HSV-1 enhances GvHR-associated parent anti-F1 alloreactivity in vivo and in vitro

The present studies were performed to demonstrate whether concurrent HSV-1 infection could enhance the immune alterations and dysfunction associated with P----F1-induced graft-versus-host reactions. Examination of phenotypic and functional parameters revealed that Gv-HR-related immune abnormalities in the (C3H.SW X H-2bm1)F1 recipient were dependent on the parental donor inoculum. Together with HSV-1 infection, virus was found to exacerbate the phenotypic changes and functional abnormalities induced in this GvHR model. In addition, the presence of concurrent HSV-1 was shown to augment the level of specific in vivo donor anti-host reactivity present in F1 recipient spleen cells. Moreover, in vitro studies demonstrated that HSV-1 also enhanced the levels of parent anti-F1 allospecific cytotoxic activity. In total, these findings support the hypothesis that viral exacerbation of GvHR is mediated by its enhancement of donor anti-host alloreactive responses.     

Suppression by cyclosporin A of murine T-cell-mediated immunity against viruses in vivo and in vitro

The immunosuppressive effect of Cyclosporin A on T-cell-mediated antiviral immune responses was examined. When administered intraperitoneally CS-A abrogated anti-vaccinia virus, anti-lymphocytic choriomeningitis virus (LCMV), and anti-vesicular stomatitis virus (VSV) T-cell responses in a dose-dependent fashion. Usually 50-60 mg/kg were efficient in suppressing primary T-cell responses completely. In contrast, 10-20 mg/kg often enhanced T-cell responses significantly when compared with controls. Suppression was observed if CS-A treatment was started before virus injection and up to 12 hr after infection; CS-A given 24 hr after the virus still suppressed T-cell activity partially. A 50 mg/kg dose of CS-A suppressed secondary anti-vaccinia virus or anti-VSV T-cell responses in vivo by a factor of about 10. This dose suppressed the primary T-cell-dependent footpad swelling induced by local LCMV infection and prevented T-cell-mediated immunopathological death due to LCM when LCMV was injected intracerebrally. In addition, clearance of LCMV was delayed drastically by CS-A treatment. When added to cultures of in vivo-primed antiviral T cells that were restimulated in vitro, CS-A inhibited both proliferation as well as generation of virus-specific cytotoxic T cells in a dose-dependent way. The results show that in CS-A-treated mice primary and secondary antiviral T-cell responses are strongly inhibited; acute viral infections with cytopathic viruses may therefore be more dramatic. In contrast immunopathological T-cell-mediated disease caused by noncytopathic viruses such as LCMV may be prevented or attenuated.     

Comparison of in vivo and in vitro SCE induction

In vivo and in vitrod sister-chromatid exchange (SCE) induction and cell replication kinetics were compared in P388 cells exposed to 4 mutagens. While concordance was observed between SCE induction and inhibition of cell replication kinetics, certain mutagens were more potent in vivo while others were more potent in vitro. These results indicate that caution should be applied before equating in vivo and in vitro mutagen exposures.     

The effect of cyclosporin A on Hg2+ -poisoning mitochondria. In vivo and in vitro studies

The protective effect of cyclosporin A on the damage induced by Hg²⁺ in kidney mitochondria was studied. Cyclosporin, added in vitro at a concentration of 0.5 μM, reversed the deleterious effects of Hg²⁺ on transmembrane potential and Ca²⁺ accumulation. However, when injected in rats, together with Hg²⁺, cyclosporin failed to protect against Hg²⁺ poisoning. Due to the low activity of cyclophilin found in kidney mitochondria, it is proposed that the protection of cyclosprin in vitro must be extered through an independent mechanism different from its binding to cyclophilin.     

Studies on the antibacterial effects of statins--in vitro and in vivo

Background

Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections.

Aim

The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract.

Methods

MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert).

Findings

The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria.

Conclusions

Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.     

