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M S Reitz

Publications and source records attributed to M S Reitz.

At least 19 recordsLinked to original sources
PubMed2002

A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice.

The rational design of new therapies against HIV-1 necessitates an improved understanding of the mechanisms underlying the production of ineffective immune responses to HIV-1 in most infected individuals. This report shows that the CD8(+) T cell responses to gp120 were greatly diminished in mice vaccinated with a bicistronic gp120-Tat DNA vaccine, compared with those induced by a DNA vaccine encoding gp120 alone. The CD8(+) T cell responses induced by the latter included strong gp120-specific IFN-gamma secretion and protective antiviral immunity against challenge by a vaccinia-env pseudotype. The degree to which Tat influenced CD8(+) T cell responses depended on the bioactivity of Tat. Thus, a bicistronic DNA vaccine that expresses gp120 and a truncated Tat defective for LTR activation elicited strong IFN-gamma -secreting CD8(+) T cell responses to gp120 but conferred only marginal protection against the vaccinia-env challenge. The effect of Tat was completely blocked, however, by immunization with inactivated Tat protein before vaccination with the bicistronic gp120-Tat DNA vaccine.

AIDS Vaccines↗
PubMed2001

Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector.

CD8(+) T-cell responses provide beneficial antiviral immunity against human immunodeficiency virus 1 (HIV-1). In this study, we show that intragastric vaccination with a Salmonella HIV-1 Env DNA vaccine vector generates Env-specific CD8(+) T-cells, both in mucosal and systemic lymphoid tissue. By contrast, intramuscular vaccination with the Env DNA vaccine alone only induced systemic CD8(+) T-cells. To our knowledge, this is the first report showing both mucosal and systemic CD8(+) T-cell responses following vaccination with a Salmonella vaccine vector. These data suggest that this mode of HIV-1 DNA vaccine delivery will be advantageous over parenterally administered HIV-1 DNA vaccines.

AIDS Vaccines↗
PubMed2001

Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER).

Depending on the nature of the costimulation of T lymphocytes, expression of regulatory cytokines and chemokines is either susceptible or resistant to cyclic AMP (cAMP)-mediated inhibition. Our data show that cAMP-mediated inhibition of endogenously expressed cytokines, which is characteristic for T helper (Th) 1- and Th 2-like phenotypes, correlates with the induction of a potent transcriptional repressor, inducible cAMP early repressor (ICER), in both subsets of T cells activated under conditions of suboptimal interleukin-2 (IL-2) expression. Importantly, Th-specific expression of certain chemokines is also susceptible to cAMP-mediated transcriptional attenuation. To determine whether ICER per se, rather than forskolin-mediated elevation of intracellular cAMP, is responsible for the observed inhibitory effect, we generated transgenic mice expressing ICER under the control of a lymphocyte-specific lck promoter. On stimulation, transgenic thymocytes overexpressing ICER exhibited reduced levels of IL-2 and interferon (IFN)-gamma and failed to express the macrophage inflammatory protein (MIP)-1alpha and MIP-1beta genes. Splenic T cells from ICER-transgenic mice showed a defect in proliferation and lacked a mixed lymphocyte reaction response, implying that ICER-mediated inhibition of cytokine and chemokine expression might play an important role in T-cell inactivation.

Animals↗
PubMed1999

Recombinant IFN-alpha (2b) increases the expression of apoptosis receptor CD95 and chemokine receptors CCR1 and CCR3 in monocytoid cells.

