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Biomedical subjects

N Ramesh

Publications and source records attributed to N Ramesh.

At least 55 records · Page 3Linked to original sources
PubMed1996

Granulocyte colony-stimulating factor expression from transduced vascular smooth muscle cells provides sustained neutrophil increases in rats.

Granulocyte colony-stimulating factor (G-CSF) regulates granulocyte precursor cell proliferation, neutrophil survival, and activation. Cyclic hematopoiesis, a disease that occurs both in humans and grey collie dogs is characterized by cyclical variations in blood neutrophils. Although the underlying molecular defect is not known, long-term daily administration of recombinant G-CSF eliminates the severe recurrent neutropenia, indicating that expression of G-CSF by gene therapy would be beneficial. As a prelude to preclinical studies in affected collie dogs, we monitored hematopoiesis in rats receiving vascular smooth muscle cells transduced to express G-CSF. Cells transduced with LrGSN, a retrovirus expressing rat G-CSF, were implanted in the carotid artery and control animals received cells transduced with LASN, a retrovirus expressing human adenosine deaminase (ADA). Test animals showed significant increases in neutrophil counts for at least 7 weeks, with mean values of 3,670 +/- 740 cells/microliter in comparison to 1,870 +/- 460 cells/microliter in controls (p < 0.001). Thus, in rats G-CSF gene transfer targeted at vascular smooth muscle cells initiated sustained production of 1,800 neutrophils/microliter, a cell number that would provide clinical benefit to patients. Lymphocytes, red cells and platelets were not different between control and test animals (p > 0.05). These studies indicate that retrovirally transduced vascular smooth muscle cells can provide sustained clinically useful levels of neutrophils in vivo.

Adenosine Deaminase↗
PubMed1996

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.

3T3 Cells↗
PubMed1995

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

3T3 Cells↗
PubMed1995

gamma/delta T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes.

T cells expressing gamma/delta T cell receptors home to epithelial tissue and may play a role in immunity to infectious agents and foreign antigens. In an effort to understand the role of gamma/delta T cells in directing B cell responses, we investigated the capacity of human gamma/delta T cells to express CD40 ligand (CD40L) and to drive immunoglobulin (Ig) isotype switching in B cells. A multiple step purification procedure resulted in the recovery of highly pure populations of peripheral blood CD4-CD8- gamma/delta T cells. Neither CD40L surface expression nor CD40L mRNA were detected in unstimulated gamma/delta T cells. Stimulation with phorbol ester and ionomycin induced CD40L mRNA and surface CD40L expression by gamma/delta T cells. Both the percentage of CD40L+ cells and the cell surface density of CD40L were significantly lower in gamma/delta T cells compared to unselected T cells. We further demonstrated that in the presence of neutralizing monoclonal antibody to interferon gamma (IFN-gamma), gamma/delta T cells could induce IgE synthesis in B cells, albeit to a lesser extent than unselected T cells. Furthermore, IgE synthesis driven by gamma/delta T cells was inhibited by monoclonal antibody to CD40L. These observations demonstrate that activated gamma/delta T cells express CD40L and can induce isotype switching in B cells.

Adult↗
PubMed1995

Long-term treatment of canine cyclic hematopoiesis with recombinant canine stem cell factor.

Grey collie dogs have cyclic fluctuations in their blood cell counts caused by a regulatory defect of hematopoietic stem cells. To examine the role of stem cell factor (SCF) or its receptor in this disorder, we investigated the stimulatory effects of recombinant canine SCF (rc-SCF) on in vitro marrow cultures, cloned and sequenced the grey collie SCF gene, and treated three grey collies with rc-SCF, either alone or in combination with recombinant canine granulocyte colony-stimulating factor (rcG-CSF). Colony-forming unit granulocyte-macrophage formation from grey collie or normal dog marrow showed similar dose-response curves for rc-SCF. Cloning and sequencing the SCF gene for two grey collies showed no evidence of mutations in the coding region of the SCF gene. Treatment with rc-SCF (10 to 100 micrograms/kg/d) did not induce neutrophilia except at the highest dose (100 micrograms/kg/d), but daily rc-SCF abrogated the neutropenic periods in doses of 20 micrograms/kg/d or greater. Combination of rc-G-CSF (0.5 to 1.0 microgram/kg/d) with rc-SCF treatment (20 to 50 micrograms/kg/d) suggested a synergistic effect, ie, the neutrophil levels on combined therapy were higher than the sum of the levels when these two cytokines were given separately. Long-term treatment of these dogs with rc-SCF in doses of 10 to 30 micrograms/kg/d was generally well tolerated, suggesting that SCF may be useful as a therapy for some chronic hypoproliferative disorders of hematopoiesis.

