Biomedical subjects

K S Nandakumar

Publications and source records attributed to K S Nandakumar.

11 recordsLinked to original sources

PubMed2005

Identification of epistasis through a partial advanced intercross reveals three arthritis loci within the Cia5 QTL in mice.

Identification of genes controlling complex diseases has proven to be difficult; however, animal models may pave the way to determine how low penetrant genes interact to promote disease development. We have dissected the Cia5/Eae3 susceptibility locus on mouse chromosome 3 previously identified to control disease in experimental models of multiple sclerosis and rheumatoid arthritis. Congenic strains showed significant but small effects on severity of both diseases. To improve the penetrance, we have now used a new strategy that defines the genetic interactions. The QTL interacted with another locus on chromosome 15 and a partial advanced intercross breeding of the two congenic strains for eight generations accumulated enough statistical power to identify interactions with several loci on chromosome 15. Thereby, three separate loci within the original QTL could be identified; Cia5 affected the onset of arthritis by an additive interaction with Cia31 on chromosome 15, whereas the Cia21 and Cia22 affected severity during the chronic phase of the disease through an epistatic interaction with Cia32 on chromosome 15. The definition of genetic interactions was a prerequisite to dissect the Cia5 QTL and we suggest the partial advanced intercross strategy to be helpful also for dissecting other QTL controlling complex phenotypes.

Animals↗

PubMed2004

Methotrexate ameliorates T cell dependent autoimmune arthritis and encephalomyelitis but not antibody induced or fibroblast induced arthritis.

OBJECTIVE: To investigate the mode of action of methotrexate (MTX) in different types of models for rheumatoid arthritis (RA) and multiple sclerosis (MS). METHODS: Models for RA and MS were selected known to have different pathogenesis--that is, fibroblast induced arthritis in SCID mice, collagen induced arthritis (CIA), anticollagen II antibody induced arthritis (CAIA), and experimental autoimmune encephalomyelitis (EAE) in (Balb/c x B10.Q)F1 and B10.Q mice, and Pristane induced arthritis in DA rats (PIA). The MTX treatment was started 1 day after the onset of disease and continued for 14 days to compare effects on the different models. RESULTS: All models known to be critically dependent on T cell activation (CIA, PIA, and EAE) were effectively down regulated by titrated doses of MTX. In contrast, no effects were seen on fibroblast induced arthritis or CAIA. No effects were seen on the levels of anticollagen II antibodies in the CIA experiment. CONCLUSION: The data show that MTX has strong ameliorative effect on both classical models of RA, like CIA and PIA, but also on a model for MS, EAE. It also suggests that MTX operates only in diseases which are preceded by, and dependent on, T cell activation. A comparison of CAIA and CIA suggested that MTX operates independently of arthritogenic antibodies. These results demonstrate that different animal models reflect the complexity of the corresponding human diseases and suggest that several models should be used for effective screening of new therapeutic agents.

Animals↗

PubMed2001

IL-10-deficient B10.Q mice develop more severe collagen-induced arthritis, but are protected from arthritis induced with anti-type II collagen antibodies.

IL-10 is a pleiotropic cytokine with stimulatory and inhibitory properties, and is thought to have a protective role in rheumatoid arthritis and collagen-induced arthritis (CIA). In this study, we investigated how IL-10 deficiency affects CIA and anti-collagen type II (CII) Ab-transferred arthritis in C57BL/10.Q (B10.Q) mice. The B10.Q.IL-10(-/-) mice had an 8-cM 129/Ola fragment around the IL-10 gene. The mice were treated with antibiotics, appeared healthy, and had no colitis. T cells from IL-10(-/-) mice expressed similar levels of IFN-gamma, IL-2, and IL-4 after mitogen stimulation; however, macrophages showed a reduced TNF-alpha production compared with IL-10(+/-) littermates. IL-10(-/-) mice had an increased incidence, and a more severe CIA disease than the IL-10(+/-) littermates. To study the role of IL-10 in T cell tolerance, IL-10(-/-) were crossed into mice carrying the immunodominant epitope, CII(256-270), in cartilage (MMC) or in skin (TSC). Both IL-10(-/-) and IL-10(+/-) MMC and TSC mice were completely tolerized against CIA, indicating that lack of IL-10 in this context did not break tolerance. To investigate whether IL-10 was important in the effector phase of CIA, arthritis was induced with anti-CII Abs. Surprisingly, IL-10(-/-) were less susceptible to Ab-transferred arthritis, as only 30% showed signs of disease compared with 90% of the littermates. Therefore, IL-10 seemed to have a protective role in CIA, but seemed to exacerbate the arthritogenicity of anti-CII Abs. These data emphasize the importance of studying IL-10 in a defined genetic context in vivo, to understand its role in a complex disease like arthritis.

Animals↗

PubMed2001

Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity.

