

Cell Transformation, Neoplastic↗
PubMed1997

Unique monocyte subset in patients with AIDS dementia.

BACKGROUND: 15-30% of patients infected with HIV will develop a debilitating dementia. Whilst HIV enters the brain soon after infection, presumably within monocyte-derived macrophages, not all patients with HIV become demented. Blood monocytes probably cross the blood-brain barrier and give rise ultimately to parenchyma macrophages. We looked for a specific monocyte subset in AIDS patients with dementia. METHODS: Peripheral blood monocytes from three groups were compared: AIDS patients with (n = 12) and without (n = 11) dementia, and ten HIV seronegative healthy controls. We used flow cytometry to analyse monocytes, and cell lysis and apoptosis assays to examine monocyte effects on human brain cells in vitro. FINDINGS: We found a unique subset of monocytes in patients with AIDS dementia. These monocytes were more dense and granular and expressed CD14/CD16 and CD14/CD69. Means (SD) for CD14/CD16 in HIV-negative controls and in AIDS non-dementia and AIDS dementia patients were 6.5% (4), 16% (13), and 37% (21), respectively (p = 0.008 between the two groups of patients). The corresponding means for CD14/CD69 were 7% (6), 8% (10), and 69% (18) (p < 0.0001). INTERPRETATION: CD69 is a member of the natural-killer-cell gene complex that is expressed after activation. Supernatants from cultures containing these dense cells can trigger apoptosis of human brain cells in vitro. The monocyte subset we found in patients with AIDS dementia might enter the brain and expose neural cells to toxic factors.

AIDS Dementia Complex↗
PubMed1997

VH gene use by HIV type 1-associated lymphoproliferations.

The pathogenesis of polyclonal HIV-associated lymphomas lacking traditional B cell cofactors (i.e., Epstein-Barr virus [EBV] infection, c-myc translocations) is poorly understood. A multistep pathogenesis model has been proposed in which polyclonal lymphomas represent an earlier stage in HIV-associated lymphomagenesis before the emergence of a dominant malignant clone. Chronically present antigens have been proposed as a likely stimulus for polyclonal B cell proliferation; if so, polyclonal lymphoma-associated immunoglobulins (Igs) should have molecular evidence of somatic hypermutation, a process by which antibody affinity maturation in response to chronic antigenic stimulation occurs. Molecular analyses of Ig heavy chain variable (V(H)) gene use by B cells in a polyclonal HIV-associated large cell lymphoma lacking EBV and c-myc rearrangement was undertaken. Eighteen randomly selected clones generated from RT-PCR yielded 15 unique V(H) sequences, all of which were most homologous to only three previously identified germline V(H)1 genes. Two sets of clones (consisting of three and two clones, respectively) had identical V(H) gene sequences, and one pair of clones had identical third complementarity determining regions (CDR3s) but different V(H) gene sequences; eight clones were <95% homologous to their most related germline V(H)1 genes. We compared these results with Ig V(H)1 gene use by B cells present in a reactive hyperplastic lymph node obtained from an HIV-1-infected individual. Fifteen clones randomly selected from RT-PCRs yielded 15 unique V(H)1 sequences, all of which were most homologous to 5 previously identified germline V(H)1 genes; 10 clones were <95% homologous to their most related germline gene. Binomial probability analysis revealed that only 1 of the 15 unique V(H)1 sequences derived from the polyclonal lymphoma (i.e., 7%), as compared with 5 of 15 unique V(H)1 sequences derived from the reactive lymph node (i.e., 33%), had a low probability of occurrence by random chance (p < 0.05). These data provide molecular evidence of polyclonality in an HIV-associated polyclonal lymphoma, demonstrate a qualitative difference in somatic hypermutations of Ig V(H) genes associated with malignant versus reactive B cell lymphoproliferations, and support an antigen-mediated multistep pathogenesis model of HIV-1-associated lymphomagenesis.

