Estrogen receptors in rat bone: their interaction with estrogen receptor modulators.

Estrogen receptors (ER) were studied in rat bone cytosol using immunoprecipitation, and Western blot technique. Ligand specificity of bone ER was studied using various known modulators of ER. Competitive experiments were performed under exchange conditions in bone tissue obtained from one day old rats. ER alpha and beta subtypes were identified using immunoblotting experiments compared with that of ovarian and uterine tissues. In competitive binding assay, maximum inhibition in specific 3H-E2 binding was shown by E2 followed by tamoxifen and diethylstilbestrol. 7-Hydroxycentchroman and 85/287 also inhibited specific 3H-E2 binding but were less potent as compared to tamoxifen and diethylstilbestrol. However, 85/287 was less effective (81%) as compared to 7-hydroxycentchroman. Polyacrylamide gel electrophoresis of cytosol and Western blot analysis revealed the presence of 55 kD and 66 kD ER immunoreactive bands corresponding to alpha and beta subtypes, respectively, in bone as well as in uterus. Interestingly, the concentration of 55 kD ER was 3-fold higher than that of 66 kD ER. Ovarian cytosol revealed the presence of a 55 kD band only in Western blot analysis. These studies suggest the action of estrogens/ER modulators on osteoblasts which contain a limited number of classical alpha as well as beta sub types of ER that are known to be structurally different in their hormone-binding domains.

Immunization with Haemophilus influenzae type b-CRM(197) conjugate vaccine elicits a mixed Th1 and Th2 CD(4+) T cell cytokine response that correlates with the isotype of antipolysaccharide antibody.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) induces protective antibodies but is T independent and poorly immunogenic in infants. Conjugate vaccines of Hib PS linked to proteins, such as CRM(197), increase the PS antibody titer and elicit immunologic memory. To define the conjugate-induced memory T cell response, 19 adults were immunized with Hib-CRM(197), and antibody titers, carrier protein-specific CD4(+) T cell proliferation, and cytokine production were measured. Hib-CRM(197) induced PS and CRM(197) antibodies, vigorous T cell recall responses, and production of cytokines, including interleukin (IL)-2, IL-5, IL-10, and interferon-gamma. There was marked variability in PS antibody titer, despite consistent CRM(197)-specific recall responsiveness, which correlated with peak IgM and IgA PS antibody titers. Correlations were also found between IL-2 and IL-5 and IgA PS antibody levels. Hib-CRM(197) induced a rapid increase in CRM(197)-specific memory T cells and mixed Th1/Th2 cytokines, which may regulate the isotype and quantity of PS antibody.

Identification of amastigote-specific antigens of Leishmania donovani using kala-azar patient sera.

Antigenic characterization of the soluble fraction of axenic amastigotes of Leishmania donovani ( strain Dd8, causative agent of Indian kala-azar) and their comparison with promastigotes is reported. The axenic amastigotes were assessed for their immunological status employing anti-A2 monoclonal antibody which is extremely specific for L. donovani amastigotes. SDS-PAGE of 35[S] methionine labeled proteins of the two parasite stages exhibited few stage specific and some conserved antigens in both the stages. An increased synthesis of heat shock proteins was observed in axenic amastigotes. Western blot experiments employing sera of kala azar positive patients identified immunodominent antigens of 116,83,26 and 12 kDa in axenic amastigotes which were not present in promastigotes. These amastigote stage specific antigens may have immense potential in immunodiagnosis and prophylaxis of kala-azar.

Glutathione-S-transferase activity in malarial parasites.

Glutathione-S-transferase (GST) activity has been detected in rodent (Plasmodium berghei, P. yoelii), simian (P. knowlesi) and human (P. falciparum) malarial parasites, and in different intraerythrocytic stages of P. knowlesi (schizont > ring > trophozoite). In chloroquine-resistant strains of rodent and human malarial parasites GST activity significantly increases compared to sensitive strains. Further, the increase in enzyme activity is directly related to drug pressure of resistant P. berghei. Complete inhibition of chloroquine-sensitive and resistant P. berghei glutathione-S-transferase activities was observed at 2.5 and 5. micrometer concentration of hemin, respectively. An inverse relationship was found between the heme level and enzyme activity of chloroquine-resistant and sensitive P. berghei. Chloroquine, artemisinin, and primaquine noticeably inhibited GST activity in P. knowlesi.