Approximately 25% of mature T cells possess two distinct cytoplasmic T cell receptor (TCR) alpha-chains, due to productive gene rearrangements of both alleles. Expression of two different alpha-chains at the cell surface is a potential risk factor for development of autoimmunity. However, it has been difficult to determine the frequency of peripheral T cells with two different alpha-chains at the surface. Our new approach is based on comparing by flow cytometry the percentage of cells that express a given Valpha-chain between wild-type mice and mice that are hemizygous for a disrupted Tcra locus (Tcra+/-) and consequently unable to express two rearranged Tcra genes. We consistently found that approximately 8% of total peripheral T cells express two surface alpha-chains. The importance of dual alpha-T cells in autoimmunity was examined in a mouse model for rheumatoid arthritis, namely collagen-induced arthritis (CIA). No significant difference was observed between Tcra+/- mice and wild-type littermates, considering arthritis incidence, day of disease onset, and maximum arthritic score. We therefore conclude that there is incomplete phenotypic allelic exclusion in TCRalpha, and that the presence of a significant number of potentially multireactive T cells does not increase the susceptibility to develop autoimmune arthritis.

Animals↗

PubMed1999

Influence of immunopotentiators on the antiporin immunoglobulin G subclass: distribution and protective immunity against murine salmonellosis.

To improve the immune potential of porin (a pore-forming protein of Salmonella sp.), different immunopotentiators such as Freund's complete adjuvant (FCA), lipopolysaccharide (LPS) and polyoxydonium (PO) were evaluated by studying the nature of the protective immune response induced against murine Salmonellosis. The nontoxic, synthetic heteropolymer polyoxydonium was as good as LPS at inducing antiporin immunoglobulin G (IgG) antibodies and protective immunity. Analysis of the antiporin IgG subclass pattern revealed a preferential increase in a particular subclass based on the immunopotentiator used. Porin, alone or emulsified in FCA, elicited predominantly antiporin IgG1 antibodies, whereas LPS preferentially evoked antiporin IgG2a, IgG2b and IgG3 antibodies. Polyoxydonium induced a clear shift towards antiporin IgG2b antibodies. The significance of these antiporin IgG subclass antibodies in protection against murine Salmonellosis was studied by passive immunization and by analysing the infected mouse sera.

Adjuvants, Immunologic↗

PubMed1999

Upregulation of antitumor immunity by IL-12 gene-transfected AK-5 tumor cells in vivo.

We have earlier demonstrated a significant role for IL-12 in the regression of a rat histiocytic tumor, AK-5. In order to analyze further the antitumor immunity induced by interleukin (IL)-12, we have established IL-12-secreting tumor cell clones by gene transfection. Significant enhancement in the lytic potential of splenocytes by the culture supernatants containing IL-12 demonstrated retention of biological activity by the tumor-cell-derived cytokine. Athymic nude mice transplanted subcutaneously with tumor cells engineered to secret IL-12 showed a significant reduction in tumor size, with enhanced antibody-dependent cellular cytotoxicity. Analysis of the serum samples from animals injected with the IL-12 gene-transfected AK-5 cells on different days revealed a significant increase in circulatory IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and antitumor antibodies, all of which contributed to the reduction in tumor mass. The enhanced proliferative capacity of splenocytes from these animals indicated the presence of highly activated immune cells in vivo. Similarly, intraperitoneal transplantation of IL-12 gene-transfected tumor cells in syngeneic Wistar rats induced a significant increase in cellular cytotoxicity, with a concomitant reduction in circulatory IL-12 (p40) protein. Administration of antibodies to IL-12 and IFN-gamma reduced the expression of the costimulatory molecules B7.1 and B7.2 and the cytolytic effectors granzyme B and Fas-L, suggesting their involvement in IFN-gamma-dependent antitumor immune response induced by IL-12. The present study thus demonstrates that IL-12 gene therapy could be among the promising approaches for an effective cancer therapy.

Animals↗

PubMed1998

Lectin-functionalized multiple emulsions for improved cancer therapy.

Fine-multiple emulsions bearing 6-mercaptopurine (6-MP) in internal aqueous phase were prepared by two step emulsification using sonication technique. It was coated with Concanavalin-A (Con-A) using carbodiimide method to obtain lectin-functionalized multiple emulsions. The Con-A coated multiple emulsion was characterized for antitumour activity on murine leukemia cell line L-1210 in vitro and compared with uncoated multiple emulsion and free drug. An increased uptake and cytotoxicity were observed for Con-A coated multiple emulsion in vitro. The IC50 was decreased upto 4-fold with Con-A coated emulsion. In vivo antitumour activity was seen by recording survival times of mice injected with L-1210 cells i.v. or i.p. The mean survival time was found to increase upon treatment with Con-A coated multiple emulsion. The tumour cell count in the peritoneal cavity was decreased significantly when animal was treated by i.p. route while there was no significant difference when it was treated by i.v. route. The normal peritoneal cells remained unaltered in number and blood parameters were also restored on treatment to tumour bearing mice. The formulation was found to be effective for the treatment of cancer.

Animals↗

PubMed1998

Role of cytokines in the maintenance of the memory immune response induced by a rat histiocytoma in syngeneic hosts.