Amino Acid Sequence↗
PubMed1997

Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection.

Antibodies, Monoclonal↗
PubMed1996

Evidence for early B-cell activation preceding the development of Epstein-Barr virus-negative acquired immunodeficiency syndrome-related lymphoma.

To investigate the origin and pathogenesis of acquired immunodeficiency syndrome (AIDS)-related lymphoma (ARL), we studied 14 cases in which Epstein-Barr virus (EBV) infection was not an etiologic factor. By histology, 8 of the specimens were of the small noncleaved cell type and 6 consisted of the large diffuse cell type. Southern analysis using a J(H) probe was consistent with a monoclonal B-cell tumor in 13 cases. To characterize the expressed Ig genes, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing of PCR products. Eight cases expressed IgM and 1 case expressed IgG. V(H)3 genes were found in 5 cases, V(H)4 genes in 3 cases, V(H)1 genes in 2 cases, and a V(H)7 gene in 1 case. The nucleotide homology to known germline V(H) genes ranged from 80% to 97%, suggesting significant somatic diversification of expressed V(H) genes. The large proportion of V(H)3-expressing lymphomas in this series corresponds to the frequency of V(H)3-expressing B cells in the peripheral blood from healthy and (recent) human immunodeficiency virus (HIV)-seropositve individuals and contrasts with the V(H)3 clonal deficit observed in late stages of HIV infection. Similar to the Ig heavy chain genes, the corresponding Ig light chain genes showed significant deviation from known germline gene sequences. The large proportion of V(H)3-expressing lymphomas as well as the high degree of somatic deviation from germline suggest that these EBV-negative lymphomas might arise from antigen-selected expanded B-cell clones before transformation. Further support for this hypothesis is provided by sequential Ig sequence analysis in 1 patient with large-cell lymphoma. It was shown that 3 years before the diagnosis of axillary lymphoma, there existed several B-cell clones in this patient's bone marrow. One of these clones present in the bone marrow expressed the same rearranged V(H) gene as the axillary lymphoma. Taken together, the current findings from Ig gene analyses suggest that activation of B cells in the early phase of HIV infection may be a predisposing factor for subsequent B-cell transformation.

Amino Acid Sequence↗
PubMed1996

The natural history and molecular heterogeneity of HIV-associated primary malignant lymphomatous effusions.

Primary malignant lymphomatous effusions arising in individuals infected with the human immunodeficiency virus, type 1 (HIV-1) represent a rare subset of HIV-associated lymphomas. Previous studies have demonstrated that the malignant cells are monoclonal (as defined by rearrangement of the immunoglobulin gene), express cell surface CD38, and are infected with Epstein-Barr virus (EBV) and human herpes virus, type 8 (HHV-8). Despite these detailed molecular and immunophenotypic studies, clinical information on this disease entity is scant, prompting us to review the clinical features of eight cases seen at our institutions. All eight patients had total peripheral CD4+ lymphocytes < 200/microliter and presented with complaints related to body cavity distension. Routine laboratory values were nondiagnostic and yielded no prognostic information. Only two patients could tolerate and thus received chemotherapy with no obvious impact on their clinical course. The mean overall survival after diagnosis was 60 days (range 6-166 days). Four patients were examined at autopsy. The primary malignant lymphomatous effusion either was the immediate cause of death or contributed significantly to the death of only two. All four patients examined post mortem, however, had lymphomatous infiltration of serosal surfaces adjacent to the site of the primary malignant effusion. Molecular and immunologic studies performed on the malignant cells and effusion fluids revealed universal expression of cell surface CD38 and the presence of HHV-8 gene sequences, but in contrast with previous studies, only four had rearranged immunoglobulin genes or EBV present: IL-6 and IL-10 levels in the malignant effusion fluids were markedly elevated. In summary, this rare subset of HIV-associated lymphomas in our eight patients arose late in the course of HIV-associated disease, had a rapid clinical course, and was molecularly heterogeneous. A pathogenetic role for HHV-8 alone in this disease process is strengthened by our observation of four cases lacking EBV but containing HHV-8.

