Specificity of anti-saliva immune response in mice repeatedly bitten by Phlebotomus sergenti.

Sand flies are bloodsucking insects transmitting parasites of genus Leishmania, the causative agents of diseases in humans and dogs. Experimental hosts repeatedly exposed to sand fly saliva can control Leishmania infection. Cell-mediated anti-saliva immune response is most likely responsible for this protective effect; however, there is no study so far concerning its antigenic specificity towards different sand fly vectors. In this study, splenocytes from BALB/c mice repeatedly exposed to the bites of Phlebotomus sergenti were challenged ex vivo with salivary gland homogenates from three different sand fly vectors -P. sergenti, P. papatasi, or P. arabicus. Mice bitten by P. sergenti had higher proliferative response to homologous antigen than splenocytes from naive mice. Splenocytes from P. sergenti bitten mice as well as anti-P. sergenti antibodies partially cross-reacted with P. papatasi saliva. In contrast, no cross-reactivity was found with P. arabicus saliva. Our data indicate that both arms of the immune system, cellular and humoral, react in a species-specific manner. Therefore, the presence of antibodies against salivary components of a certain species indicates the specificity of cell-mediated immune response as well. The data suggest that unique transmission-blocking vaccine would be required for each vector -Leishmania combination.

On the role of interleukin-10 in the induction and maintenance of specific transplantation tolerance.

The role of a cytokine synthesis inhibitory factor, interleukin-10 (IL-10), in the induction and maintenance of neonatal transplantation tolerance was studied in mice. We showed that neonatal spleen cells (NSC) significantly inhibited interleukin-2 (IL-2) production by activated T cells from adult mice. Simultaneously we demonstrated a high expression of the IL-10 gene in stimulated spleen cells from newborn mice. However, neutralizing monoclonal antibody (mAb) anti-IL-10 did not abolish the NSC-mediated suppression of IL-2 production. IL-10, therefore, does not appear to be the principal inhibitory molecule responsible for the suppression of IL-2 production. Similarly, specific alloantigen-activated spleen cells from adult tolerant animals were profoundly hyporeactive in IL-2 production. This hyporeactivity was not reversed to a positive reactivity in the presence of mAb anti-IL-10. In addition, anti-IL-10 antibody enhanced proliferation in mixed lymphocyte cultures of cells from both control and tolerant animals, but the antibody did not abrogate specific hyporeactivity of cells from tolerant mice. These results thus showed that newborn animals were nonspecifically and tolerant animals specifically deficient in IL-2 production, but that IL-10 in neither case appeared to be responsible for this IL-2 hyporeactivity.

Induction of specific cell-mediated cytoxicity in vitro by means of RNA isolated from allografted mice.

Ribonucleic acid extracts were isolated by cold phenol extraction procedure from lymphoid organs of mice immunized with allogeneic skin grafts. Cultivation of lymphocytes from normal mice with these RNA extracts in vitro led to the induction of specific immunological reactivity which was determined by microcytotoxicity assay. Pretreatment of the RNA extracts with DNase enhanced their ability to induce cytotoxicity, while pronase had no effect, and RNase completely inhibited the ability of extracts to sensitize lymphocytes. The results show that specific cell-mediated cytotoxicity can be induced in vitro in lymphocytes from normal animals by a transfer of "immune" RNA.

Transfer of immunological tolerance by means of RNA.

Ribonucleic acid extracts were isolated from the lymph nodes and spleens of mice bearing tolerated skin allografts for long periods after neonatal tolerance induction. Lymphoid cells from control, untreated mice were incubated in vitro with these RNA extracts and then transferred into normal newborn mice. Skin allografts applied to the RNA-treated animals tn adult life survived significantly longer when compared with the non-treated mice or mice treated with RNA extracted from normal, non-tolerant mice. The results indicate that RNA from tolerant animals, like suppressor cells, is effective in prolonging skin allograft survival.

Isolation, partial characterization and translation of mRNAs for chymosin and pepsin, the two main aspartyl proteinases of bovine stomach.

Poly(A) RNA from the mucosa of the fundal region of the fourth stomach of suckling calf and adult cattle was isolated by the phenol or guanidine thiocyanate procedure. The mRNAs for chymosin and pepsin were present in the 15S fraction of poly(A) RNA. They were active both in cell-free translation systems and in oocytes of Xenopus laevis and directed the synthesis of either chymosin or pepsin precursor, depending upon the age of the donor animal. In the reticulocyte and wheat germ system only preprochymosin or prepepsinogen were synthesized. In the oocyte system only the synthesis and secretion of prochymosin or pepsinogen could be detected. Both proenzymes, prochymosin and pepsinogen, present in oocytes or secreted into the medium, were converted to active enzymes, chymosin and pepsin, respectively, at pH 3.0, as shown by their proteolytic and milk-clotting activity.

Lack of synthesis of pentamer IgM in Xenopus oocytes after injection of poly(A)+ RNA from hybridoma cells.