IFN-alpha-2b, known as potent immune modulator, can either inhibit or enhance immune cell activity within the tightly regulated microenvironment of inflammation, depending upon the concentration of the cytokine and the activation stage of the cell. Chemokine receptors, which not only mediate chemotaxis of immune cells to the site of inflammation but also affect cellular activation by transferring corresponding signals, represent yet another level of immune regulation. Here we demonstrate that IFN-alpha increases the expression of CCR1 and CCR3 in primary mononuclear phagocytes, as well as in the monocytoid cell line U937. Enhanced receptor mRNA expression correlated with functional readouts such as increased intracellular calcium mobilization and cell migration in response to ligands. Expression of CCR2b, CCR4, CCR5, and CXCR4 was unchanged or decreased after IFN-alpha treatment. These observations indicate a differentially regulated cellular signaling relationship of IFN-alpha pathways and chemokine receptor expression. We also provide evidence that, under these conditions, IFN-alpha treatment increased the expression of CD95 (Fas, Apo1), resulting in enhanced susceptibility to apoptosis. Taken together, these data add important information for the rational application of IFN-alpha (2b) in immune and cancer therapies.

Apoptosis↗
PubMed1999

Comparison of genetic variability at multiple loci across the genomes of the major subtypes of Kaposi's sarcoma-associated herpesvirus reveals evidence for recombination and for two distinct types of open reading frame K15 alleles at the right-hand end.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0. 35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized "orphan" region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results suggest that an original recombination event with a related primate virus from an unknown source introduced exogenous right-hand side ORF-K15(M) sequences into an ancient M form of HHV8, followed by eventual acquisition into the subtype C lineage of the modern P-form of the HHV8 genome and subsequent additional, more recent transfers by homologous recombination events into several subtype A and B lineages as well.

Alleles↗
PubMed1998

Progressive and persistent downregulation of surface CXCR4 in CD4(+) T cells infected with human herpesvirus 7.

We have previously shown that infection of CD4(+) T lymphocytes with the T-lymphotropic human herpesvirus 7 (HHV-7) downregulates surface CD4, which represents the high-affinity receptor for HHV-7. In this study, we report that HHV-7 infection also causes a progressive loss of the surface CXC-chemokine receptor 4 (CXCR4) in CD4(+) T cells, accompanied by a reduced intracellular Ca2+ flux and chemotaxis in response to stromal cell-derived factor-1 (SDF-1), the specific CXCR4 ligand. Moreover, CXCR4 is downregulated from the surface of HHV-7-infected T cells independently of CD4. Because intracellular CXCR4 antigen and mRNA levels are unaffected in productively HHV-7-infected cells, the downregulation of CXCR4 apparently does not involve a transcritional block. Since CXCR4 functions in association with CD4 to permit entry of several human immunodeficiency virus (HIV) isolates, the potential of HHV-7 to persistently downregulate the surface expression of CXCR4 may provide novel strategies for limiting HIV infection.

CD4 Antigens↗
PubMed1998

Interferon-gamma increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells.

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-gamma (IFN-gamma) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-gamma with those that regulate chemokine receptor expression.

Antineoplastic Agents↗
PubMed1998

A novel sensitive assay to define immune status using short-term peripheral blood derived cell culture and dual-color flow cytometry.

In this study we describe a novel and highly sensitive in vitro system to determine the functionality of immune cells based on short term culture of peripheral blood derived mononuclear cells (PBMCs) and subsequent analysis of cellular proliferation and surface marker expression by automated dual-color flow cytometry. The standardized mild stimuli introduced into the culture system by supplemented medium (containing exogenous interleukin-2 (IL-2), and fetal bovine serum (FBS)) allow a more physiological interaction of the different cell subsets contained in PBMCs (including CD14+ accessory cells) than other methods that are based on potent and harsh cell activators, such as phytohemagglutinin (PHA) or anti-CD3 antibodies. Measurement of T-cell proliferation and cell surface marker (CD3, C25, CD26, CD71, HLA-DR) analysis revealed that activation response capacity in our assay depends on both the status of the obtained cells and their ability to interact in culture with CD14+ cells. This in vitro assay proved to be very sensitive in detecting changes in the status of T-cell activation and proliferation capacity, and avoid the use of radioactive reagents.

Animals↗
PubMed1998

Inhibition of HIV-1 replication by combined expression of gag dominant negative mutant and a human ribonuclease in a tightly controlled HIV-1 inducible vector.

