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Biomedical subjects

M E Klotman

Publications and source records attributed to M E Klotman.

At least 19 recordsLinked to original sources
PubMed2004

Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Cells, Cultured↗
PubMed2003

Development of topical microbicides for prevention of human immunodeficiency virus and herpes simplex virus.

Topical microbicides, designed for vaginal or rectal administration, are needed to prevent human immunodeficiency virus (HIV) and other sexually transmitted infections (STI). Presently marketed topical microbicides are cytotoxic and damage the vaginal epithelium with frequent use. Rational development of new candidate compounds should build on knowledge of the pathways of microbial invasion. The establishment of assays and models that predict efficacy and safety is critical. Comprehensive pre-clinical evaluation of promising microbicides should include rigorous assessment of the effects of repeated application of topical agents on mucosal inflammatory cells, cytokines, and the genital tract virus population. These studies will lay the groundwork for future clinical trials.

Anti-Infective Agents, Local↗
PubMed2002

Circular viral DNA and anomalous junction sequence in PBMC of HIV-infected individuals with no detectable plasma HIV RNA.

Closed circular (cc) forms of extrachromosomal HIV DNA are detected in patients with high viral loads; however, it is unclear whether these forms remain if virus replication is suppressed to undetectable levels by combination antiretroviral therapy. A nested primer polymerase chain reaction amplification assay was used to detect the presence of ccDNA containing two long terminal repeat sequences (2-LTR) in PBMC of patients with low or undetectable plasma HIV RNA. Fifty percent of patients with plasma RNA levels <50 copies/ml of blood had detectable 2-LTR DNA. Sequencing of the products identified normal LTR--LTR junctions in the minority of cases with the majority containing anomalies including deletions and insertions. The persistence of HIV ccDNA in patients with no detectable plasma RNA could be consistent with ongoing de novo infection of dividing cells or with stability of this form of DNA in nondividing cells.

Base Sequence↗
PubMed2002

Mandelic acid condensation polymer: novel candidate microbicide for prevention of human immunodeficiency virus and herpes simplex virus entry.

Presently marketed vaginal barrier methods are cytotoxic and damaging to the vaginal epithelium and natural vaginal flora when used frequently. Novel noncytotoxic agents are needed to protect men and women from sexually transmitted diseases. One novel candidate is a mandelic acid condensation polymer, designated SAMMA. The spectrum and mechanism of antiviral activity were explored using clinical isolates and laboratory-adapted strains of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). SAMMA is highly effective against all CCR5 and CXCR4 isolates of HIV in primary human macrophages and peripheral blood mononuclear cells. SAMMA also inhibits infection of cervical epithelial cells by HSV. Moreover, it exhibits little or no cytotoxicity and has an excellent selectivity index. SAMMA, although not a sulfonated or sulfated polymer, blocks the binding of HIV and HSV to cells by targeting the envelope glycoproteins gp120 and gB-2, respectively, and also inhibits HSV entry postattachment. SAMMA is an excellent, structurally novel candidate microbicide that warrants further preclinical evaluation.

Antiviral Agents↗
PubMed2001

HIV envelope gp120 activates human arterial smooth muscle cells.

There have been increasing reports of acute coronary thrombotic events in patients with HIV. Although these clinical events have been attributed primarily to dyslipidemia associated with protease inhibitor therapy, autopsy studies in children with HIV suggest the presence of an underlying arteriopathy. This study demonstrates that the HIV envelope protein, gp120, activates human arterial smooth muscle cells to express tissue factor, the initiator of the coagulation cascade. The induction of tissue factor by gp120 is mediated by two biologically relevant coreceptors for HIV infection, CXCR4 and CCR5, and is also dependent on the presence of functional CD4. Induction of tissue factor by gp120 requires activation of mitogen-activating protein kinases, activation of protein kinase C, and generation of reactive oxygen species, signaling pathways that have protean effects on smooth muscle cell physiology. The activation of smooth muscle cells by gp120 may play an important role in the vascular, thrombotic, and inflammatory responses to HIV infection.

CD4 Antigens↗
PubMed2000

Single-dose safety, pharmacology, and antiviral activity of the human immunodeficiency virus (HIV) type 1 entry inhibitor PRO 542 in HIV-infected adults.

PRO 542 (CD4-IgG2) is a recombinant antibody-like fusion protein wherein the Fv portions of both the heavy and light chains of human IgG2 have been replaced with the D1D2 domains of human CD4. Unlike monovalent and divalent CD4-based proteins, tetravalent PRO 542 potently neutralizes diverse primary human immunodeficiency virus (HIV) type 1 isolates. In this phase 1 study, the first evaluation of this compound in humans, HIV-infected adults were treated with a single intravenous infusion of PRO 542 at doses of 0.2-10 mg/kg. PRO 542 was well tolerated, and no dose-limiting toxicities were identified. Area under the concentration-time curve, and peak serum concentrations increased linearly with dose, and a terminal serum half-life of 3-4 days was observed. No patient developed antibodies to PRO 542. Preliminary evidence of antiviral activity was observed as reductions in both plasma HIV RNA and plasma viremia. Sustained antiviral effects may be achieved with repeat dosing with PRO 542.

