Characterization of granuloma T lymphocyte function from Schistosoma mansoni-infected mice

This study was undertaken to characterize and compare T lymphocyte function from the vigorous and modulated liver granulomas of Schistosoma mansoni-infected mice. Although both types of lesion contained equal percentages of T lymphocytes, the T cell subset distribution was different. For vigorous lesions, the ratio of helper/effector to suppressor/cytotoxic T cells was 2 to 3:1. For modulated lesions the ratio was lower (1:1). Differences in the phenotypic profiles of vigorous and modulated granuloma (Gr) T cells were reflected in their functional activity. Vigorous Gr T cells were more active in lymphoproliferation, IL-2 production, and granuloma formation than those from modulated lesions. Moreover, modulated Gr T cells suppressed the functional activity of vigorous Gr T cells in a dose-dependent manner. The selective depletion of T cell subsets showed that phenotypically, the Gr delayed-hypersensitivity T cell is L3T4+, Lyt-1+ whereas the Gr Ts cell is an Lyt-2+ lymphocyte. Both of these T cell subsets are present in vigorous and modulated lesions. During acute infection, delayed-hypersensitivity T cell lymphocyte functions predominate, whereas Ts lymphocyte functions appear to prevail during chronic infection.     

Modulation of granulomatous hypersensitivity. I. Characterization of T lymphocytes involved in the adoptive suppression of granuloma formation in Schistosoma mansoni-infected mice.

The cellular basis of the spontaneous modulation of the granulomatous response to schistosome eggs was analyzed in mice infected with Schistosoma mansoni. Spleen cells of 20 or 32 week-infected mice undergoing modulation, when transferred to 6 week-infected recipients, suppressed the maximal granulomatous response at 8 weeks. Suppression of both naturally forming asynchronous liver and synchronously induced lung lesions was achieved. Specificity of this effect was demonstrated by the suppression of egg granulomas but not antigen-coated bead granulomas developing simultaneously in the lungs of cell recipients. Further characterization showed that suppression was abrogated by pretreating transferred cells with either anti-Thy 1.2 or anti-Iak alloantisera and C. Transfer of macrophage-depleted or fractionated T and B spleen cells confirmed that T cells alone transferred suppression. Moreover, an Ia antigen-bearing (Ia+) subpopulation of T cells was required in the transferred suppression. Moreover, an Ia antigen-bearing (Ia+) subpopulation of T cells was required in the transferred population. An examination of T cells obtained from isolated, dispersed lung granulomas from control and adoptively suppressed mice revealed an increased proportion of Ia+ cells in the latter. It is suggested that Ia+ T cells may be involved in the local modulation of the granulomatous response.     

Strain dependence of muramyl dipeptide-induced LAF(IL 1) release by murine-adherent peritoneal cells

The capacity of MDP to stimulate LAF(IL 1) production by adherent peritoneal cells (APC) from different strains of mice was examined. It was observed that MDP could stimulate thioglycollate-induced APC from DBA/2, C57B1/6, and CBA/CA mice, but not from C3H mice. Resident APC from DBA/2 or C3H/HeJ mice were also responders and nonresponders, respectively, to MDP for LAF(IL 1) production, the positive effect of MDP being more marked on DBA/2 cells in the presence of indomethacin. Because a LPS high-responder C3H substrain (C3HeB/Fe) did not respond to MDP, it was concluded that the strain dependence with respect to the induction of LAF(IL 1) seen with both MDP and LPS was not linked. Moreover, the unresponsiveness of C3H mice to MDP was not linked to their MHC haplotype, because a second H-2k strain (CBA/CA) was a good responder to MDP stimulation.     