An HIV-1-based expression vector has been constructed that produces protective genes tightly regulated by HIV-1 Tat and Rev proteins. The vector contains either a single protective gene (HIV-1 gag dominant negative mutant (delta-gag)) or a combination of two different protective genes (delta-gag and eosinophil-derived neurotoxin (EDN), a human ribonuclease) which are expressed from a dicistronic mRNA. After stable transfection of CEM T cells and following challenge with HIV-1, viral production was completely inhibited in cells transduced with the vector producing both delta-gag and EDN and delayed in cells producing delta-gag alone. In addition, cotransfection of HeLa-Tat cells with an infectious HIV-1 molecular clone and either protective vector demonstrated that the HIV-1 packaging signals present in the constructs were functional and allowed the efficient assembly of the protective RNAs into HIV-1 virions, thus potentially transmitting protection to the HIV-1 target cells.

Blotting, Northern↗
PubMed1998

Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV8.

Strong serologic and molecular probe correlations indicate that the newly discovered gamma herpesvirus KSHV or HHV8 is the likely etiologic agent of all forms of Kaposi's sarcoma as well as BCBL/PEL and MCD in patients with acquired immunodeficiency syndrome (AIDS). Two large segments of HHV8 DNA from an AIDS-associated BCBL tumor covering genomic positions 0-52 kilobase [kb] and 108-140 kb have been cloned, mapped, and partially sequenced. Our studies have focused on novel viral proteins encoded within a 13-kb divergent locus (DL-B) by nine captured homologues of cellular genes, including vIL-6, vDHFR, vTS, vBcl-2, three C-C beta chemokines (vMIP-1A, vMIP-1B, and vBCK), and two LAP/PHD subclass zinc finger proteins (IE1A and IE1B). The HHV-8 vIL-6, vDHFR, vTS, and vBcl-2 proteins have all been shown to be active in a variety of appropriate functional assays, and transcripts from vIL-6, vMIP-1B, vIE1-A, vIE1-B, and vDHFR genes are all expressed as abundant single messenger RNA species after butyrate or phorbol ester (TPA) induction of the lytic cycle in HHV8-positive BCBL cell lines. All of these genes lie within a divergent transcriptional domain that contains a single central enhancer and associated untranslated leader region plus seven distinct proximal promoters, some of which are negatively regulated through AP-1 and ZRE motifs by the EBV ZTA transactivator. This region also encompasses a predicted complex oriLyt domain of 1050 bp that is duplicated in inverted orientation adjacent to the T0.7 latency RNA in another large divergent locus (DL-E). We have previously described three distinct subtypes of the HHV8 genome that differ by 1.0%-1.5% at the nucleotide level within the ORF26 and ORF75 genes. Certain strains or clades appear to have preferential geographic distributions, but it is not known as yet whether there are any specific disease associations. Interestingly, the A, B, and C subtypes of HHV-8 also proved to differ dramatically in coding content at both the extreme left and right ends of the unique segment of the genome as well as in the positions of the junctions with the terminal repeats. On the left-hand side, the receptor-like ORF-K1 protein is highly variable with A-strain subtypes displaying 15% amino acid differences from C strains and up to 30% differences from B strains. On the right-hand side, two unrelated alternative types of the putative multiple membrane spanning ORF-K15 protein are found.

Amino Acid Sequence↗
PubMed1998

CD4 promoter transactivation by human herpesvirus 6.