ADP-ribosyl Cyclase↗
PubMed1996

Monokine products as predictors of AIDS dementia.

OBJECTIVE: To determine whether or not soluble factors produced by peripheral blood mononuclear cells (PBMC) can predict AIDS dementia. DESIGN AND METHODS: PBMC were isolated from individuals with and without AIDS dementia complex (ADC) to determine if the levels of cytokines tumour necrosis factor (TNF)-alpha and interleukin (IL)-6, or the production of a neurotoxic substance, were significantly different. PBMC were studied after determining that the numbers of monocyte-derived macrophages isolated by adherence were highly variable from patients with ADC compared with individuals without ADC. We prospectively studied 16 AIDS dementia patients, 13 healthy HIV-seropositive individuals, and eight sero-negative controls. Supernatants from PBMC were assayed for TNF-alpha, IL-6 and alone for neurotoxicity on human neural cells in vitro. RESULTS: We observed a trend towards worse cognitive and motor performance in patients suffering from ADC but who had no opportunistic infections ('pure dementia'; n = 8). Levels of PBMC IL-6 were significantly higher in 'pure dementia' patients. There was a trend towards lower levels of PBMC TNF-alpha in the group of patients who had both dementia and opportunistic infections compared with "pure dementia' patients. Supernatant from PBMC of ADC patients was significantly more neurotoxic than that from healthy HIV-seropositive individuals. CONCLUSIONS: Macrophage isolation from PBMC of patients with ADC was altered. Soluble factors produced from PBMC were significantly more neurotoxic than soluble factors from PBMC of healthy HIV-seropositive individuals. PBMC production of TNF-alpha and IL-6 was not a significant predictor of ADC.

AIDS Dementia Complex↗
PubMed1995

Influence of molecular characteristics on clinical outcome in human immunodeficiency virus-associated non-Hodgkin's lymphoma: identification of a subgroup with favorable clinical outcome.

The relationship between clinical and molecular characteristics of 45 treated individuals with histologically-documented human immunodeficiency virus (HIV)-associated B-cell non-Hodgkin's lymphoma was examined to determine whether differences in molecular features of lymphoma were associated with differences in clinical outcome. Tissue specimens from these tumors were evaluated for evidence of Ig heavy-chain gene rearrangements using both Southern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Lymphomas were also evaluated for the presence of Epstein-Barr virus (EBV) DNA sequences and c-myc gene rearrangements. Twenty-five lymphomas were characterized as polyclonal and 20 as monoclonal. PCR amplification of expressed Ig variable (V)-region genes confirmed polyclonality in three extensively studied polyclonal lymphomas. The median CD4 count was significantly higher in the group with polyclonal disease (277/microL) than in the group with monoclonal disease (123/microL), P = .04. The complete response rate to therapy was significantly higher in patients with polyclonal disease (78%) and CD4 greater than 200/microL (81%) than in those with monoclonal disease (31%) and CD4 less than 200/microL (33%). CD4 count, clonality, and presence of EBV DNA sequences were the most important predictors of survival. Both Kaplan-Meier and Cox proportional hazards analyses showed a markedly prolonged survival in those patients with both CD4 > or = 200/microL and polyclonal disease. Histologically the polyclonal lymphomas were high grade in appearance and contained prominent macrophages. All seven surviving patients were in this group. Median survival for those individuals whose tumors contained EBV sequences was only 3.2 months (range, 0.4 to 19.5), whereas those with EBV- tumors survived for a median of 9.0 months (range, 0.7 to 65.2), P = .0007. These data indicate that molecular features of HIV-associated lymphomas may be important predictors of clinical outcome. These characteristics define a distinct subset of patients with polyclonal EBV- tumors and CD4 counts greater than 200/microL that appear to have a less aggressive clinical course.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

Identification of a clonal form of HIV in early Kaposi's sarcoma: evidence for a novel model of oncogenesis, "sequential neoplasia".