The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements.

To determine the molecular mechanisms underlying the "cross talk" between the activity of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), which binds to arylhydrocarbon receptor (AHR) and estradiol (E2)-liganded estrogen receptor (ER), we first examined the initial step of estrogen action, ligand binding to ER. None of the AHR ligands tested, i.e. TCDD, benzo[a]pyrene, 3,3',4,4',5-pentachlorobiphenyl, beta-naphthoflavone, or alpha-naphthoflavone, bound to ER alpha. We report the first examination of TCDD interaction with ER beta: TCDD did not displace E2 from ER beta. We then examined a second possible mechanism, i.e. direct inhibition of ER alpha binding to estrogen response elements (EREs) by the AHR/AHR nuclear translocator (ARNT) complex. The AHR/ARNT heterodimer did not bind either a full or half-site ERE. However, AHR/ARNT bound specifically to oligomers containing naturally occurring EREs derived from the human c-fos, pS2, and progesterone receptor (PR) gene promoters that include xenobiotic response element (XRE)-like sequences. In contrast, neither purified E2-liganded-ER from calf uterus or recombinant human ER alpha bound a consensus XRE. TCDD inhibited E2-activated reporter gene activity from a consensus ERE and from EREs in the pS2, PR, and Fos genes in transiently transfected MCF-7 human breast cancer cells. However, this inhibition was not reciprocal since E2 did not inhibit TCDD-stimulated luciferase activity from the CYP1A1 promoter in transiently transfected MCF-7 or human endometrial carcinoma HEC-1A cells. We propose that at least part of the mechanism by which the AHR/ARNT complex inhibits estrogen action is by competitively inhibiting ER alpha binding to imperfect ERE sites, adjacent to or overlapping XREs.

Current trends in research on tissue stage of malaria.

The tissue stage or the exoerythrocytic (EE) stage of the malaria parasite, for many years remained the most neglected form mainly because of its inaccessibility being located in the liver. The advent of in vitro techniques resulting in the successful cultivation of these forms in primary hepatocyte cultures and a variety of cell lines has greatly augmented research on these stages and have provided unique in vitro systems which can be used as primary screens for candidate chemotherapeutic and immunoprophylactic agents and have facilitated better understanding of the sporozoite-hepatocyte interactions. Sensitive and specific nucleic acid probes (DNA and ribosomal RNA) have been developed to quantify EE stages in infected livers. Efforts to establish SCID mouse as a model for cultivation of EE stages of human malaria parasites have been encouraging. The earlier assumptions that these tissue stages are free from immune attack have been proven wrong and the hepatic phase itself now appears to be essential for the induction of protection against the pre-erythrocytic stages. Liver stage specific antigens have been identified in recent years. Despite its intracellular position, this 'hidden' form has been found to constitute a target for antibodies, cytokines, and cytotoxic T cells. The present review focuses on the advances in research on the 'silent' stage of malaria parasites.

Heme metabolism in promastigotes of Leishmania donovani.

Promastigotes of Leishmania donovani (Dd-8 strain) showed presence of important key enzymes of heme synthesizing (delta-aminolevulinic acid synthase and ferrochelatase) and degrading (heme oxygenase and biliverdin reductase) systems, classical leishmanicidal drugs viz allopurinol, amphotericin B, pentamidine and CDRI compound 93/202 inhibited the heme oxygenase activity of the parasite, whereas, delta-aminolevulinic acid synthase activity practically remained unaffected. The Km, Vmax and pH values of heme oxygenase of promastigotes were found to be 1666 microM hemin, 625 nmol of bilirubin formed h-1 mg protein-1 and 7.5 respectively. The findings suggest the presence and importance of heme metabolism in the de novo synthesis of different hemoproteins of the Leishmania parasite as well as the detoxification and its defence against biological insults.