Schistosoma mansoni: schistosomulicidal activity of macrophages isolated from liver granulomas of infected mice

Mice infected with Schistosoma mansoni develop T cell-mediated granulomatous reactions around disseminated parasite eggs. In this study, granuloma-derived leucocytes have been examined for schistosomulicidal capacity by the use of in vitro cytotoxicity assays. Adherent macrophages, that were shown by electron microscopy to exhibit no gross morphological abnormalities, were unable to mediate significant mortality in the absence of serum factors. When cocultured with immune serum and complement, however, these cells killed +/- 26% of the larvae at a cell:target ratio of 5000:1. In contrast, granuloma-derived cell populations that were enriched for eosinophils (50-70% eosinophil content) showed only minimal cytotoxic potential. This may be related to observed structural changes in the eosinophil lysosomal granules, or perhaps to blocking of the cell-surface receptors by immune complexes. It is concluded that granuloma macrophages, activated by egg antigen-sensitised T lymphocytes, may serve as effector cells in immunity to schistosomules.     

Changes in the expression of cytochrome P450 isozymes and related carcinogen metabolizing enzyme activities in Schistosoma mansoni-infected mice

Mixed-function oxidase enzymes metabolize most xenobiotic agents. Western blotting was used to investigate the effect of Schistosoma mansoni infection on the expression of various cytochrome P450 (CYP) isozymes and specific enzyme assays to study related metabolic functions in mouse liver microsomes. Male BK-TO mice were infected with 200 cercariae per mouse and their livers were assayed at 6, 15, 30 and 45 days post-infection (p.i.) and compared with appropriately matched controls. The expression of each of the CYP isozymes (1A1, 2B1/2, 2C6, and 4A) was either unaffected or transiently increased up to 30 days post-infection. By 45 days, a significant loss of signal was observed, particularly for CYP 1A1 and 2B1 /2 where no signal could be detected. Evidence supporting these findings was obtained from enzyme assays specific for particular CYP isozymes. The activity of ethoxyresorufin O-deethylase (CYP 1A1) was reduced by 97% and that of pentoxyresorufin O-depentylase (CYP 2B1 /2) by 96% at 45 days p.i. Similarly, the activity of ethoxycoumarin hydroxylase was progressively reduced over the period under study. It is believed that N-nitrosamines are activated principally by N-nitrosodimethylamine N-demethylase I which was significantly increased at both 30 and 45 days p.i. To further investigate metabolic competency following S. mansoni infection, the in vitro binding of benzo(a)pyrene metabolites to DNA was measured, using isolated liver microsomes to activate benzo(a)pyrene. Benzo(a)-pyrene-DNA adduct formation was markedly increased at 6,15 and 30 days with a maximum at 15 days, but decreased at 45 days p.i. It was concluded that S. mansoni infection changes the expression of different CYP isozymes and also the activity of phase I drug-metabolizing enzymes at different periods of infection and may thus change the liver's capacity to activate or detoxify many endogenous and exogenous compounds. Such alterations may also change the therapeutic actions of drugs that are primarily metabolized by the P450 system, when administered to patients with schistosomiasis.     

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.     

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.     

Characterization of murine interferon-alpha 12 (MuIFN-alpha12): biological activities and gene expression

Interferon alpha (IFN-alpha) belongs to the type I interferon family and consists of multiple subtypes in many species. In the mouse, there are at least 14 IFN-alpha genes and 3 IFN-alpha pseudogenes, the most recently identified of which are murine interferon-alpha 12 (MuIFN-alpha12), MuIFN-alpha13 and MuIFN-alpha14. To further study the biological activities of MuIFN-alpha12, we have produced a recombinant MuIFN-alpha12 (rMuIFN-alpha12) protein using COS-1 cells. rMuIFN-alpha12 was found to inhibit the growth of murine myeloid leukemia JCS cells. Flow cytofluorometric analysis with propidium iodide staining showed that the growth inhibitory activity of rMuIFN-alpha12 may be caused by the induction of apoptosis. Flow cytofluorometric analysis also revealed that rMuIFN-alpha12 was able to up-regulate the expression of MHC-I on both JCS cells and primary macrophages. Functional studies indicated that a MuIFN-alpha12 transgene could induce an anti-viral state in L929 cells against Influenza A virus. Moreover, expression of MuIFN-alpha12 was not detectable by RT-PCR in untreated, Influenza A virus infected, polyI:polyC induced L929 cells, or in a wide range of normal murine tissues. Taken together, this data shows that MuIFN-alpha12 is a protein with all the biological traits of a type I IFN.     