Animals↗
PubMed1995

CD40-CD40 ligand (CD40L) interactions and X-linked hyperIgM syndrome (HIGMX-1).

Interactions between the B cell surface antigen CD40 and its ligand (CD40L) expressed on activated T cells play a critical role in isotype switching. This is illustrated by failure of isotype switching in patients with X-linked hyperIgM syndrome in whom the CD40L gene is mutated and by failure of isotype switching of CD40-deficient mice in response to T-cell-dependent antigens. We review these findings and discuss the signaling mechanisms of CD40 and the developmental control and transcriptional regulation of CD40L expression.

Animals↗
PubMed1995

High-level expression from a cytomegalovirus promoter in macrophage cells.

To identify vectors that provide optimal gene expression in human hematopoietic cells, we investigated retroviruses containing the adenosine deaminase (ADA) gene under the transcriptional control of the promoters/enhancers of Moloney murine leukemia virus and the human cytomegalovirus (CMV) immediate-early gene. ADA expression was monitored in transduced human multipotential promyelocytic leukemic HL-60 cells and human monocytic leukemic THP-1 cells. HL-60 cells can be induced by phorbol ester to differentiate into macrophage lineage cells and by retinoic acid into granulocytic cells. THP-1 cells undergo phorbol ester-induced differentiation to macrophage cells. In LNCA-transduced HL-60 derived macrophage cells, ADA controlled by the CMV promoter was expressed at 100.0 mumol/hr.mg, in contrast to 1.2 mumol/hr.mg from LN-transduced control cells. LNCA-transduced THP-1 macrophage cells showed a similar increase in ADA activity over control cells. These elevated enzyme activities were confirmed by Northern blots, which showed substantial increases in ADA mRNA derived from the CMV promoter. This suggests use of the CMV promoter for gene therapy targeted at macrophages, as, for example, in the treatment of lysosomal storage disorders such as Gaucher disease. These inducible cell lines have allowed the evaluation of transduced gene expression in proliferating and differentiating hematopoietic cells that may serve as an in vitro model of bone marrow-targeted gene therapy.

Adenosine Deaminase↗
PubMed1995

Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.

Amino Acid Sequence↗
PubMed1995

Bacterial superantigens induce the proliferation of resting gamma/delta receptor bearing T cells.

We observe that highly purified (> or = 97% pure) gamma/delta T cells isolated from normal peripheral blood proliferate to bacterial toxin supperantigens SEA, SEB, SED, and TSST-1. MHC class II molecules were necessary and sufficient for the recognition of superantigens by gamma/delta T cells because MHC Class II deficient B cell line failed to support the proliferation of gamma/delta T cells to toxins and murine L cells transfected with HLA-DR but not untransfected cells were capable of presenting toxins to gamma/delta T cells. As in the case with alpha/beta T cells, bacterial superantigens synergized with PMA in causing the proliferation of purified gamma/delta T cells rigorously depleted of accessory cells. Together, our findings suggest that gamma/delta T cells recognize and respond to bacterial superantigens in a manner similar to alpha/beta T cells.

Animals↗
PubMed1994

Constitutive mucin secretion linked to CFTR expression.

Mucus plugging is a hallmark of cystic fibrosis, but the link between the defective gene product, the cystic fibrosis transmembrane conductance regulator, and the abnormal mucus phenotype is unclear. To demonstrate CFTR involvement in mucin glycoprotein secretion in epithelial cells, a retroviral vector was used to overexpress CFTR in gallbladder epithelial cells, and constitutive mucin labeling and secretion were monitored. Achievement of high-level vector expression was confirmed by transduction with marker genes. Cells transduced with vectors carrying CFTR cDNA showed 5-fold increased expression of CFTR by Western blotting. Mucin labeling and secretion were 4-fold elevated in transduced cells. These results suggest constitutive mucin synthesis and secretion in gallbladder epithelial cells are regulated by CFTR.

Amino Acid Sequence↗
PubMed1994

Structure of the murine CD40 ligand gene.

