Thermoluminescence study of CaNa2 (SO4 )2 phosphor doped with Eu3+ and synthesized by combustion method.

In the present study, the thermoluminescence (TL) properties of an Eu3+ -doped CaNa2 (SO4 )2 phosphor were studied. The Eu3+ -doped CaNa2 (SO4 )2 phosphor was synthesized using the combustion method. The samples were well crystallized in the monoclinic phase. The TL glow curve of the Eu3+ -doped CaNa2 (SO4 )2 phosphor showed a single prominent peak at around 210°C with showed linearity on increasing exposure. The response curve of the synthesized phosphor showed linearity in the range 500-7000 Gy. Trapping parameters of synthesized phosphors such as activation energy, frequency factor, and order of kinetics were calculated in the study. These trapping parameters were determined by different methods such as Chen's peak method, the initial rise method, and Ilich's method. Each characteristic of these outcomes demonstrated that the synthesized Eu3+ -doped CaNa2 (SO4 )2 phosphor had outstanding TL properties and might be valuable for TL dosimetry application.

ZnAl2O4:Eu novel phosphor: SEM and mechanoluminescence characterization synthesized by solution combustion technique.

Eu doped ZnAl2O4 phosphors were synthesized by the solution combustion technique using carbohydrazide as a fuel. Mechanoluminescence (ML) was excited impulsively by dropping a piston of 0.7 kg onto the phosphors. Two distinct peaks were observed in the ML glow curve of the γ-ray irradiated ZnAl2O4 :Eu phosphors. Dependence of ML on various parameters as impact velocity of the piston dropped on to it, mass of the sample, gamma ray doses given to the sample and ML spectra have been studied. ML emission spectrum showed the characteristic emission of Eu(3+) ion in this system. ML is observed to be optimum for the sample having 0.2 mol% of Eu in the ZnAl2O4 phosphor. XRD result confirms formation of the phosphors. SEM characterization shows its surface morphology. This novel phosphor may be a potential candidate for dosimetric use due to its linear dose response.

Stem cell pharmacogenomics.

Therapeutic stem cell applications represent a newly evolving approach for the treatment of several genetic and degenerative diseases. The advent of pharmacogenomics too, holds promise for an individualized, optimal treatment regime for a large variety of medical conditions. A combination of the benefits of these two technologies creates a new niche in therapeutic medicine research viz. that of stem cell pharmacogenomics (SCP). The development of this approach requires the application of existing technologies in genomics, proteomics and bioinformatics to resolve the various issues involved in advancing the therapeutic applications of stem cell medicine. In this brief overview of the subject, we attempt to provide fresh insights into the exclusive niche of stem cell pharmacogenomics and discuss some of the priority issues that need to be targeted, based on the existing principles of pharmacogenomics, stem cell characteristics and transplantation medicine. Advances in these areas are imperative in realizing the dream of stem cell therapies contributing towards the improvisation of the quality of human life.

Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene haplotype SVMNT in P. falciparum malaria in India.

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) and the P. falciparum multidrug resistance 1 (Pfmdr1) mutation (N86Y) were analyzed as markers of chloroquine resistance in the DNAs of 73 blood samples from patients with P. falciparum malaria in India. Seventy of the 73 DNAs had the Pfcrt K76T mutation. Of these, 66 had the SVMNT haplotype and four had CVIET, the African/Southeast Asian haplotype. Only 20 of 69 DNAs had the Pfmdr1 N86Y mutation. It is surprising that the Pfcrt haplotype in India is predominantly SVMNT, rather than that seen in Southeast Asia. The widespread prevalence of the Pfcrt K76T mutation is a cause for concern.

Th1-specific bystander costimulation imparts resistance against Mycobacterium tuberculosis infection.

The protection against Mycobacterium tuberculosis infection is mediated by T helper type-1 (Th1) cells. Infection of BALB/c mice with M. tuberculosis downregulates expression of a Th1-specific costimulatory molecule, M150, on the surface of infected macrophages. The proliferation of Th cells and Th1-cytokine production by these cells are higher in case of M. tuberculosis antigen presentation by uninfected macrophages than by infected macrophages. The difference in inducing interleukin(IL)-2 and interferon (IFN)-gamma secretion is abolished by providing bystander costimulation through M150 on liposomes.

