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Biomedical subjects

A Jani

Publications and source records attributed to A Jani.

At least 19 recordsLinked to original sources
PubMed2001

Foreign antigenic peptides delivered to the tumor as targets of cytotoxic T cells.

Cytotoxic T cells (CTL) are readily activated by immunogenic peptides and they exert potent anti-tumor activity if the same peptides are displayed on class I major histocompatibility complex (MHC) of the tumor cells. A handful of tumor-associated antigens have been identified and many of them are weak antigens. As an alternative strategy, strongly antigenic foreign peptides are delivered to the tumor, marking them for CTL recognition. To establish the principle of this new strategy, in vitro and in vivo tumor destruction was tested with BALB/c CTL to L(d)-associated beta-galactosidase (beta-gal) peptide p876. In vitro, anti-p876 CTL destroyed tumor cells in a single-cell suspension or in 3-D tumor boluses when exogenous p876 was added. Exogenous IL-2 was required to sustain CTL activity for complete destruction of tumor boluses. In vivo, BALB/c mice were immunized with p876 and a CD4 activating Pan DR reactive epitope (PADRE). PADRE, which binds to several different MHC class II antigen and activates CD4 T cells, induced delayed-type hypersensitivity and stimulated T cell proliferation. Immunized mice were injected with tumor cells loaded with p876 and mixed with PADRE. Starting from the day after tumor injection, mice received five rounds of peptide injection at the tumor sites and all tumors were rejected. Injection with saline had no effect. Injection with PADRE had minor anti-tumor activity. Immunization and treatment with p876 alone was not protective. Therefore, by delivering CD4 and CD8 reactive foreign peptides to the tumor, peptide-specific T cells rejected the tumors as demonstrated by the in vitro and in vivo tests.

Animals↗
PubMed2001

Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation.

Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.

Acute Disease↗
PubMed2000

Inducible nitric oxide synthase transcription in human lung transplantation.

BACKGROUND: Recent animal data suggest that inducible nitric oxide synthase (iNOS) mRNA expression in the bronchoalveolar lavage (BAL) may be useful for the diagnosis of lung rejection. The aim of this study was to evaluate iNOS mRNA transcription in the BAL fluid of human lung allografts. METHODS: iNOS mRNA transcription was quantified by competitive reverse transcription-polymerase chain reaction in 51 BAL cell pellets of lung transplant patients. According to bacteriological and histological results, BAL samples were divided into three groups: normal (n=21), acute rejection (AR, n=15), and infection (INF, n=15). RESULTS: Compared with the control group, iNOS transcription increased significantly with INF (P=0.0005) but only slightly with AR (P>0.05). INF values were significantly higher than AR values (P=0.0029). CONCLUSION: BAL iNOS mRNA transcript determination by competitive reverse transcription-polymerase chain reaction may be useful in differentiating infected from normal and/or acutely rejecting allografts.

Analysis of Variance↗
PubMed2000

Quantification of cytotoxic T-cell gene transcripts in human lung transplantation.

BACKGROUND: Differentiating between acute rejection and cytomegalovirus (CMV) infection is one of the major challenges of lung transplantation. The aims of this study were to: (1) quantify the transcription of the cytotoxic T lymphocyte (CTL) effector molecules in the bronchoalveolar lavage (BAL) of lung transplant recipients and (2) evaluate the clinical usefulness of this technique. METHODS: Sixty-six single-lung, double-lung, or heart-lung transplant patients were prospectively enrolled in the study. BAL was performed either for routine surveillance or for acute graft dysfunction. RNA was extracted from BAL cell pellets and underwent competitive reverse transcription-assisted polymerase chain reaction (RT-PCR) for perforin, granzyme B, granulysin, and Fas ligand. Gene transcript analysis was compared to clinical diagnosis established by conventional methods [BAL microbiological and transbronchial biopsy (TBB) analyses]. RESULTS: After exclusion of several BAL according to the study criteria, 62 BAL were submitted for data analysis. Significantly higher expression of all the analyzed transcripts was found during CMV infection, compared with each of the other defined diagnostic categories, namely nonsignificant pathology, acute rejection, and nonviral pulmonary infection. CONCLUSION: Quantification by competitive RT-PCR of the CTL effector molecule transcripts (perforin, granzyme B, granulysin, and Fas ligand) could represent a valuable tool for the differential diagnosis of graft dysfunction in lung transplantation.

Adult↗
PubMed2000

Charcot-Marie-Tooth disease type 1: molecular pathogenesis to gene therapy.

Charcot-Marie-Tooth disease type 1 (CMT1) is caused by mutations in the peripheral myelin protein, 22 kDa (PMP22) gene, protein zero (P0) gene, early growth response gene 2 (EGR-2) and connexin-32 gene, which are expressed in Schwann cells, the myelinating cells of the peripheral nervous system. Although the clinical and pathological phenotypes of the various forms of CMT1 are similar, including distal muscle weakness and sensory loss, their molecular pathogenesis is likely to be quite distinct. In addition, while demyelination is the hallmark of CMT1, the clinical signs and symptoms of the disease are probably produced by axonal degeneration, not demyelination itself. In this review we discuss the molecular pathogenesis of CMT1, as well as approaches to an effective gene therapy for this disease.