Anti-HIV Agents↗
PubMed2000

CD8+ cell lines isolated from HIV-1-infected children have potent soluble HIV-1 inhibitory activity that differs from beta-chemokines.

CD8+ cells from human immunodeficiency virus type 1 (HIV-1) infected individuals have been shown to suppress HIV-1 replication both through a major histocompatibility complex (MHC)-restricted cytolytic pathway as well as through a noncytolytic pathway mediated through soluble factors. To characterize this soluble activity and its potential role in disease progression further, we studied the HIV-1 inhibition by supernatants derived from herpesvirus saimiri-transformed CD8+ cells isolated from infected children. Three of the six CD8+ cell lines derived had a phenotype consistent with an unusual natural killer (NK) cells phenotype with low CD3, high CD56, and low CD16. Supernatants from some of the cell lines derived from children with rapid progression as well as long-term nonprogressors exhibited broad HIV-1-inhibitory activity in primary CD4+ cells as well as in primary macrophages. In contrast to a cocktail of beta-chemokines, the supernatants inhibited T-tropic as well as M-tropic viruses, efficiently inhibited infection in primary macrophages, and inhibited HIV-1 activation in the chronically infected U1 cell line. The HIV-1-inhibitory activity was heat stable and active over a broad pH range. Fractionation of the supernatant by size and ion exchange chromatography demonstrated activity in the complete absence of RANTES as well as interferons-alpha, beta, and gamma and in a size range of less than 10 kD and greater than 3 kD. CD8+ cell supernatants contain additional unidentified factors that have anti-HIV activity to account for this broad phenomenon.

CD4-Positive T-Lymphocytes↗
PubMed1999

HIV-associated nephropathy is a late, not early, manifestation of HIV-1 infection.

BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) can be the initial presentation of HIV-1 infection. As a result, many have assumed that HIVAN can occur at any point in the infection. This issue has important implications for appropriate therapy and, perhaps, for pathogenesis. Since the development of new case definitions for acquired immunodeficiency syndrome (AIDS) and better tools to assess infection, the relationship of HIVAN to the time of AIDS infection has not been addressed. In this study, we reassessed the stage of infection at the time of HIVAN diagnosis in 10 patients, and we reviewed all previously published cases applying the new case definitions to assess stage of infection. METHODS: HIVAN was confirmed by kidney biopsy in HIV seropositive patients with azotemia and/or proteinuria. CD4+ cell count and plasma HIV-1 RNA copy number were measured. We also reviewed all published cases of HIVAN to determine if AIDS-defining conditions, by current Centers for Disease Control definitions, were present in patients with biopsy-proven HIVAN. RESULTS: Twenty HIV-1 seropositive patients with proteinuria and an elevated creatinine concentration were biopsied. HIVAN was the single most common cause of renal disease. CD4+ cell count was below 200/mm3 in all patients with HIVAN, fulfilling Centers for Disease Control criteria for an AIDS-defining condition. HIV-1 plasma RNA was detectable in all patients with HIVAN. In reviewing previous reports, an AIDS-defining condition was present in virtually all patients with HIVAN. CONCLUSION: HIVAN develops late, not early, in the course of HIV-1 infection following the development of AIDS. This likely accounts for the poor prognosis noted in previous publications and has implications for pathogenesis. In addition, given the detectable viral RNA levels, highly active antiretroviral therapy is indicated in HIVAN. Highly active antiretroviral therapy may improve survival as well as alter the natural history of HIVAN.

AIDS-Associated Nephropathy↗
PubMed1999

Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.

There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Animals↗
PubMed1998

Effect of UV-B phototherapy on plasma HIV type 1 RNA viral level: a self-controlled prospective study.

OBJECTIVE: To study the plasma human immunodeficiency virus type 1 (HIV-1) RNA levels of 12 patients seropositive for HIV who were undergoing UV-B phototherapy to determine if UV-B phototherapy upregulates HIV activity in humans. DESIGN: A self-controlled prospective cohort of HIV-infected patients seen for the treatment of a skin disorder responsive to UV-B phototherapy. Viral levels were measured weekly for 8 weeks of phototherapy. Follow-up viral levels were measured for patients who continued phototherapy beyond 8 weeks, those who had a significant change in their viral level, or both. SETTING: Outpatient clinic of an academic hospital. PATIENTS: Patients with HIV disease and a skin disorder responsive to UV-B phototherapy. Inclusion criteria for patients in this study were those receiving a stable antiviral regimen for at least 6 weeks and who had no major illness or immunization in the 2 months before starting phototherapy. Of 72 patient volunteers screened, 15 met the criteria, 2 declined to participate, and 13 entered the study. One patient was dropped from the study because an accurate baseline measurement could not be obtained. Twelve patients were analyzed, 2 of whom left the study early, 1 at 6 weeks and 1 at 7 weeks. INTERVENTIONS: Ultraviolet-B phototherapy. MAIN OUTCOME MEASURE: Plasma HIV-1 RNA viral level. RESULTS: Plasma HIV-1 RNA levels showed no significant increase or decrease in most of the patients, defined as a 3-fold change from baseline (mean fold change from baseline after 8 weeks of phototherapy, -1.1; 95% confidence interval, 2.9 to -5.0). Trend analysis indicated no significant pattern of change in viral levels (slope, -0.013 log; P > .25). The CD4+ cell counts also remained unchanged (mean before therapy, 277 x 10(9)/L; mean after therapy, 285 x 10(9)/L; P = .67). CONCLUSION: No significant effect of UV-B exposure was seen on plasma HIV-1 levels.