Total poly(A)+ RNA was isolated from B cell hybridomas producing monoclonal antibodies of the IgM class specific for sheep red blood cells. These RNAs were microinjected into Xenopus oocytes and the translation products were analyzed. Unlike the hybridoma cells, the oocytes synthetized only the light chains and oligomer IgM, but evidence for assembly of functional pentamer IgM was not obtained. The possible reasons for these differences are discussed.

Molecular cloning and identification of cDNA recombinants coding for bovine prochymosin in E. coli.

Poly(A) RNA was isolated from the gastric mucosa of the bovine fourth stomach (the abomasum) using and analysing several calves not older than 12 days. The amount of the preprochymosin mRNA in the mucosa of those animals at best reaches about 5-10% of the poly(A) RNA as estimated by in vitro translation and immunoprecipitation. Starting from that material double-stranded complementary DNA was synthesized, inserted by dG dC tailing into the PstI site of the vector plasmid pBR322 and used for transformation of E. coli. Tetracycline resistant clones containing DNA sequences coding for the full length of prochymosin were recognized by colony hybridization with five specific d-oligonucleotides corresponding either to the N-terminal, the middle or the C-terminal part of prochymosin. Six recombinants were detected by screening of 1 500 recombinants with an oligonucleotide which corresponds to positions 649 to 663 of the nucleotide sequence published by Harris et al. (1982). Two of them were found to cover together the complete prochymosin sequence as evidenced by both positive colony hybridization with either the N-terminal or the C-terminal oligonucleotide probe, as well as by the restriction pattern of the selected plasmids.

[Repair of mitochondrial DNA after x-ray irradiation of teleost fish loach Misgurnus fossillis eggs]. / Reparatsiia mitokhondrial'noi DNK posle rentgenovskogo oblucheniia iaitsekletok v'iuna Misgurnus fossilis.

X-ray irradiation of loach Misgurnus fossilis mature eggs induces breaks in mtDNA molecules with efficiency about 100 ev per break. The yield of damaged molecules of mtDNA reduces after fertilization or activation of irradiated eggs and subsequent incubation. Two-fold reduction in the relative amount of broken DNA molecules in mitochondria is observed after an 6-7 hour incubation. X-ray irradiation largely accelerates mtDNA synthesis in eggs. Activation of mtDNA synthesis is observed in whole irradiated eggs and in mitochondria isolated from them as well. Appearance of nascent DNA in the broken mtDNA molecules indicates the repair nature of mtDNA synthesis induced by X-irradiation.

Leishmaniasis: prevention, parasite detection and treatment.

Leishmaniasis remains a public health problem worldwide, affecting approximately 12 million people in 88 countries; 50 000 die of it each year. The disease is caused by Leishmania, obligate intracellular vector-borne parasites. In spite of its huge health impact on the populations in vast areas, leishmaniasis is one of the most neglected diseases. No safe and effective vaccine currently exists against any form of human leishmaniasis. The spectrum and efficacy of available antileishmanial drugs are also limited. First part of this review discusses the approaches used for the vaccination against leishmaniasis that are based on the pathogen and includes virulent or attenuated parasites, parasites of related nonpathogenic species, whole killed parasites, parasites' subunits, DNA vaccines, and vaccines based on the saliva or saliva components of transmitting phlebotomine vector. Second part describes parasite detection and quantification using microscopy assays, cell cultures, immunodetection, and DNA-based methods, and shows a progress in the development and application of these techniques. In the third part, first-line and alternative drugs used to treat leishmaniasis are characterized, and pre-clinical research of a range of natural and synthetic compounds studied for the leishmanicidal activity is described. The review also suggests that the application of novel strategies based on advances in genetics, genomics, advanced delivery systems, and high throughput screenings for leishmanicidal compounds would lead to improvement of prevention and treatment of this disease.

Genetics of susceptibility to leishmaniasis in mice: four novel loci and functional heterogeneity of gene effects.

Symptoms of human leishmaniasis range from subclinical to extensive systemic disease with splenomegaly, hepatomegaly, skin lesions, anemia and hyperglobulinemia, but the basis of this variation is unknown. Association of progression of the disease with Th2 lymphocyte response was reported in mice but not in humans. As most genetic studies in Leishmania major (L. major)-infected mice were restricted to skin lesions, we analyzed the symptomatology of leishmaniasis in mice by monitoring skin lesions, hepatomegaly, splenomegaly and seven immunological parameters. We detected and mapped 17 Leishmania major response (Lmr) gene loci that control the symptoms of infection. Surprisingly, the individual Lmr loci control 13 different combinations of pathological and immunological symptoms. Seven loci control both pathological and immunological parameters, 10 influence immunological parameters only. Moreover, the genetics of clinical symptoms is also very heterogeneous: loci Lmr13 and Lmr4 determine skin lesions only, Lmr5 and Lmr10 skin lesions and splenomegaly, Lmr14 and Lmr3 splenomegaly and hepatomegaly, Lmr3 (weakly) skin lesions, and Lmr15 hepatomegaly only. Only two immunological parameters, IgE and interferon-gamma serum levels, correlate partly with clinical manifestations. These findings extend the paradigm for the genetics of host response to infection to include numerous genes, each controlling a different set of organ-specific and systemic effects.