The observation that human herpesvirus 6 (HHV-6) can induce CD4 gene transcription and expression in CD4(-) cells was reported several years ago (P. Lusso, A. De Maria, M. Malnati, F. Lori, S. E. DeRocco, M. Baseler, and R. C. Gallo, Nature 349:533-535, 1991) and subsequently confirmed (P. Lusso, M. S. Malnati, A. Garzino-Demo, R. W. Crowley, E. O. Long, and R. C. Gallo, Nature 362:458-462, 1993; G. Furlini, M. Vignoli, E. Ramazzotti, M. C. Re, G. Visani, and M. LaPlaca, Blood 87:4737-4745, 1996). Our objective was to identify the mechanisms underlying such phenomena. Using reporter gene constructs driven by the CD4 promoter, we report that HHV-6 can efficiently transactivate such genetic elements. Activation of the CD4 promoter occurs in the presence of the viral DNA polymerase inhibitor phosphonoformic acid, which limits expression to the immediate-early and early classes of viral genes. Using deletion mutants and specific CD4 promoter mutants, we identified an ATF/CRE binding site located at nucleotides -67 to -60 upstream of the CD4 gene transcription start site that is important for HHV-6 transactivation. The ATF/CRE site is also essential for CD4 promoter activation by forskolin, an activator of adenylate cyclase. Using electrophoretic mobility shift assays and specific antibodies, we showed that CREB-1 binds specifically to the -79 to -52 region of the CD4 promoter. Last, we have identified two open reading frames (ORFs) of HHV-6, U86 and U89 from the immediate-early locus A, that can transactivate the CD4 promoter in HeLa cells. However, transactivation of the CD4 promoter by ORFs U86 and U89 is independent of the CRE element, suggesting that additional HHV-6 ORFs are likely to contribute to CD4 gene activation. Taken together, our results will help to understand the complex interactions occurring between HHV-6 and the CD4 promoter and provide additional information regarding the class of transcription factors involved in the control of CD4 gene expression.

Base Sequence↗
PubMed1997

Characterization of a chemokine receptor-related gene in human herpesvirus 8 and its expression in Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) is a recently discovered, virus that is highly associated with Kaposi's sarcoma (KS) and AIDS-associated body cavity lymphomas, although it is also found in some normal individuals. HHV-8 is related by nucleotide sequence homology to herpesvirus saimiri (HVS), which causes T cell lymphomas in some New World monkeys, and to Epstein-Barr virus (EBV), a human herpesvirus linked etiologically with Burkitt's lymphoma and nasopharyngeal carcinoma. We report that, like HVS but unlike EBV, HHV-8 contains a gene (ORF74) with significant sequence homology to the high-affinity IL-8 receptor, a member of the alpha (CXC) chemokine receptor family of transmembrane G protein-coupled receptors. We also show by reverse transcription PCR that the chemokine receptor-related HHV-8 gene is detectable in some RNA samples from KS tissue, and that its expression varies independently from that of ORF26, a minor capsid protein. The presence of a potential chemokine receptor in HHV-8 and its expression in KS tissue suggests that it may be important in the regulation of viral gene expression and may play a role in the etiology of KS and AIDS-related body cavity lymphomas.

AIDS-Related Opportunistic Infections↗
PubMed1997

A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak.

The Bcl-2 protein family is characterized by the ability to modulate cell death, and members of this family share two highly conserved domains called Bcl-2 homology 1 (BH1) and 2 (BH2) which have been shown to be critical for the death-repressor activity of Bcl-2 and Bcl-xL. Through sequence analysis we identified a novel viral Bcl-2 homolog, designated KSbcl-2, from human herpesvirus 8 (HHV8) or Kaposi sarcoma-associated herpesvirus. The overall amino acid sequence identity between KSbcl-2 and other Bcl-2 homologs is low (15-20%) but concentrated within the BH1 and BH2 regions. Overexpression of KSbcl-2 blocked apoptosis as efficiently as Bcl-2, Bcl-xL, or another viral Bcl-2 homolog encoded by Epstein-Barr virus, BHRF1. Interestingly, KS-bcl-2 neither homodimerizes nor heterodimerizes with other Bcl-2 family members, suggesting that KSbcl-2 may have evolved to escape any negative regulatory effects of the cellular Bax and Bak proteins. Furthermore, the herpesvirus Bcl-2 homologs including KSbcl-2, BHRF1, and ORF16 of herpesvirus saimiri contain poorly conserved Bcl-2 homology 3 (BH3) domains compared with other mammalian Bcl-2 homologs, implying that BH3 may not be essential for anti-apoptotic function. This is consistent with our observation that amino acid substitutions within the BH3 domain of Bcl-xL had no effect on its death-suppressor activity.