Adult↗
PubMed1998

Sites of recombinant adeno-associated virus integration.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.

Dependovirus↗
PubMed1998

Management of antiretroviral therapy.

In a very short period of time, availability of antiretroviral drugs has increased from one drug with very modest activity to 12 approved drugs with remarkable potency, particularly when used in combination. The additional availability of absolutely quantitative assays with a broad dynamic range to measure plasma HIV-1 RNA has dramatically changed the evaluation of these new antivirals and the management of patients infected with the human immunodeficiency virus (HIV). The approved antiretroviral drugs represent three novel classes including nucleoside analog reverse transcriptase inhibitors, non-nucleoside analog reverse transcriptase inhibitors as well as protease inhibitors. Clinical trials evaluating different combinations of these drugs have resulted in the generation of some basic guidelines for their appropriate use. These guidelines focus on using these drugs in complex, multidrug regimens with the ultimate goal to keep the viral burden, as measured by plasma viral RNA levels, as low as possible and for as long as possible on a drug regimen that is compatible with long-term tolerance and compliance. To maximize the potential of these new drugs and to avoid significant associated toxicities and drug interactions, the treating physician must be completely aware of the pharmacokinetics and unique antiviral properties of these drugs. This review focuses on these unique properties as well as provides general guidelines for their appropriate use.

Anti-HIV Agents↗
PubMed1997

Differential expression of alternatively spliced pX mRNAs in HTLV-I-infected cell lines.

Human T cell leukemia/lymphotropic virus (HTLV) is a complex 9 kb human retrovirus with at least eight alternatively spliced mRNAs expressed from the 3' or pX region of the genome. These mRNAs allow for the expression of novel proteins from the previously recognized pX open reading frames I and II in addition to Tax, Rex and p21rex encoded from orf III and IV. These alternatively spliced messages have been detected using reverse-transcriptase polymerase chain reaction (RT/PCR) amplification in HTLV-I-transformed T cell lines as well as in peripheral blood mononuclear cells (PBMC) from infected patients with and without disease. To gain insight into the role of these alternatively spliced mRNAs in pathogenesis, we developed a semi-quantitative non-PCR-based RNase protection assay to detect and quantitate their presence in HTLV-I-infected cells. Analysis of RNA from HTLV-I-infected cells established from patients with adult T cell leukemia (ATL) as well as tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and both IL-2-dependent and IL-2-independent HTLV-I-infected cell lines by RNase protection has confirmed the existence of all of the alternatively spliced messages in each cell line analyzed. However, the relative quantity of each message was significantly different among these lines suggesting that splice site utilization is an important viral regulatory pathway.

Adult↗
PubMed1996

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Base Sequence↗
PubMed1996

Molecular therapy for renal diseases.

The introduction of molecular therapy through the delivery of nucleic acids either as oligonucleotides or genetic constructs holds enormous promise for the treatment of renal disease. Significant barriers remain, however, before successful organ-specific molecular therapy can be applied to the kidney. These include the development of methods to target the kidney selectively, the definition of vectors that transduce renal tissue, the identification of appropriate molecular targets, the development of constructs that are regulated and expressed for long periods of time, the demonstration of efficacy in vivo, and the demonstration of safety in humans. As the genetic and pathophysiologic basis of renal disease is clarified, obvious targets for therapy will be defined, for example, polycystin in polycystic kidney disease, human immunodeficiency virus (HIV) type 1 in HIV-associated nephropathy, alpha-galactosidase A in Fabry's disease, insulin in diabetic nephropathy, and the "minor" collagen IV chains in Alport's syndrome. In addition, several potential mediators of progressive renal disease may be amenable to molecular therapeutic strategies, such as interleukin-6, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta(TGF-beta). To test the in vivo efficacy of molecular therapy, appropriate animal models for these disease states must be developed, an area that has received too little attention. For the successful delivery of genetic constructs to the kidney, both viral and nonviral vector systems will be required. The kidney has a major advantage over other solid organs since it is accessible by many routes, including intrarenal artery infusion, retrograde delivery through the uroexcretory pathways, and ex vivo during transplantation. To further restrict expression to the kidney, tropic vectors and tissue-specific promoters also must be developed. For the purpose of inhibition of endogenous or exogenous genes, current therapeutic modalities include the delivery of antisense oligodeoxynucleotides or ribozymes. For these approaches to succeed, we must gain a much better understanding of the nature of their transport into the kidney, requirements for specificity, and in vivo mechanisms of action. The danger of a rush to clinical application is that superficial approaches to these issues will likely fail and enthusiasm will be lost for an area that should be one of the most exciting developments in therapeutics in the next decade.

Animals↗