Charcot-Marie-Tooth Disease↗
PubMed1999

Myophosphorylase gene transfer in McArdle's disease myoblasts in vitro.

McArdle's disease is due to a genetic deficiency of glycogen phosphorylase and results in a lack of glucose mobilization from glycogen during anaerobic exercise. A genetic defect in Merino sheep produces a similar picture. We constructed a first-generation adenoviral recombinant containing the full-length human phosphorylase cDNA under the control of the Rous sarcoma virus promoter. Primary myoblast cultures from phosphorylase-deficient human and sheep muscle were efficiently transduced with this vector, resulting in restoration of the phosphorylase activity. A similar correction of the genetic defect in muscles of McArdle's patients in vivo appears feasible, preferably with the use of an adeno-associated viral vector.

Animals↗
PubMed1999

Overcoming cellular immunity to prolong adenoviral-mediated gene expression in sciatic nerve.

In a previous report, we demonstrated that a first generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenoviral-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral gene expression over time. In our current work we have optimized adenoviral-mediated gene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old animals, decreases thereafter, and there is minimal associated tissue injury. In contrast, very few adenoviral-infected Schwann cells are found in nerves of adult animals 10 days after injection, probably due to immune clearance of viral-infected cells. Consistent with this notion, high levels of lacZ are found in sciatic nerve 30 days after injection of adult SCOD mice, which have a genetic defect in both cellular and humoral immunity, of adult beta 2 microglobulin-deficient mice (beta 2 M-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenoviral-infected Schwann cells co-cultured with axons in vitro, in the absence of a host immune response, ensheath axons and express lacZ for at least 8 weeks. These data thus demonstrate that expression of first generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Adenoviridae↗
PubMed1999

Modulation of cell-mediated immunity prolongs adenovirus-mediated transgene expression in sciatic nerve.

In a previous report, we demonstrated that a first-generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenovirus-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral transgene expression over time. In our current work we have optimized adenoviral vector-mediated transgene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old rats, decreases thereafter, and there is minimal associated tissue injury. In contrast, few lacZ-expressing Schwann cells are found in nerve of adult animals 10 days after injection, probably owing to immune clearance of virus-infected cells. Consistent with this notion, high levels of LacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult beta2-microglobulin-deficient mice (beta2M4-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenovirus-infected Schwann cells cocultured with axons in vitro, in the absence of a host immune response, ensheathe axons and express lacZ for at least 8 weeks. These data thus demonstrate that lacZ transgene expression of first-generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Adenoviridae↗
PubMed1998

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells.

Recombinant adenovirus (AVR) promises to be an efficient vector in gene therapy for neuromuscular diseases, but in preclinical experiments the expression of therapeutic genes is shorter lived in immunocompetent animals than in immunocompromised hosts. Interferons (IFN), which are known to have a role both in early antiviral activity and in late cytotoxic immunoreaction against the virus or transduced cells, may influence the efficiency of gene transfer. In this study we investigated the role of IFNs in determining the efficiency of gene transfer by AVR. AVRs expressing beta-galactosidase (beta-gal) from either a cytomegalovirus (CMV) or a troponin-I promoter were used. Muscle cells were infected by AVR after exposure to various IFNs. The alphaIFN treatment significantly reduced (up to fivefold) the CMV promoter-driven gene expression in muscle cells in vitro and in immature muscles in vivo, while the least effective inhibitor was betaIFN. The decrease in gene expression by IFNs was more pronounced with the CMV-driven transgene than troponin-I promoter-driven one and was due to a decrease in transcript level. Intrinsic IFNs that are triggered by AVR administration can decrease the efficiency of gene transfer in muscle cells. Therefore the use of muscle specific promoters in AVR and/or IFN inhibitory agents will likely improve the prospects of effective gene therapy by AVR.

3T3 Cells↗
PubMed1997

Generation, validation, and large scale production of adenoviral recombinants with large size inserts such as a 6.3 kb human dystrophin cDNA.

Human, serotype 5 (Ad 5), replication-defective recombinant adenoviruses (AdVs) expressing a 6.3 kb partial dystrophin cDNA (Becker) under the control of either the CMV early or the RSV LTR promoter/enhancer in combination with various polyadenylation sequences (polyA), were developed for gene transfer studies aimed at Duchenne muscular dystrophy. Based on previous experience, a strategy for generation, screening and validation of AdVs with relatively large size gene expression cassette inserts was established. Here we focus on some aspects of stability and safety of such AdVs as gene therapeutic tools based on relevant molecular biological methods. Furthermore, the quality of our best AdV-minidystrophin construct was validated following its large scale production and purification as well as its delivery in mdx mice. These results are of interest for establishing other AdVs, where the combined length of a tissue specific promoter, the gene of interest and the polyA sequences reach the upper limit of the packaging capacity of first generation AdVs.