Amino Acid Sequence↗
PubMed1997

Humanization of an antibody recognizing a breast cancer specific epitope by CDR-grafting.

BACKGROUND: Muc1-H23 is a cell surface mucin that is expressed on normal breast luminal epithelial cells and over-expressed in most breast tumors. In addition, Muc-1 expressed by malignant cells is glycosylated differently than Muc-1 expressed by normal cells. This difference in glycosylation exposes a peptide epitope on malignant cells which is not exposed on normal cells. Murine monoclonal antibody H23 recognizes this epitope and stains 91% of breast cancers, but only 1/56 non-malignant breast tissue samples. OBJECTIVE: To create a human antibody that was equivalent to H23 for potential uses in imaging and/or the therapy of breast cancer. STUDY DESIGN: We decided to humanize H23 by CDR-grafting using overlap PCR, and to this end, designed and constructed a bacterial expression vector that would allow V-regions, cloned via unique restriction sites, to be expressed as Fab fragments. In this way, we hoped to be able to rapidly evaluate Fab constructs for binding to Muc-1 and to cells and tissue sections that expressed the antigen. RESULTS: A fully humanized Fab fragment was able to bind Muc-1 peptide, as well as breast cancer cells known to express the epitope and tissue sections, generally showing the same reactivity as the native antibody. In addition, an analysis of sFab expressed with a [His]6 tag preceded by a factor Xa proteolytic cleavage site suggested that E. coli periplasmic signal peptidase was able to cleave the factor Xa site, thereby removing the [His]6 tag. CONCLUSION: We have generated a human antibody that is capable of recognizing a tumor specific epitope expressed by 91% of breast cancers.

Antigens, Neoplasm↗
PubMed1997

Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6.

Human herpesvirus-8 (HHV-8) has been detected in Kaposi's sarcoma (KS) lesions of all types (AIDS-related, classical and endemic), in body-cavity-based B-cell lymphomas (BCBLs) and in lesions of multicentric Castleman's disease (MCD). We have identified a major gamma-herpesvirus-divergent locus (DL-B) in HHV-8 DNA encoding several HHV-8 unique open reading frames (ORFs), including a homologue of interleukin-6 (IL-6) and two homologues of macrophage inflammatory protein MIP-1. We show that the HHV-8-encoded IL-6 homologue (vIL-6) shares functional properties with endogenous IL-6 proteins and that both vIL-6 and vMIP-1 transcripts are present at high levels following butyrate induction of an HHV-8' BCBL cell line. Low amounts of constitutive vIL-6, but not vMIP-1, mRNA were also detected. The presence of a functional IL-6 homologue encoded by HHV-8 may provide a mechanistic model for the hypothesized role of HHV-8 in KS, MCD and BCBL that involves the mitogenic effects of vIL-6 on surrounding cells. MIP-1 proteins may enhance these effects through the chemotactic recruitment of endogenous cytokine-producing cells into affected tissues and could potentially influence HIV disease progression in coinfected individuals through interactions with the HIV co-receptor CCR-5.

Amino Acid Sequence↗
PubMed1997

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Adenoviridae↗
PubMed1997

New insight on the role of extrachromosomal retroviral DNA.

During infection with different retroviruses, high levels of unintegrated extrachromosomal DNA accumulate in infected cells. While extrachromosomal linear DNA is the immediate precursor of the integrated provirus, the function, if any, of extrachromosomal circular DNA has been unclear. Several groups have attempted to address the possible function, activity, and importance of this unintegrated DNA during the life cycle of retroviruses and the course of retroviral-associated diseases. This review summarizes recent work in this field and tries to analyze some aspects of extrachromosomal forms of retroviral DNA and their possible application as a molecular biological tool.

Animals↗