Suppressive effect of the blast cells of AKR strain mice on antibody formation in vivo and lymphocyte proliferation in vitro

Blast cells obtained from the "erythropoietic spleen" of FG-stimulated young mice and cells accumulating in the spleens of preleukemic AKR mice have a marked suppressive effect on spontaneous and mitogen-induced proliferation of young mouse splenocytes in vitro and suppress the development of humoral immune response in immunized recipients during syngeneic transfer in vivo. Some disturbances in erythron system in preleukemic AKR mice manifested in the accumulation of immature erythroid precursors which are suppressors of immunocompetent lymphocytes are suggested to be a pathogenetic link in the development of leukemia.     

Suppressive effects of co-stimulatory molecule expressions on mouse splenocytes by anti-allergic agents in vitro

The influence of anti-allergic drugs, epinastine hydrochloride (EP) and disodium cromoglycate (DSCG), on the co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with haemocyanin absorbed to aluminium hydroxide were cultured in the presence of 100.0 microg/ml haemocyanin and various concentrations of the agents. Low concentrations (<1.5 x 10(-4)M) of EP and DSCG did not influence spleen cell blastic activity induced by antigenic stimulation, whereas these agents caused significant inhibition of spleen cell activation when 2 x 10(-4) M of the agents were added to cell cultures. EP and DSCG also did not affect blastic activity of sensitized splenic T cells by anti-CD3 monoclonal antibody stimulation even when these cells were cultured in the presence of 2 x 10(-4) M of the agents. We next examined the influence of EP and DSCG on the expression of co-stimulatory molecules on spleen cells in response to antigenic stimulation. Sensitized spleen cells were cultured in the presence of 2 x 10(-4)M of the agents and the expression of molecules were examined by flow cytometer 24h later. EP and DSCG suppressed the expression of costimulatory molecules, CD40 and CD80, but not CD86, on splenic B cells which were enhanced by antigenic stimulation in vitro.     

Suppressive effect of cyclosporin A on the development of Leishmania tropica-induced lesions in genetically susceptible BALB/c mice

The effect of cyclosporin A (CyA) application on the development of cutaneous lesions was analyzed in genetically susceptible BALB/c mice infected s.c. with Leishmania tropica promastigotes. Daily i.p. injections of CyA, beginning 2 days before or at the day of the infection, dose dependently inhibited the development of parasite-induced lesions; no effect on the lesions was observed, however, if CyA application was started 14 days after the infection. Cessation of CyA administration after having successfully suppressed the cutaneous lesions for a period of 42 days, resulted in the development of lesions within 3 days. CyA had no inhibitory effect on lesions developing in L. tropica infected hypothymic BALB/c nu/nu mice. CyA or CyA-containing mouse serum did not directly affect the viability and the growth rate of L. tropica promastigotes, suggesting that the effect of the agent was imposed on the cells participating in the formation of the cutaneous lesions. Quantitative analysis of the cell distribution in the spleens of infected mice revealed that CyA markedly suppressed the infection-associated numerical increase of splenocytes. Within the Thy-1+ lymphocyte compartment, CyA had its most pronounced effect on the Lyt-1+ T lymphocyte subset. Early in the disease, the frequency of splenic cells proliferating in response to L. tropica antigen in vitro was clearly inhibited by CyA; in the later stages of the infection, however, this effect could not be observed, indicating the presence of L. tropica-inducible T cells being relatively resistant to CyA. Taken together, our findings indicate that CyA reversibly inhibits or delays the parasite-induced expansion of Lyt-1+ splenic T lymphocytes, and thus suppresses the biological function of those T cells that are instrumental for the formation of cutaneous lesions in L. tropica-infected BALB/c mice.     

Suppressive activity of a macrolide antibiotic, roxithromycin, on pro-inflammatory cytokine production in vitro and in vivo

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro-inflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL-1beta and TNF-alpha contents by enzyme-linked immunosorbent assay. RXM suppressed the in vitro production of IL-1beta and TNF-alpha in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 microg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL-1beta and TNF-alpha in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5-12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL-1beta and TNF-alpha in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.     