Leishmania donovani infection of a susceptible host results in CD4+ T-cell apoptosis and decreased Th1 cytokine production.

The disease visceral leishmaniasis is caused by a protozoan parasite, Leishmania donovani and is characterized by depressed cell-mediated immunity (CMI) and unhindered parasite growth in a susceptible host. The opposite trend is observed in a resistant host. However, the mechanism of this loss of CMI during the progressive disease is unknown as yet. In this report, we demonstrate that more than 40% of CD4+ T cells from a susceptible host undergo apoptosis resulting in a significant decrease in interleukin (IL)-2 and interferon (IFN)-gamma secretion, leaving IL-4 secretion unaffected. These changes are not apparent in the case of CD4+ T cells derived from a resistant host. The data reported here suggest that experimental Leishmania donovani infection leads to selective deletion of the IL-2 and IFN-gamma-secreting cells but not Th2-like cells in a susceptible but not a resistant host.

Apoptosis of Th1-like cells in experimental tuberculosis (TB).

Th1 cell-induced anti-mycobacterial immunity is lost during a progressive Mycobacterium tuberculosis infection in a susceptible host. This study was designed to test the mechanism of the loss of anti-mycobacterial cell-mediated immune response. We demonstrate that M. tuberculosis infection results in increased Fas expression and decreased Bcl-2 expression in CD4+ T cells. When CD4+ T cells are stimulated in vitro, they show increased apoptosis and decreased production of IL-2 and interferon-gamma (IFN-gamma) but not of IL-4. These changes may result in selective apoptosis of Th1-like cells, leading to the loss of cell-mediated immune response against M. tuberculosis.

M150 modulates the costimulatory signals delivered by B cells to T cells and enhances their ability to help B cells.

There is a prerequirement of at least two sets of signals delivered by the antigen-presenting cell (APC) for the optimal activation of T helper (Th) cells. The first signal is provided by the engagement of T cell receptor with the antigen-MHC class II complex, followed by a second stimulus in the form of costimulatory signals. In the present study, we provide evidence that in a T-dependent antigen-driven system, the signals generated by hapten-specific B cells to stimulate Th cells for the secretion of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and IL-4 were differentially modified by M150, a 150-kDa molecule expressed on the surface of macrophages. When ovalbumin-specific Th cells were cultured in the presence of 2,4,6 trinitrophenol (TNP)-specific B cells, M150 significantly increased the proliferation of Th cells and the secretion of IL-2 and IFN-gamma and decreased the production of IL-4. Further, Th cells stimulated with M150 acquired improved ability to help B cells, resulting in an increase in the number of antibody-secreting cells and in the production of TNP-specific IgG2a antibodies. M150 possibly promotes Th1-like cell activity, as evidenced by predominant secretion of IL-2, IFN-gamma, and IgG2a but not IL-4 and IgG1.

Differential effect of anti-B7-1 and anti-M150 antibodies in restricting the delivery of costimulatory signals from B cells and macrophages.

B7-1 and M150 are potent costimulatory molecules expressed on B cells and macrophages. We have examined the capacity of Abs against B7-1 and M150 in differentially inhibiting the costimulatory signals delivered by macrophages and B cells to OVA-specific CD4+ T cells. The anti-B7-1 Ab significantly blocked the proliferation of Th cells, MLR, T cell help to B cells, and secretion of IFN-gamma when B cells were used to provide costimulation, but not when macrophages were used. In contrast, anti-M150 Ab significantly decreased the proliferation of Th cells, MLR, and production of IFN-gamma, when macrophages were utilized to provide costimulatory signals, but not when B cells were used as APC. However, when macrophages activated with IFN-gamma were used as a source of costimulation, like anti-M150 Ab, Ab to B7-1 also down-regulated the activation of Th cells. The significance of this finding is that M150 is a potent first costimulatory signal for initiating proliferation and secretion of IFN-gamma and providing cognate help for B cells by Th cells when the macrophage is used as an accessory cell. M150-induced IFN-gamma production induces the expression of B7-1 on the surface of macrophages, which then delivers a second cosignal for Th cells. B7-1 works efficiently when B cell provides cosignal. Both of the molecules promote Th1 activity, as evidenced by the inhibition of the secretion of IFN-gamma but not IL-4 by Th cells with anti-M150 and B7-1 Abs.