Adenoviruses, Human↗
PubMed1997

Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.

Adenoviridae↗
PubMed1996

Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer.

We have generated high-titer adenoviral recombinants (AVR) expressing a 6.3-kb partial dystrophin cDNA insert under the control of either the Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter. These AVR preparations were free of both E1-containing AVR and AVR with a nonfunctional dystrophin expression cassette. With these optimal AVR preparations, we have obtained a high degree of short-term (10 days) expression of a truncated (approximately 200 kD) dystrophin in dystrophin-deficient mdx muscles injected in the neonatal period; a lesser degree of expression of dystrophin was found in muscles injected in the young adult age and in old animals. Microscopic indices of muscle damage revealed that the truncated dystrophin provided a significant protection of the transduced muscle fibers. However, by 60 days post-injection, a substantial reduction of the number of dystrophin-positive fibers was noted, even in the neonatally injected muscles, and near-total elimination of dystrophin-positive fibers occurred in muscles injected in the adult age. These effects appeared to be brought about by the activity of CD8+ cytotoxic lymphocytes directed against the transduced cells, leading to their eventual elimination. In severe combined immunodeficiency (SCID) mice, lacking both humoral and cellular immune competence, muscles transduced (either in the neonatal or adult age) by AVR containing a CMV-LacZ expression cassette maintained the early (10 day) transduction level up to 30 days post-injection. Systemic administration of AVR (i.e., into the left ventricle of the heart) led in 5 days to a high number of dystrophin-positive fibers in heart, diaphragm, and intercostal muscles but not in limb muscles.

Adenoviruses, Human↗
PubMed1996

Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue.

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules. In vivo, in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers [> or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100-1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.

Adenoviridae↗
PubMed1995

Differential short-term transduction efficiency of adult versus newborn mouse tissues by adenoviral recombinants.

We demonstrated different transduction efficiency in several major organs of the immature (newborn) versus mature (adult) mice using adenoviral recombinants containing expression cassettes for either firefly luciferase or bacterial beta-galactosidase reporter genes. The studied tissues included skeletal muscle, heart, brain, lung, kidney, and liver. The transduction efficiency in all tissues, especially skeletal muscle, was significantly less in adults than in newborns, with two exceptions. In the heart, transduction efficiency was the same in newborns and adults, while in brain, it was greater in the adult than in the newborn. The cited differences in transduction efficiencies between newborn and adult tissues applied approximately equally to both reporter genes. The alpha v integrin level showed the same trend as the transduction efficiency in all tissues, except the heart. Polymerase chain reaction showed a specific adenoviral product in proportion to the reporter gene expression in muscle, heart, and brain. The results of this study should be considered in designing gene therapy strategies in genetic diseases.

Adenoviridae↗
PubMed1995

Efficiency and functional consequences of adenovirus-mediated in vivo gene transfer to normal and dystrophic (mdx) mouse diaphragm.

The protein dystrophin is absent in muscles of patients with Duchenne muscular dystrophy (DMD) as well as in mdx mice. The mdx mouse diaphragm closely resembles the human DMD phenotype and should serve as an appropriate model for future studies of dystrophin gene replacement. In this regard, recombinant adenovirus (AV) holds great promise as a vector for delivering a functional dystrophin gene to muscle. However, the use of AV is hampered by the development of an immune response against transduced cells, resulting in short-lived transgene expression as well as possible adverse effects on organ function. In the present study, sensitive reporter genes were employed to determine the efficiency and functional consequences of AV-mediated gene transfer to the diaphragm in both normal and mdx adult mice. One week after direct intramuscular injection of AV into the diaphragm, the level of transgene expression was significantly increased in mdx compared with normal diaphragms. In addition, small-caliber fibers (< 500 microns2) demonstrated preferential transduction in both groups of mice. Normal diaphragms receiving AV exhibited a substantial reduction in maximal twitch and tetanic force generation, whereas no significant effect on diaphragm contractility was found in the mdx group at 1 wk after injection. At 1 mo after AV administration, however, there was a significant decrease in force production by both normal and mdx diaphragms. Immunosuppression with cyclosporine A over 1 mo did not augment the level of transgene expression, but a beneficial effect on diaphragm force-generating capacity was observed in both groups of animals. We conclude the following: (1) short-term transduction of the diaphragm is more efficient in mdx than in normal mice; (2) AV leads to reduced force production by the diaphragm, with this effect being more pronounced in normal than in mdx in the early (but not the late) postinjection period; and (3) immunosuppressive therapy with cyclosporine has a partially protective effect on muscle function after AV administration, which is apparently unrelated to sparing of transduced fibers from elimination by the host immune system. These findings have important implications for the application of AV-mediated dystrophin gene transfer to the treatment of DMD.

Adenoviruses, Human↗