IL-4 bypasses the immune suppressive effect of cyclosporin A during the in vitro induction of murine cytotoxic T lymphocytes

We have analyzed at the clonal level the effect of IL-4 on the immune suppressive action of cyclosporin A (CsA) during the in vitro primary activation of anti-MHC alloantigen-reactive murine CD8+ CTL. Although neither IL-4 nor IL-2 alone were able to overcome the CsA-mediated suppression, the addition of IL-4 in the presence of IL-2 restored in a dose-dependent manner the induction of cytolytic activity. On the other hand, CsA greatly impaired proliferative responses of alloantigen-reactive CD8 T cells, thereby operationally dissecting proliferative responsiveness from acquisition of cytolytic activity during primary activation of alloantigen-reactive CD8+ T cells. The existence of a CsA-resistant induction pathway for Ag-specific CD8+ T cell-mediated cytolytic activity may be of relevance for experimental and clinical organ transplantation.     

Suppressive effect of Withania somnifera root powder on experimental gouty arthritis: An in vivo and in vitro study

The effect of Withania somnifera L. Dunal root powder on paw volume and serum lysosomal enzyme activities was investigated in monosodium urate crystal-induced rats. The levels of beta-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal incubated polymorphonuclear leucocytes (PMNL). A significant increase in the level of paw volume and serum lysosomal enzymes was observed in monosodium urate crystal-induced rats. The increased beta-glucuronidase and lactate dehydrogenase level were observed in untreated monosodium urate crystal incubated polymorphonuclear leucocytes. On treatment with the W. somnifera root powder (500/1000 mg/kg body weight), the above changes were reverted back to near normal levels. W. somnifera also showed potent analgesic and antipyretic effect with the absence of gastric damage at different dose levels in experimental rats. For comparison purpose, non-steroidal anti-inflammatory drug (NSAID) indomethacin was used as a standard. These results provide evidence for the suppressive effect of W. somnifera root powder by retarding amplification and propagation of the inflammatory response without causing any gastric damage.     

GRP78 induction by cyclosporin A in human HeLa cells

Immunosuppressive drugs such as cyclosporin A (CsA) and FK506 are known to have pleiotropic effects on cells. Here we demonstrate that treatment of HeLa cells with low concentrations of CsA (but not of FK506) induces the synthesis of a stress protein, GRP78, located inside the endoplasmic reticulum. High concentrations of CsA lead to a general decrease in protein synthesis. When cells are stressed (heat-shocked) during the CsA treatment, the synthesis of heat shock proteins is reinforced. FK506 has no detectable effects at any concentration. The mechanism of induction of GRP78 by CsA remains presently unknown. Whatever the mechanism involved, GRP78 overexpression might be responsible for some of the physiological effects of CsA.     

Suppressive effects of soluble histocompatibility antigens on the in vitro generation of cytotoxic T cells to D-end alloantigens

Murine histocompatibility antigens were solubilized from the spleens and lungs of C57BL/6 (H-2b) animals with hypertonic salt (3 M KC1). Aggregate-free soluble antigens were incubated with nonadherent lymph node cells from BALB/c (H-2d) mice for 18 hr prior to their use as responder cells in the mixed-lymphocyte reaction (MLR). It was found that the generation of cytotoxic cells was suppressed while the proliferative response was not affected. The observed suppression was not due to a shift in the kinetics of the generation of cytotoxicity as determined throughout a 10-day culture period. The suppression was specific in that the response in MLR to unrelated H-2f stimulator cells and the subsequent generation of cytotoxic cells were unchanged. Using various H-2 recombinant strains as target cells in the assay of cell-mediated lympholysis, suppression of cytotoxicity was observed when the D end, but not the K end, was shared with the C57BL/6 strain from which the antigens were derived.