Immune response against the non-repeat region (293-310) of the circumsporozoite protein of Plasmodium vivax.

Immunization by peptides based on the repeat sequences of Plasmodium falciparum or P. vivax antigen(s) have shown inconsistent results during clinical trials in humans. This could be attributed to the lack of T-cell help or antigenic polymorphism. Thus, attention has been focused towards the more conserved non-repeat regions. The present study was undertaken to map the antigenic determinant in the vicinity of region II (outside the repeat) of CS protein of P. vivax. The immunogenicity of the peptide was studied alone and after linking with polytuftsin (PT), using alum and Freund's adjuvant, in inbred strains of mice with different genetic backgrounds. The humoral response and antigen induced T-cell proliferation assays clearly demonstrated the immunomodulatory activity of PT. Comparable results were observed with antigen(s) administered either in alum or Freund's adjuvant. The induction of IgG2a and IgG2b antibody isotypes by both, peptide as well as the conjugate, may indicate that the T-helper response involved is of Th1 type. Further the immunofluorescence studies have shown that antibodies recognized the air dried sporozoites of P. cynomolgi. The results thus show that the above sequence has overlapping B and T-cell determinants and that alum can be substituted for Freund's adjuvant in generating an effective immune response.

New lymphocyte stimulating monocot lectins from family Araceae. II.

Two lectins purified from the tubers of Arisaema consanguineum Schott (ACA) and A. curvatum Kunth (ACmA) belonging to the monocot family Araceae were mitogenic for human peripheral blood mononuclear cells (PBMC) in the [3H]-thymidine uptake assay. ACA and ACmA had an optimum stimulatory concentration of 10-25 micrograms/ml and 50-100 micrograms/ml, respectively, as observed in PBMC from five different individuals. The mitogenic response of PBMC was inhibitable in a dose-dependent manner by asialofetuin. The lectins were T-cell specific, and stimulation kinetic studies using ACA and ACmA showed that they induce maximum thymidine uptake in PBMC at day 4 and 3, respectively.

In-vitro cultivation of exoerythrocytic stages of Plasmodium cynomolgi in hepatocytes of Macaca radiata.

Hepatocytes from bonet monkey (Macaca radiata) obtained by perfusion of a liver biopsy were infected in-vitro with Plasmodium cynomolgi bastianellii sporozoites raised in Anopheles stephensi. The development of exoerythrocytic (EE) stages was seen under phase contrast microscope and by Giemsa staining. Multinucleated EE-stages were seen in the cultured hepatocytes on day 7-8 post-sporozoite inoculation.

Studies on heme and heme oxygenase during Plasmodium berghei infection in golden hamsters.

Heme and heme degrading enzymes namely heme-oxygenase (HO) and biliverdin reductase (BR) were monitored in liver and spleen during Plasmodium berghei infection in golden hamsters. There was a sequential rise in the levels of heme and HO with the rise in parasitaemia. BR was also significantly increased in these organs following infection.

Purification and properties of four monocot lectins from the family Araceae.

Four new monocot lectins from the tubers of araceous plants, namely, Arisaema consanguineum Schott (ACA), A. curvatum Kunth (ACmA) and Sauromatum guttatum Schott (SGA) from the tribe Areae, and Gonatanthus pumilus D. Don (GPA) from the tribe Colocasieae have been purified by affinity chromatography on asialofetuin-linked amino activated silica beads. These lectins possess similar physicochemical and biological properties. All the lectins gave a single peak on HPLC size exclusion and cation exchange columns, and a single band on PAGE, (pH 4.5). In SDS-PAGE, all the lectins gave a single band corresponding to a subunit of M(r) 1,3000. All the lectins yielded multiple peaks on anion-exchange column, multiple bands on non-denatured PAGE (pH 8.3) and a family of bands on isoelectric focusing. The lectins agglutinate rabbit, rat and sheep red blood cells (RBCs) but are inactive towards human ABO erythrocytes. The haemagglutination activity of these lectins is inhibited by asialofetuin only, while simple sugars/derivatives including chitin, porcine mucin and fetuin did not react. In serological studies against rabbit anti-SGA serum, all four lectins produced immunoprecipitin lines. The lectins within each tribe were identical but the lectins belonging to the tribe Areae were only partially identical to the lectins from the tribe Colocasieae.