The nature of immune memory induced by a rat histiocytoma, AK-5, in syngeneic hosts was studied. AK-5 tumor when transplanted intraperitoneally (i.p.) into naive animals grows as ascites and is 100% fatal. However, spontaneous regression of AK-5 tumor was observed in 60% of animals upon s.c. transplantation. Interestingly, all the tumor-rejected animals (immune) were found to resist further i.p. challenges with AK-5 cells. The immunity thus developed is specific for AK-5 tumor, since other tumors grow in these animals. In order to understand the tumor-specific immune memory induced after AK-5 tumor transplantation, we have evaluated circulatory-cytokine profiles of i.p. tumor-transplanted naive and immune animals. Our results show an increase in the levels of IL-2, IL-12 and IL-4 in tumor-injected immune animals compared with normal animals, whereas the interferon-gamma levels were totally reversed in these two sets of animals. We also found elevated levels of circulating immune complexes in the sera from AK-5-rechallenged immune animals. We have also evaluated the cytotoxic potential of splenocytes and pure natural killer cells from immune animals rechallenged with AK-5 cells, and have found a significant increase in antibody-dependent cellular cytotoxicity. Similarly, in vitro proliferation of total splenocytes and nylon-wool non-adherent cells from immune animals was much higher compared with the normal animals. The present study thus suggests antigen-independent maintenance of clonal burst size, which could be the form of immune memory induced by AK-5 tumor in the syngeneic host.

Animals↗

PubMed1997

Early expression of interleukin-12, p40 subunit and IFN-gamma inhibits regression of AK-5 tumor.

Differential immune response of syngeneic animals to a rat histiocytoma AK-5 based on the route of transplantation was investigated. Spontaneous regression of subcutaneous tumor was observed in 55-60% of animals. On the other hand, when the tumor cells were injected intraperitoneally, none of the animals survived. Earlier studies from this laboratory indicated upregulation of Th-1-type cytokines, leading to early tumor regression when the tumor was transplanted subcutaneously. Hence we evaluated and compared the circulatory-cytokine profiles in both s.c. and i.p. tumor-injected animals. Our results show an early increase in the p40 subunit of IL-12, prolific increase in IFN-gamma and lower levels of IL-2 in i.p. tumor-injected animals. However, there were no significant differences in the levels of transcripts for these cytokines in either of the groups. Significantly, a lower level of cytotoxicity was observed with splenocytes from i.p. tumor-transplanted animals. Moreover, the cytotoxicity of IL-12-activated but not IL-2-activated NK cells was inhibited by sera (rich in IL-12, p40 subunit) from i.p. tumor-transplanted animals, suggesting the participation of p40 subunit in the regulation of tumor regression. Thus the present study suggests a possible translational regulation of Th-1-type cytokines in AK-5 tumor-host interaction.

Adjuvants, Immunologic↗

PubMed1993

Diagnosis of typhoid fever: detection of Salmonella typhi porins-specific antibodies by inhibition ELISA.

Porins are highly immunogenic outer membrane proteins of Salmonella. Sera from typhoid patients contained a high level of IgG antibodies directed to porins of Salm. typhi. Since porins are highly conserved proteins, anti-porins antibodies both from typhoid patients and healthy normals reacted with porins from several Gram-negative bacteria. Therefore, in order to improve the specificity of detecting Salm. typhi porins-specific antibodies, an inhibition ELISA was developed using enzyme-conjugated MoAbs (MP1 and MPN4) specific to Salm. typhi porins. Sera from typhoid patients with positive haemoculture (16 out of 17) inhibited the binding of MP1 to porins, thus showing a positive test for typhoid, whereas sera from patients with other Gram-negative bacterial infections (n = 7) and from healthy volunteers (66 out of 67) were found to be negative. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this assay were 94.1, 98.7, 97.8, 94.1 and 98.7% respectively. The validity of our inhibition ELISA for typhoid was higher than that of the Widal test. The diagnosis of typhoid fever as early as 3 days after the onset of fever, using a single specimen is possible.

Adolescent↗

PubMed1992

Monoclonal antibodies against Salmonella porins: generation and characterization.

Monoclonal antibodies (mAbs) were generated against porins, one of the major outer membrane proteins of Salmonella typhi. Six clones, designated MP1, MP2, MP3 (IgG2ak), MPN4, MPN6 (IgG1k) and MPN5 (IgG2bk) were characterized by enzyme immunoassay (ELISA) for their reactivity to porins from S. typhi, Salmonella paratyphi A, S. paratyphi B, S. paratyphi C, Salmonella choleraesuis, Salmonella enteritidis, Salmonella krefeld, Salmonella panama, Salmonella typhimurium, Escherichia coli B, Shigella flexneri 1b and Pseudomonas aeruginosa. All the clones positive for S. typhi porins showed varying reactivity towards several Salmonella species. However, none of them was positive for porins from other Gram-negative bacteria or for lipopolysaccharide (LPS). The affinity constant of these mAbs, except MPN4, was found to be in the higher range. Dot ELISA revealed that the mAbs recognized porins only in their native form. The results of inhibition ELISA using horseradish peroxidase (HRP)-conjugated MP1 suggest that the clones MP1, MP2, MP3, MPN5 and MPN6 secreted antibodies to identical epitope(s) of a 36-kDa peptide and MPN4 to a different epitope of a 35-kDa peptide. The possible applications of these mAbs were discussed.

Animals↗