The mouse CD40 ligand (CD40L) gene was cloned, sequenced and characterized. DNA sequence analysis showed that the CD40L gene comprises five exons and four intervening introns, spread over 13-14 kb of genomic DNA. The putative site for initiation of mRNA transcription was identified at 67 bp upstream of the translation initiation (ATG) codon. The nucleotide sequence of the 5'-flanking region of this gene revealed the presence of several regulatory regions including a TATA-like box, an Sp1-like box and six potential NF-AT-like motifs. The 3'-untranslated region of the murine CD40L gene contained two ATTTA-elements which are thought to confer instability to the mRNA of many cytokines and two adjacent dinucleotide repeates, (CT)25 and (CA)45. These elements may play a role in the post-transcriptional regulation of CD40L gene expression.

Amino Acid Sequence↗
PubMed1994

Cyclosporin A inhibits CD40 ligand expression in T lymphocytes.

The ligand for CD40 is expressed on activated T lymphocytes and delivers contact-dependent activation signals to B lymphocytes. The mechanisms regulating CD40 ligand gene expression are largely unknown. Optimal expression of CD40 ligand required activation of protein kinase C and a rise in intracellular calcium concentration. CD40 ligand expression was inhibited by pretreatment of T cells with cyclosporin A. Cyclosporin A analogues inhibited CD40 ligand expression with a potency mirroring the ability of each compound to inhibit calcineurin activity, indicating that calcineurin plays a key role in CD40 ligand gene expression. Cyclosporin A inhibited IL-4-driven CD40 ligand-dependent IgE isotype switching in PBMC but did not inhibit IgE synthesis induced by CD40 mAb plus IL-4. PBMC derived from transplant patients receiving cyclosporin A failed to express CD40 ligand upon stimulation. These results suggest that patients receiving cyclosporin A may be deficient in CD40 ligand-dependent T cell help.

Adolescent↗
PubMed1994

A toxic shock syndrome toxin-1 peptide that shows homology to amino acids 180-193 of mycobacterial heat shock protein 65 is presented as conventional antigen.

Peripheral blood mononuclear cells (PBMC) from the majority of adults (12 out of 18 subjects tested) showed an in vitro proliferative response to a 20 amino acid long peptide (peptide 1, a.a 18-37) derived from TSST-1. In contrast, thymocytes and PBMC from cord blood did not proliferate to this peptide. TSST-1 peptide 1 did not induce IL-1 beta mRNA in monocytes indicating that it does not behave as a superantigen. Proliferation of PBMC to peptide 1 could be blocked by anti-HLA-DR, but not by anti-HLA DP or DQ monoclonal antibodies suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide. Peptide 1 shows homology to a.a 180-193 of mycobacterial hsp 65 and was shown to stimulate the proliferation of T cell lines and T cell clone specific for the purified protein derivative (PPD) of Mycobacterium tuberculosis. This cross reactivity may confer on TSST-1 the potential to trigger self reactivity and may also contribute to the natural immunity against TSST-1.

Adult↗
PubMed1993

Sequential switching from mu to epsilon via gamma 4 in human B cells stimulated with IL-4 and hydrocortisone.

The molecular events leading to IgE synthesis in human B cells stimulated with IL-4 and hydrocortisone were analyzed. IL-4, but not hydrocortisone, induced C epsilon germ line transcription. However, hydrocortisone increased the levels of IL-4-induced germ line C epsilon transcripts by twofold and delivered the signal required for transcription of mature C epsilon mRNA. Nested primer polymerase chain reaction of high m.w. DNA revealed deletional switch recombination occurring in B cells sorted for lack of expression of surface IgE and stimulated with both IL-4 and hydrocortisone, but not in B cells stimulated with IL-4 alone or hydrocortisone alone. DNA sequence analysis of 10 switch fragments revealed direct joining of S mu to S epsilon in eight fragments, one of which exhibited an 876-bp deletion in S mu. The ninth fragment contained a 50-bp insertion at the S mu/S epsilon junction, which was likely to be derived from S gamma 4. The sequence of the 10th fragment was consistent with either a 17-bp insertion at the S mu/S epsilon junction derived from S gamma 4 or with a complex 38-bp deletion within S epsilon. Mapping of the switch junction sites showed "hot spots" for recombination within S mu but not within S epsilon. These findings indicate that hydrocortisone induces S mu-S epsilon deletional switch recombination in IL-4-treated B cells, and support a model of sequential isotype switching from IgM to IgE via IgG4.

B-Lymphocytes↗
PubMed1993

Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM.

B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease.

Adolescent↗