Leishmania donovani infection of a susceptible host results in apoptosis of Th1-like cells: rescue of anti-leishmanial CMI by providing Th1-specific bystander costimulation.

A protective immune response against Leishmania donovani infection is mediated by T-helper type 1 (Th1) cells. Th1 induced cell-mediated immunity (CMI), as assessed by anti-leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down-regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite down-regulates the expression of a Th1-specific costimulatory molecule, M150, on the surface of infected BALB/c mice-derived macrophages. Th cells are rendered unresponsive to anti-CD3 Ab-mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL-2, IL-4 and IFN-gamma compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti-CD3 Ab induced unresponsiveness and IFN-gamma but not IL-4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.

Antigen incorporation into liposomes results in the enhancement of IL-4 and IgG1 secretion: evidence for preferential expansion of Th-2 cells.

Liposomes have been used to modify the immunological behaviour of a number of antigens. The present study was designed to evaluate the effect of liposomization of ovalbumin on the induction of Th-1 and Th-2-cell response by monitoring the secretion of lymphokines and IgG Isotypes. Liposomes having varied physicochemical properties (positively and negatively charged, neutral and pH-sensitive) were used for this purpose. Ovalbumin delivered in this way induced preferential secretion of IL-4 and production of antigen-specific IgG1 isotypes. This was observed irrespective of the surface charge properties of the liposomes. Further, the concentration of antigen required for the activation of Th cells was 10(2)- to 10(3)-fold lower after encapsulating it in liposomes. These results suggest that liposomes may prove useful adjuvants to prime Th2-like immune responses.

Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application.

The development of a microgravimetric immunobiosensor using a piezoelectric quartz crystal as a detector requires a stable and reproducible immobilization method for ligand binding. The method of silanization using 3-aminopropyltriethoxysilane (APTES) has been widely used for activating the carrier surface. In the present study, APTES deposition on a piezoelectric crystal surface was studied under various solvent conditions. A fluorescence method, using fluorescence isothiocyanate as a dye, was demonstrated for the quantification of amino groups on the silanized piezoelectric crystal surface. The optimum binding conditions of APTES deposition on a piezoelectric crystal surface were incorporated for the covalent immobilization of protein on the crystal surface in developing a stable and sensitive microgravimetric immunobiosensor. Determination of immunoglobulin G (IgG) concentration was performed using APTES modified piezoelectric crystals coated with protein G. The resonant frequency shift, resulting from the formation of protein G-IgG complex on the crystal surface, correlated with the concentration of IgG in the range 10 ng/ml to 0.1 mg/ml. The APTES modified, protein G coated crystal were found to be quite stable and did not show a significant loss of sensitivity even after 12 weeks of storage at 4 degrees C in a desiccator.

Characterization of a novel co-stimulatory molecule: a 155-160kD B cell surface protein provides accessory help to CD4+ T cells to proliferate and differentiate.

Optimal activation of T cells to clonally expand requires at least two distinct biological signals; one is generated by the interaction of the T cell receptor (TcR) with peptides bound to MHC molecules. The other signal(s) is (are) generated by a functionally defined event called the co-stimulatory pathway. We have characterized the co-stimulatory property of a murine B lymphocyte membrane protein (155-160 kD) on resting CD4+ T cells. The study involved the isolation of a 155-160 kD protein (B1) from the membranes of LPS-stimulated B cells. When reconstituted into lipid vesicles, B1 exerted a dose-dependent proliferative response to CD4+ T cells, resulting in the predominant secretion of IL-4 and IL-5 after cross-linking receptors with anti-CD3 mAb. This protein is a phosphoglycoprotein which gives a single spot on two-dimensional gel electrophoresis under reducing conditions and as a distinct peak on reverse phase-HPLC. The B1 binds to the T cell surface as is demonstrated by electron microscopic autoradiography and scanning electron microscopy, as well as competitive binding assays. It does not cross-react with antibodies directed against ICAM-1, LFA-1 alpha, B7, HSA and VCAM-1, suggesting the novelty of the protein. Activation of CD4+ T cells with B1 in the presence of anti-CD3 resulted in the translocation of protein kinase C (PKC). The B1 is barely detectable on the surface of resting B cells and digestion of this protein with V8 protease and peptide N-glycosidase F resulted in distinct protein bands on an autoradiogram.

Characterization of novel costimulatory molecules. A protein of 38-42 kDa from B cell surface is concerned with T cell activation and differentiation.

Optimal activation of T cells often requires signals delivered by the ligation of T cell receptor (TcR) and those resulting from costimulatory interaction between certain T cell surface accessory molecules and their respective counter receptors on antigen presenting cells. The molecular events underlying the co-stimulatory activity are still not understood fully. Here we describe a 38-42-kDa (B3) protein, present on the surface of lipopolysaccharide-activated B cells, which can provide co-stimulation to resting T cells leading to a predominant release of interleukin (IL)-4 and IL-5 and negligible amounts of IL-2 and interferon-gamma. Binding assay and electron microscopic autoradiography data suggest that this molecule binds T cells, and the same can be competed by unlabeled B3. Characterization experiments point out that B3 shows up as a single prominent peak on reverse phase-high performance liquid chromatography, runs as a single spot in reducing two-dimensional gel electrophoresis, and is a phosphoglycoprotein. The Western analysis indicate that it does not cross-react with antibodies directed against murine ICAM-1, LFA-1 alpha, VCAM-1, HSA, and B7 suggesting the novelty of the protein. The internal amino acid sequence of this molecule suggests that it does not belong to a known category of murine B cell surface molecules.

Macrophage-T cell interaction in experimental visceral leishmaniasis: failure to express costimulatory molecules on Leishmania-infected macrophages and its implication in the suppression of cell-mediated immunity.

The most important immunopathological consequence of infection with Leishmania seen in murine and human hosts is the suppression of T cell-mediated immune responses to both mitogens and leishmanial antigens. It has been suggested that this suppression is mediated by macrophages, either by defective antigen processing and presentation or by the elaboration of suppressive mediators like prostaglandins. Optimum activation of T helper cells requires not only T cell receptor occupancy by the antigen-Ia complex, but also costimulatory signals provided by the antigen-presenting cells. We investigated the status of several costimulatory molecules on infected macrophages from both genetically susceptible BALB/c and resistant C57BL/6 mice. Our results demonstrate that upon parasitization, the macrophages become unable to deliver costimulatory signals to T helper cells, and that this effects is mediated by prostaglandins, as the inhibition of its synthesis by indomethacin recovered the defect. Upon infection with L. donovani, B7-1 expression was decreased, while ICAM-1 was marginally increased in BALB/c macrophages and there was no significant change in the expression of B7-1 and ICAM-1 in Leishmania-infected C57BL/6 macrophages. Expression of VCAM-1 did not change during infection. This selective alteration in the expression of costimulatory molecules on L. donovani-infected BALB/c macrophages was caused by the living parasite, as shown by the fact that killing of the parasites by stibogluconate led to no alteration in the levels of costimulatory molecules. We found that the change in B7-1 expression on the surface of infected macrophages resulted in the inhibition of delayed-type hypersensitivity-mediating functions of T helper cells from BALB/c mice. The results described in this study not only throw light on the possible mechanism of leishmanial pathogenesis, but also open up the possibility of immunotherapy of leishmaniasis by selective manipulation of costimulatory molecules.

Development of piezoelectric crystal based microgravimetric immunoassay for determination of insulin concentration.

A microgravimetric, piezoelectric crystal based immunoassay for the quantification of insulin concentration is described. The method utilizes a modified piezoelectric crystal device having an antibody specific to insulin bound to its surface. The antibody to insulin was immobilized on the surface of crystal electrode by using either 3-aminopropyltriethoxy silane (3-APTES), polyethyleneimine (PEI) or covalently coupled protein A-gold immobilization method. Coating an electrode with a cross linked protein A-antibody complex gave better results in terms of sensitivity and stability. Using the system described, the insulin concentration up to 1 ng ml-1 could be detected. The stability and reusability of the system was further improved by using a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized insulin antibody. Scanning tunneling microscopic (STM) study was also done to confirm the surface coverage and orientation of insulin and antibody molecules on the modified piezoelectric crystal electrode surface. A comparison between the present study and the well-established radioimmunoassay technique (RIA) revealed that the described microgravimetric immunoassay technique (MIA) could successfully be developed as an alternative of RIA.

A 38-kDa antigen of Mycobacterium tuberculosis predominantly induces the secretion of interleukin-2, interferon-gamma and IgG2a antibodies.

In the present study, mice of 3 different haplotypes (H-2d, H-2k and H-2b) were sensitized subcutaneously with heat-killed H37Ra or 38-kDa antigen of Mycobacterium tuberculosis. Lymphocytes obtained from immunized animals were challenged in vitro with 38-kDa antigen in both cases. The dominant pattern of Th1-like lymphokines (IL-2 and IFN-gamma) and preferential production of 38-kDa specific IgG2a-type antibody were observed. It was noted that 38-kDa antigen was recognized permissively by all 3 strains of mice used in the present study. It was interesting to note that C3H/HeJ mice, which express BCG-resistant alleles showed a higher level of proliferative as well as cytokine response as compared to BALB/c and C57BL/6 mice, which bear BCG-susceptible alleles. These results suggest that not only in recall responses but also during the induction as well as expression phase of the immune response mediated by 38-kDa antigen of M. tuberculosis the Th1-like immune response predominates.

Macrophage-T cell interaction in experimental mycobacterial infection. Selective regulation of co-stimulatory molecules on Mycobacterium-infected macrophages and its implication in the suppression of cell-mediated immune response.

The most important immunopathological consequence of experimental mycobacterial infection is the suppression of T cell-mediated immune response to both mitogens and mycobacterial antigens. We registered that there was decreased concanavalin A-induced spleen cell proliferation in infected susceptible BALB/c mice as compared to normal mice. In resistant (C3H/HeJ) mice, infection with the bacteria did not induce any suppression in the mitogen-induced lymphoproliferation. Likewise, delayed-type hypersensitivity (DTH) responses, to keyhole limpet hemocyanin and mycobacterial crude soluble antigen were suppressed in infected BALB/c mice but not in C3H/HeJ mice. This depressed T helper cell function may either be due to defective T cell-receptor occupancy by antigen-Ia complex or altered co-stimulatory signals provided by antigen-presenting cells. In the present study, we have investigated the status of certain co-stimulatory molecules on the infected macrophages from both susceptible and resistant mice. Our results demonstrate that upon mycobacterial infection, the macrophages are rendered incapable of delivering the co-stimulatory signals to T helper cells, possibly due to the involvement of prostaglandin, as inhibition of its biosynthesis by indomethacin reversed the defect. Furthermore, the selective regulation was bacteria-induced as killing of the bacteria by rifampicin abrogated the derangements in the expression of co-stimulatory molecules on the Mycobacterium-infected macrophages. Our observations revealed that upon infection with Mycobacterium tuberculosis, B7 was down-regulated while ICAM-1 was increased only in BALB/c but not in C3H/HeJ mice. Expression of VCAM-1 did not change during the infection in either strain of mice. We found that these changes in ICAM-1 and B7 expression on the surface of infected macrophages resulted in inhibition of DTH-mediating functions of T helper cells from BALB/c mice. The results obtained in this study describe not only a novel immune evasion strategy adopted by Mycobacterium, but also open up the possibility of immunotherapy of mycobacterial infection by selective manipulation of co-stimulatory molecules.

A 150-kDa molecule of macrophage membrane stimulates interleukin-2 and interferon-gamma production and proliferation of ovalbumin-specific CD4+ T cells.

In the present study, we describe the potential co-stimulatory role of a macrophage membrane-associated protein of 150 kDa (M150). The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was found to be a single molecule on two-dimensional gel electrophoresis. The molecule was re-constituted in phosphatidyl choline vesicles and tested for its ability to promote the proliferation and the secretion of lymphokines from T helper (Th) cells. The reconstituted M150 induced a significant proliferation of anti-CD3 monoclonal antibody (mAb)-stimulated ovalbumin-specific CD4+ T cells. Further, Th cells activated with this molecule in the presence of anti-CD3 mAb mainly secreted interleukin (IL)-2 and interferon-gamma but not IL-4. M150 could not promote the proliferation of Th cells, or lymphokine secretion in the absence of anti-CD3 mAb. These observations suggest that M150 acts by selectively activating a Th1-like immune response.