Modulation of murine cellular immune response and cytokine production by salivary gland lysate of three sand fly species.

Saliva of sand flies (Diptera: Phlebotominae) plays an important role in transmission of Leishmania parasites by modulating host immune response. However, because of the different protein compositions of saliva, the immunomodulatory effects may vary among sand fly species. We have therefore analysed and compared the immunomodulation effects of salivary gland lysate (SGL) of three different sand flies. Spleen cells from BALB/c mice were incubated with SGL of Phlebotomus papatasi, P. sergenti or Lutzomyia longipalpis. Concanavalin A-stimulated lymphocyte proliferation was significantly suppressed with SGLs of all three sand fly species and all SGL doses tested. This result indicates that saliva from different sand fly species is able to suppress host proliferative response even to the potent mitogen. In parallel experiments, we analysed the effect of SGL on IFN-gamma, IL-2, and IL-4 production; in mitogen-stimulated cells SGLs markedly inhibited IFN-gamma production in all intervals tested (reduced up to 31%) and to a lesser degree impaired production of the other two cytokines as well. Despite some species-specific differences in the intensity of immunomodulatory effects, saliva of all sand fly species modulated cell proliferation as well as cytokine production in a similar way.

Antigen recognition and IL-2 receptor gene expression as evidence against clonal deletion in mice with neonatally induced transplantation tolerance.

Neonatal transplantation tolerance was induced in B10.A mice by the injection of spleen and bone marrow cells from semiallogeneic [C57BL/10(B10) x B10.A] F1 donors. The neonatally treated mice accepted skin grafts from B10 donors. Spleen cells from tolerant animals did not respond by proliferation to tolerated B10 antigens in vitro. However, spleen cells from tolerant mice recognized specific (B10) antigens and synthesized mRNA for the inducible 55-kDa interleukin-2 receptor (IL-2R) as did cells from normal animals. Maintenance of this early phase of cell activation upon contact with tolerated antigens is direct evidence against clonal deletion as a mechanism, in this particular model of neonatally induced transplantation tolerance.

Expression of genes for interleukin-1 alpha and tumour necrosis factor-alpha in newborn mice.

Spleen cells from newborn mice are immunologically non-reactive and do not respond by proliferation to T- or B-cell mitogens. However, they synthesize significant levels of mRNA for interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) after mitogen stimulation. Since these two cytokines mediate many protective activities in the body, they may be important in ensuring the survival of immunologically immature newborns.

Tumour-bearing animals synthesize a decreased level of mRNA for the inducible 55,000 MW interleukin-2 receptor.

Spleen cells from mice bearing progressive growing methylcholanthrene-induced syngenic tumours were deeply hyporeactive in response to T-cell mitogens. This hyporeactivity was associated with decreased ability to synthesize mRNA for the inducible 55,000 MW interleukin-2 receptor (IL-2R). Since the expression of functional IL-2R represents one of the early and pivotal events in lymphoid-cell activation, it is suggested that the defect in effective IL-2R expression may be one of the primary factors responsible for the immunological hyporeactivity of tumour-bearing hosts.

Interleukin-1 production by immunologically hyporeactive tumour-bearing mice.

Mice bearing progressively growing syngeneic methylcholanthrene-induced sarcomas are immunologically hyporeactive. However, both basal (steady-state) and bacterial lipopolysaccharide (LPS)-induced synthesis of mRNA for interleukin-1 (IL-1) in peritoneal exudate cells (PEC) or spleen cells were comparable in control and tumour-bearing animals. Furthermore, the production of IL-1 by PEC stimulated with LPS in the presence of indomethacin was same in control and tumour-bearing mice. The results thus demonstrate that LPS-stimulated cells from animals bearing progressively growing syngeneic sarcomas synthesise the same quantities of mRNA for IL-1 and produce comparable amounts of IL-1 as do cells from normal animals, in spite of the profound immunological hyporeactivity of the former.

Interleukin-2 activates the gamma-interferon gene in newborn mice.

Spleen cells from newborn mice are immunologically nonreactive and do not respond by proliferation upon stimulation with the T cell mitogen concanavalin-A (Con-A) or with recombinant interleukin-2 (IL-2). We have found that, in spite of the observed non-reactivity in the proliferative tests, cells from newborn mice were able to synthesize a significant level of mRNA for gamma-interferon (gamma-IFN) after stimulation with IL-2, but did not synthesize gamma-IFN upon stimulation with Con-A. Since gamma-IFN is of prime importance for antiviral and fungicidal activities and has complex regulatory functions for the cells of the immune system, we suggest that it could play an important role in the survival of newborns.