We recently reported clonal human immunodeficiency virus (HIV involvement in four acquired immune deficiency syndrome (AIDS)-associated non-B-cell lymphoproliferations. In three of these cases HIV expression was localized to tumor-associated macrophages. Because one of the cases had a major component involved in angioproliferation, we speculated that some form of Kaposi's sarcoma (KS), which also has a major component of angioproliferation, might be involved clonally with HIV. The current study is an evaluation of four cases of KS and control tissues taken from four patients who died with complications of HIV disease. With use of the inverse polymerase chain reaction technique to identify clonal forms of HIV, a clonal form of HIV was found in one of four KS cases. The HIV-positive tumor was an early KS lesion of the bowel, and uninvolved bowel from the same patient showed no clonal HIV. Immunohistochemical analysis demonstrated the presence of prominent HIV-expressing macrophages that also coexpressed high levels of HIV tat, basic fibroblast growth factor, and interleukin-6. These data provide evidence for a pathogenic process termed "sequential neoplasia," wherein a clonal macrophage provides a growth factor milieu stimulating the proliferation of a responder cell population that ultimately becomes autonomous. In the current case, the macrophages expressing HIV were located adjacent to the KS tumor tissue and were found to be producing known KS growth factors. The absence of finding clonal HIV in three more advanced KS lesions suggests that the clonal macrophage may be required only for early pathogenesis and that sequential neoplastic changes occurring in the endothelial cells gave rise to autonomous KS.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads.

An oligonucleotide (I100-15) composed of only deoxyguanosine and thymidine was able to inhibit human immunodeficiency virus type-1 (HIV-1) in culture assay systems. I100-15 did not block virus entry into cells but did reduce viral-specific transcripts. As assessed by NMR and polyacrylamide gel methods, I100-15 appears to form a structure in which two stacked guanosine tetrads are connected by three two-base long loops. Structure/activity experiments indicated that formation of intramolecular guanosine tetrads was necessary to achieve maximum antiviral activity. The single deoxyguanosine nucleotide present in each loop was found to be extremely important for the overall antiviral activity. The toxicity of I100-15 was determined to be well above the 50% effective dose (ED50) in culture which yielded a high therapeutic index (> 100). The addition of a cholesterol moiety to the 3' terminus of I100-15 (I100-23) reduced the ED50 value to less than 50 nM (from 0.12 microM for I100-15) and increased the duration of viral suppression to greater than 21 days (versus 7-10 days for I100-15) after removal of the drug from infected cell cultures. The favorable therapeutic index of such molecules coupled with the prolonged suppression of HIV-1, suggest that such compounds further warrant investigation as potential therapeutic agents.

3T3 Cells↗
PubMed1995

Cytokine expression in large cell lymphoma associated with acquired immunodeficiency syndrome.

Cytokine expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in a retrospective sampling of 16 AIDS-associated large cell lymphomas (LCL). IL-6 receptor (IL-6R) and IL-10 expression was detected in a majority of the tumor specimens tested, IL-6 expression was detected in 5 of 16 lymphomas that also expressed IL-6R, suggestive of an autocrine mechanism of disease. A subset of tumor samples described as mixed immunophenotype contained large numbers of infiltrating T lymphocytes and macrophages. Immunoperoxidase staining of a representative tumor of mixed immunophenotype demonstrated the presence of HIV-infected macrophages that also stained with anti-IL-6. This finding suggests that IL-6 produced by nonlymphoid cells may act as a paracrine growth factor for tumor cells that express IL-6R. Although earlier studies of AIDs burkitt's lymphoma cell lines suggested that IL-10 expression required EBV infection, 7 of 12 AIDS LCLs that expressed IL-10 did so in the absence of EBV by EBER in situ hybridization. Because AIDS LCLs frequently express cell surface CD5, we speculate that IL-10 may act as an autocrine or paracrine growth factor for this class of lymphoma. These studies suggest that IL-6 and IL-10 are involved in the pathogenesis of AIDS-associated large cell and mixed immunophenotype lymphoma.