JQ-1 ameliorates schistosomiasis liver granuloma in mice by suppressing male and female reproductive systems and egg development of Schistosoma japonicum

Schistosomiasis is a serious and widespread parasitic disease caused by infection with Schistosoma. Because the parasite’s eggs are primarily responsible for schistosomiasis dissemination and pathogenesis, inhibiting egg production is a potential approach to control the spread and severity of the disease. The bromodomain and extra-terminal (BET) proteins represent promising targets for the development of epigenetic drugs against Schistosoma. JQ-1 is a selective inhibitor of the BET protein family. In the present study, JQ-1 was applied to S. japonicum in vitro. By using laser confocal scanning microscopy and EdU incorporation assays, we showed that application of JQ-1 to worms in vitro affected egg laying and the development of both the male and female reproductive systems. JQ-1 also inhibited the expression of the reproductive-related genes SjPlk1 and SjNanos1 in S. japonicum. Mice infected with S. japonicum were treated with JQ-1 during egg granuloma formation. JQ-1 treatment significantly reduced the size of the liver granulomas and levels of serum alanine aminotransferase and aspartate aminotransferase in mice and suppressed both egg laying and the development of male and female S. japonicum reproductive systems in vivo. Moreover, the mRNA expression levels of some proinflammatory cytokines were decreased in the parasites. Our findings suggest that JQ-1 treatment attenuates S. japonicum egg–induced hepatic granuloma due at least in part to suppressing the development of the reproductive system and egg production of S. japonicum. These findings further suggest that JQ-1 or other BET inhibitors warrant additional study as a new approach for the treatment or prevention of schistosomiasis.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Schistosoma mansoni: suppression of carbamoyl phosphate synthetase (ammonia) and ornithine carbamoyltransferase activities in the liver of infected mice

ICR female mice infected with cercariae of Schistosoma mansoni exhibited a significant decrease in both total and specific activities of carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine carbamoyltransferase (EC 2.1.3.3), and also in the serum urea level. Intraperitoneal administration of the S. mansoni egg granulomas or 15,000g X 30 min supernatant fluid of their extract into the uninfected, normal mice also significantly decreased the total and specific activities of both enzymes without any appreciable histopathological influence on their livers. S. mansoni viable eggs caused a significant decrease in the total and specific activities of carbamoyl phosphate synthetase (ammonia) alone as well as active intraperitoneal inflammation when inoculated into the normal mice by the same route. There was no difference in the amount of food intake between the control and these experimental mice. These findings suggest that the granuloma or inflammatory cells induced by schistosome eggs produce some factor(s) which may be responsible for reduction of these enzymatic activities in experimental schistosomiasis mansoni.     

Species-specificity of T cell stimulating activities of IL 2 and BSF-1 (IL 4): comparison of normal and recombinant, mouse and human IL 2 and BSF-1 (IL 4)

Mouse and human interleukin 2 (IL 2) both cause proliferation of T cells of the homologous species at high efficiency. Human IL 2 also stimulates proliferation of mouse T cells at similar concentrations, whereas mouse IL 2 stimulates human T cells at a lower (sixfold to 170-fold) efficiency. In contrast, the T cell stimulating activities of mouse and human B cell stimulatory factor 1 (interleukin 4; IL 4) appear to be species specific over the range of concentrations tested; we detected no activity of mouse IL 4 on human T cells, or human IL 4 on mouse T cells.     