New lymphocyte stimulating monocot lectins from family Araceae.

Three monocot lectins from underground tubers of plants belonging to the family Araceae were investigated for their mitogenic potential towards human peripheral blood lymphocytes. All the three lectins turned out to be potent mitogens in the [3H]-thymidine uptake assay. Gonatanthus pumilus lectin was mitogenic at an optimum concentration of 25 micrograms/ml while Alocasia indica and Sauromatum guttatum lectins were most effective at a concentration of 50 micrograms/ml. [3H]-thymidine incorporation studies further revealed that the lectins were T-cell mitogens and did not induce any appreciable DNA synthesis in B-enriched lymphocytes. The proliferation kinetic studies detected maximum incorporation on day 3 and the mitogenic response was shown to be inhibited by asialofetuin in a concentration-dependent manner.

Amaranthus hypochondriacus and A. tricolor lectins: isolation and characterization.

Asialofetuin-linked amino activated silica was used for the affinity purification of lectins from Amaranthus hypochondriacus Linn (AHL) and A. tricolor Linn (ATL). Like a few other Amaranthus lectins, these lectins were also inhibited by N-acetyl-D-galactosamine, fetuin and asialofetuin; they agglutinated human and different animal erythrocytes. The purified lectins yielded a single band on PAGE pH 8.3, pH 4.5 and SDS-PAGE, pH 8.3. These also gave a single peak in gel exclusion on Biogel P-200, HPLC 300 SW and cation exchange columns. However, both lectins gave multiple peaks in anion exchange column and multiple bands in isoelectric focusing. AHL and ATL are dimeric proteins in which the subunits having M(r) 29,000 and 39,000, respectively, are not held together by disulphide linkages. The pure lectins are glycoproteins and do not require Ca2+, Mn2+ and Mg2+ for their agglutination activity.

Role of calcium in biphasic immunomodulation by gamma-HCH (lindane) in mice.

gamma-HCH (Lindane) is reported to cause a biphasic immunomodulation-stimulation followed by suppression-after oral administration in mice. Role of calcium in this biphasic immunomodulation was assessed after 4, 12 and 24 wks of gamma-HCH administration. 45Ca-uptake was enhanced during the initial immunostimulation followed by decrease concomitant with immunosuppression. Lymphocyte proliferation was inhibited during both the phases of immune response by verapamil, a calcium channel blocker, and by trifluoperazine, a calmodulin inhibitor. These findings show an impairment of calcium homeostasis in lymphocytes culminating into the biphasic immunomodulatory effects of gamma-HCH.

Infectivity studies on anopheles stephensi using Plasmodium cynomolgi B infection in rhesus monkeys.

The characteristics of primary and secondary asexual peak parasitaemia during sporozoite induced Plasmodium cynomolgi B infections in 40 rhesus monkeys have been studied. Colony bred Anopheles stephensi were fed on different days on the gametocyte carrying monkeys and the infectivity of mosquitoes as determined by oocyst count on day 8 post-feeding was recorded. Following the day of sporozoite inoculation, the mean prepatent period was 8.58 +/- 0.87 days. The primary asexual peak was attained on day 8.38 +/- 2.27 and the secondary peak on day 15.15 +/- 2.63 after patency. Infectivity rate was 100% in 144 batches of mosquitoes fed three days before to three days after the secondary asexual peak. Judging from the day of patency, the high oocyst count was obtained between day 12-18 after patency. Infective gametocyte peak coincided with the secondary asexual peak parasitaemia, with the result that maximum oocyst number was observed in batches fed on the day of secondary peak or one day prior to or one day after the secondary peak. Infectivity during the period of primary asexual peak was inconsistent.

In-vitro exoerythrocytic development of Plasmodium cynomolgi bastianellii: inhibitory activity of monoclonal antibodies against sporozoites of different P. cynomolgi strains and of P. knowlesi.

The inhibitory effect of anti-sporozoite monoclonal antibodies (MoAb) on the in-vitro development of liver stages of Plasmodium cynomolgi bastianellii (NIH strain) was evaluated using primary cultures of rhesus monkey hepatocytes. MoAbs against the circumsporozoite proteins of five strains of P. cynomolgi (NIH, London, Gombak, Ceylon, Berok), and of P. knowlesi (H strain) were used. Incubation of sporozoites of P. cynomolgi bastianellii with the anti-NIH strain MoAbs entirely prevented liver-stage development; MoAbs produced against the other four strains had no apparent activity. The anti-P. knowlesi MoAbs had a partially inhibitory effect on parasite development. These functional studies complement previous immunological studies on P. cynomolgi strain specificity, and confirm the cross-reactivity observed previously between sporozoites of P. cynomolgi bastianellii and P. knowlesi (H strain).

Association of microneme antigens of Plasmodium brasilianum merozoites with knobs and other parasite-induced structures in host erythrocytes.

The localization of Plasmodium brasilianum antigens, common to merozoite micronemes and parasite-induced structures in the host erythrocyte, was determined by means of immunogold electron microscopy and monoclonal antibodies directed against blood stages of this parasite. All monoclonal antibodies reacted with micronemes. In addition, some reacted with either knob protrusions or caveolae of the host erythrocyte membrane; one reacted with a parasite-derived antigen present in the erythrocyte cytoplasm. Gold particles appeared over the membranes of ring-infected cells before the appearance of knobs and caveolae. We hypothesize that at least some knob- and caveolae-associated antigens of P. brasilianum are inserted into the erythrocyte membrane at the time of merozoite invasion.

Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies.

Monoclonal antibodies (MoAb) were produced against both salivary gland sporozoites (SGS) and oocyst sporozoites (OS) of Plasmodium gallinaceum, an avian malaria parasite. By indirect immunofluorescence, all of the MoAbs reacted with both SGS and OS of P. gallinaceum and two of the MoAbs cross-reacted weakly with P. berghei sporozoites. None of the MoAbs reacted with sporozoites of six additional species of mammalian plasmodia. In Western blot analysis of extracts of either SGS or OS of P. gallinaceum, these MoAbs identified two polypeptides with molecular weights of approximately 76,000 and 64,000 D. The results of a MoAb inhibition of binding assay and a two-site one-antibody immunoradiometric assay indicate that the circumsporozoite protein of P. gallinaceum, like those of mammalian malaria parasites, contains a repetitive immunodominant epitope. Two of the anti-P. gallinaceum MoAbs were tested in a sporozoite neutralization assay and decreased, but did not abolish, the infectivity of sporozoites for chickens, indicating that the polypeptide of P. gallinaceum identified by immunoblot is probably the protective antigen.

Membrane-associated antigens of blood stages of Plasmodium, brasilianum, a quartan malaria parasite.

The localization of Plasmodium brasilianum-derived antigens in short and long clefts within the cytoplasm of infected erythrocytes and in association with knobs of the host cell membrane was demonstrated by immunoelectron microscopy with monoclonal antibodies. Our results document that malaria-induced short and long clefts, previously distinguishable only by morphology, differ also in antigenic composition. Another parasite-derived antigen was found to be associated with the parasitophorous vacuole space in schizonts. In segmenters, this antigen was present in large amounts between merozoites and in the cytoplasm of infected cells. These antigens were characterized by biosynthetic labeling and gel electrophoresis.