Base Sequence↗
PubMed1994

Identification of a common clonal human immunodeficiency virus integration site in human immunodeficiency virus-associated lymphomas.

Infection with human immunodeficiency virus type 1 (HIV-1) is associated with a high incidence of lymphoma. Typically, the lymphomas are B-cell in origin, and although they occur in the setting of HIV-1 infection, historical studies have found no evidence for the presence of HIV-1 within the transformed B-cells. We describe a new class of large cell lymphoma wherein HIV p24 expression within the tumor specimens was found to be extremely high. In the first case, HIV was expressed in the tumor-associated transformed T-cells. In three other cases, HIV was found to be highly expressed in tumor-associated macrophages. These tumors exhibited a mixed immunophenotype histologically. Analysis by inverse polymerase chain reaction, using HIV long terminal repeat primers, demonstrated monoclonal HIV integration sites for all four tumors. Direct sequencing of the T-cell lymphoma inverse polymerase chain reaction products identified the HIV integration site within the fur gene, just upstream from the c-fes/fps protooncogene. Using segments of the fur gene as a probe, the other three monoclonal integration sites mapped to the same region. Although the integration and up-regulation of c-fes/fps was localized to the tumor cells within the T-cell lymphoma, the cells containing the monoclonal HIV in the other mixed immunophenotype lymphomas are currently unknown. These observations suggest that HIV may contribute directly to lymphomagenesis and identify a common site of HIV integration within a subset of acquired immunodeficiency syndrome lymphoma.

Base Sequence↗
PubMed1994

HIV infection of monocyte-derived macrophages in vitro reduces phagocytosis of Candida albicans.

HIV-1 infection of peripheral blood monocyte-derived macrophages (MDMs) is unrelated to the level of CD4 expression on the surface of the cell, is associated with considerable donor variability, causes minimal cytopathology, and results in peak viral antigen production after 2 weeks of infection. Phagocytosis of opsonized Candida albicans by MDMs infected in vitro with several strains of HIV was compared with that of uninfected cells from the same donors; the proportion of MDMs containing the fluorescein isothiocyanate-labeled yeast was determined by flow cytometry and phase contrast microscopy. The intracellular localization of C. albicans was confirmed by confocal microscopy. Using paired MDMs from nine donors, 81% of uninfected and 53% of HIV-infected MDMs phagocytosed C. albicans. In addition, the number of yeast per cell was significantly higher in uninfected MDMs than in HIV-infected cells (mean 6.1 versus 2.5). These findings may partially explain the high incidence of mucocutaneous candidiasis in HIV-infected patients with advanced disease.

AIDS-Related Opportunistic Infections↗
PubMed1994

Investigation of HIV-infected macrophage neurotoxin production from patients with AIDS dementia.

The mechanism for AIDS dementia may involve the production of toxic cytokines. Since macrophage/microglia are the infected cells in the brain, we were interested in the production of some of the same cytokines by HIV-infected macrophages from patients with AIDS dementia. HIV-infected macrophages from 11 patients with AIDS dementia were cultured and evaluated for p24, cytomegalovirus (CMV) DNA, and the production of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF alpha) and other neurotoxic factors. The percentage of HIV macrophage infectivity did not correlate with the severity of dementia nor did the presence of CMV. The production of IL-6 and an unidentified neurotoxin did not correlate with HIV macrophage infectivity suggesting that these soluble factors are strain specific. Production of TNF alpha by HIV-infected macrophages was relatively low (< 1-35 pg/ml) and may not be a significant factor in toxicity.

AIDS Dementia Complex↗