Interactive sites in the MyD88 Toll/interleukin (IL) 1 receptor domain responsible for coupling to the IL1beta signaling pathway

Myeloid differentiation factor MyD88 is the essential adaptor protein that integrates and transduces intracellular signals generated by multiple Toll-like receptors including receptor complex for interleukin (IL) 1beta, a key inflammatory cytokine. IL1beta receptor complex interacts with MyD88 via the Toll/IL1 receptor (TIR) domain. Here we report structure-function studies that help define the MyD88 TIR domain binding sites involved in IL1beta-induced protein-protein interactions. The MyD88 TIR domain, employed as a dominant negative inhibitor of IL1beta signaling to screen MyD88 TIR mutants, lost its suppressing activity upon truncation of its Box 3. Accordingly, mutations of Box 3 residues 285-286 reversed the dominant negative effect of the MyD88 TIR domain on IL1beta-induced and NFkappaB-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helix alphaA, 195-197 in Box 2, and 275 in betaE-strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and IL1 receptor accessory protein (IL1RAcP), whereas substitution of neighboring canonical Pro200 by His was without effect. Mutations in Box 2 and 3 prevented homotypic MyD88 oligomerization via TIR domain. Based on this structure-function analysis, a three-dimensional docking model of TIR-TIR interaction between MyD88 and IL1RAcP was developed.     

Characterization of integrin beta6 and thrombospondin-1 double-null mice

To identify overlapping and non-overlapping functions for TSP-1 and alphavbeta6, we crossed TSP-1-null and beta6-null mice and compared the phenotype of the double-null mice with those of wild-type and single-null mice. The double-null mice exhibited focal acute and organizing pneumonia that was more severe than the wild-type and single-null mice as well as a significantly higher incidence of inflammation in tissues other than the lung. The TSP-1-null and beta6-null mice exhibited a five to eight-fold increase in granulocyte recruitment to the lung three days after exposure to lipopolysaccharide. They also had abnormalities that were infrequently observed in the wild-type and single-null mice, including heart degeneration (8.35% in wild-type and 28.1% in double-null mice), hyperplasia of the glandular of the stomach (2.8% in wild-type and 21.1% in double-null mice) and endometrial hyperplasia (0% in wild-type and 38.5% in double-null females). Furthermore, the beta6-null and double-null mice displayed a significant elevation in benign and malignant cancers. Stomach papillomas, squamous cell carcinomas of the ear and stomach, and adenocarcinomas of the lungs, vagina/cervix and colon were observed with the highest frequency. These data demonstrate that TSP-1 and alphavbeta6 are involved in regulation of the immune system and epithelial homeostasis. They also indicate that alphavbeta6 functions as a tumor suppressor gene and that activation of TGFbeta by TSP-1 and alphavbeta6 contributes to normal tissue architecture and function.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

(Xeno)estrogen sensitivity of smooth muscle BK channels conferred by the regulatory beta1 subunit: a study of beta1 knockout mice

Estrogen and xenoestrogens (i.e. agents that are not steroids but possess estrogenic activity) increase the open probability (P(o)) of large conductance Ca(2+)-activated K(+) (BK) channels in smooth muscle. The mechanism of action may involve the regulatory beta1 subunit. We used beta1 subunit knockout (beta1-/-) mice to test the hypothesis that the regulatory beta1 subunit is essential for the activation of BK channels by tamoxifen, 4-OH tamoxifen (a major biologically active metabolite), and 17beta-estradiol in native myocytes. Patch clamp recordings demonstrate BK channels from beta1-/- mice were similar to wild type with the exception of markedly reduced Ca(2+)/voltage sensitivity and faster activation kinetics. In wild type myocytes, (xeno)estrogens increased NP(o) (P(o) x the number of channels, N), shifted the voltage of half-activation (V(12)) to more negative potentials, and decreased unitary conductance. These effects were non-genomic and direct, because they were rapid, reversible, and observed in cell-free patches. None of the (xeno)estrogens increased the NP(o) of BK channels from beta1-/- mice, but all three agents decreased single channel conductance. Thus, (xeno)estrogens increase BK NP(o) through a mechanism involving the beta1 subunit. The decrease in conductance did not require the beta1 subunit and probably reflects an interaction with the pore-forming alpha subunit. We demonstrate regulation of smooth muscle BK channels by physiological (steroid hormones) and pharmacological (chemotherapeutic) agents and reveal the critical role of the beta1 subunit in these responses in native myocytes.     

Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (Sephadex bead)-type granuloma macrophages. Evidence for sequential production of IL-1 and tumor necrosis factor

The present study examined the potential role of IL-1 and TNF in granuloma formation. Mice were given i.v. injections of Schistosoma mansoni eggs or Sephadex beads to induce synchronous immune T cell-mediated (hypersensitivity type) or nonimmune (foreign-body type) granulomas, respectively. Granuloma macrophages isolated at 2, 4, 8, 16, and 32 days of granuloma formation were evaluated for their capacity to produce IL-1 and TNF in response to 1 microgram/ml LPS. This was related to circulating levels of the acute phase protein, serum amyloid P (SAP) and expression of Ia Ag by monocytes and macrophages. Macrophages from nonimmune bead lesions were generally weak producers of IL-1 and TNF. In contrast, those from T cell-mediated egg lesions produced significant levels of both monokines. Moreover, there was a clear pattern of sequential monokine production such that IL-1 was produced in greatest amounts early (2 to 4 days), whereas TNF was produced later (8 to 16 days). Levels of SAP showed an initial sharp rise following particle embolization, then decreased rapidly in bead injected animals. However, mice with immune granulomas showed a prolonged elevation in SAP levels that corresponded to the period of maximal IL-1 production (2 to 4 days). Macrophage/monocyte Ia Ag expression was greatest at 8 to 16 days, corresponding to the period of TNF production. Bead injected animals showed low levels of Ia expression over the study period. These findings suggest that IL-1 may be important in the early recruitment stages of granuloma formation while TNF may take part in later maintenance or effector functions. The extent of production of both is likely influenced by the local or systemic milieu of lymphokines.     

Expression of mRNA for interleukin-1beta, interleukin-6, tumor necrosis factor-alpha and macrophage migration inhibitory factor in HPA-axis tissues in Schistosoma mansoni-infected baboons (Papio cynocephalus)

Cytokines may regulate the function of the hypothalamic-pituitary-adrenal axis during schistosomiasis. This possibility was investigated in baboons experimentally infected with Schistosoma mansoni. Serum levels of corticotrophin-releasing hormone, adrenocorticotrophin, cortisol and dehydroepiandrosterone were confirmed to be decreased in infected baboons as previously shown. To explore if this effect is associated with specific expression of cytokines with endocrine activity, and are also associated with the pathology of the disease, Northern blots for interleukin-1beta, interleukin-6, tumor necrosis factor-alpha and macrophage migration inhibitory factor in hypothalamic-pituitary-adrenal axis tissues were performed. Infection induced interleukin-1beta gene expression in the hypothalamus, while interleukin-6 and migration inhibitory factor mRNAs were induced only in the pituitary and adrenal glands. Tumor necrosis factor-alpha gene expression was induced in the hypothalamus and the pituitary gland. Histopathological analysis of the hypothalamic-pituitary-adrenal axis tissues in infected and control baboons revealed no morphological differences between them. These results suggest that specific cytokines expressed in hypothalamic-pituitary-adrenal axis tissues could regulate hormone secretion during schistosomiasis.     

Characterization of beta 1----4 galactosyltransferase purified from rat liver microsomes.

beta 1----4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound beta 1----4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53,000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse beta 1----4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the NH2-terminal 12 amino acids from a precursor polypeptide.     

Characterization of potassium channels in pancreatic beta cells from ob/ob mice

The patch-clamp technique in the cell-attached mode was used to study the K channels present in the membrane of cultured pancreatic beta cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP-sensitive potassium channel from normal beta cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage-activated potassium channels described in normal rodent beta cells. Thus, potassium channels in ob/ob pancreatic beta cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability.