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Biomedical subjects

R G Titus

Publications and source records attributed to R G Titus.

At least 37 records · Page 2Linked to original sources
PubMed1998

The vasoactive peptide maxadilan from sand fly saliva inhibits TNF-alpha and induces IL-6 by mouse macrophages through interaction with the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor.

Maxadilan is a vasodilatory peptide encoded by a gene cloned from Lutzomyia longipalpis salivary glands. In this study we investigated the effects of maxadilan on macrophage functions. Maxadilan treatment of LPS-stimulated BALB/c macrophages inhibited TNF-alpha release but increased IL-6. Further, it also induced IL-6 release in a dose-dependent manner from unstimulated macrophages. Maxadilan increased production of PGE2, and the inhibition of TNF-alpha was completely abrogated by indomethacin. Others have recently shown that maxadilan is a selective agonist of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor. Treatment with the receptor antagonist PACAP 6-38 blocked maxadilan activities on macrophages. The natural endogenous ligand, PACAP 38, had the same effects as maxadilan on TNF-alpha and IL-6 production. Finally, in a dose- and time-dependent fashion, maxadilan induced the intracellular accumulation of cAMP in macrophages. Taken together, the results presented here indicate a modulatory effect of ligands of PACAP type I receptor on cytokine production by macrophages and suggest that activation of this receptor, with the subsequent elevation of intracellular cAMP in macrophages, could participate in a negative-feedback mechanism that controls certain inflammatory responses.

Adenylyl Cyclases↗
PubMed1998

The PACAP-type I receptor agonist maxadilan from sand fly saliva protects mice against lethal endotoxemia by a mechanism partially dependent on IL-10.

Sand fly saliva contains maxadilan, a peptide that causes vasodilation and modifies the secretion of pro-inflammatory cytokines by macrophages. We show that 1 to 10 microg maxadilan protected BALB/c mice against a lethal dose of LPS. Maxadilan reduced serum levels of TNF-alpha by approximately tenfold, while it caused a threefold increase in IL-6 and IL-10. The protective effect of maxadilan is partially dependent on its ability to induce IL-10 production since maxadilan did not prevent death from endotoxic shock in IL-10(-/-) mice. Finally, maxadilan is a selective agonist of the pituitary adenylate cyclase-activating peptide (PACAP) type I receptor, and we found that the natural ligand of this receptor (PACAP 38) also protected mice against lethal endotoxemia. These results indicate that activation of the PACAP type I receptor may contribute to the control of systemic inflammation by a mechanism that is partially dependent on IL-10.

Animals↗
PubMed1998

Histologic characterization of experimental cutaneous leishmaniasis in mice infected with Leishmania braziliensis in the presence or absence of sand fly vector salivary gland lysate.

Leishmania braziliensis is the causative agent of human cutaneous leishmaniasis in parts of the New World. In the murine model of infection, L. braziliensis does not produce severe or lasting cutaneous lesions in either BALB/c or C3H mice. However, when the parasites are injected into BALB/c mice with salivary gland lysate of the sand fly vector for the parasite, infection is significantly enhanced, as measured by lesion size, parasite burden, and the outcome of infection. Histologic examination of these cutaneous lesions showed that initially, nodular and diffuse dermal infiltrates of neutrophils, eosinophils, and histiocytes occurred in all mice. Over time, the saliva-free lesions progressed to small organized granulomas of epithelioid macrophages that contained few parasites, with eventual resolution of inflammation and mild dermal fibrosis. The saliva-associated lesions progressed to extensive, poorly organized accumulations of heavily parasitized epithelioid macrophages, with persistent neutrophils and eosinophils, and minimal fibroplasia. These results indicate that sand fly salivary gland lysate markedly modifies the inflammatory response to infection with L. braziliensis.

Animals↗
PubMed1997

A simple method for quantifying Leishmania in tissues of infected animals.

In experimental animals infected with Leishmania major, the size of cutaneous lesions of the parasite often does not correlate with the number of parasites within the lesion. Indeed, cutaneous lesions can heal, but still contain parasites. Thus, the ability to determine parasite burden in infected animals becomes important, especially when assessing vaccines that are intended to induce sterilizing immunity. Here, Hermenio Lima, Julie Bleyenberg and Richard Titus describe a simple technique for enumerating Leishmania in infected tissue. It is hoped that this technique will allow all researchers working with Leishmania (especially those in countries where leishmaniasis is endemic) to determine parasite burden easily in infected animals.

Journal Article↗
PubMed1997

The influence of antigen-presenting cell type and interferon-gamma on priming and cytokine secretion of Leishmania major-specific T cells.

A Leishmania major-specific primary in vitro system that mimics the immune response of infected mice was used to determine the role that dendritic cells, B cells, and macrophages play in L. major T cell priming. Their relative priming potential (in order) was dendritic cells, B cells, and macrophages. Initiating primary in vitro responses with cell populations depleted of either B or dendritic cells modestly enhanced interferon (IFN)-gamma production; deleting both cells markedly enhanced IFN-gamma production. Thus, macrophages were the most effective cell for eliciting L. major Th1 cells. The effects of exogenously added IFN-gamma or neutralizing anti-IFN-gamma were also studied. With cells from genetically susceptible BALB/c mice, IFN-gamma inhibited proliferation and interleukin-4 secretion by T cells, whereas with resistant C57BL/6 cells, IFN-gamma enhanced IFN-gamma secretion. These results could not be explained by differences in IFN-gamma receptor expression.

Animals↗
PubMed1997

An in vitro model for infection with Leishmania major that mimics the immune response in mice.

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).

Animals↗
PubMed1997

Borrelia burgdorferi-pulsed dendritic cells induce a protective immune response against tick-transmitted spirochetes.

Borrelia burgdorferi-pulsed dendritic cells and epidermal cells were able to initiate the production of anti-outer surface protein A (OspA) antibody in vitro with normal T and B cells from either BALB/c or C3H/HeJ mice. Inhibition of anti-B. burgdorferi antibody production was observed after 3 days, but not after 2 days, of exposure of the antigen-presenting cells to tumor necrosis factor alpha +/- granulocyte-macrophage colony-stimulating factor. Furthermore, splenic dendritic cells pulsed in vitro with live B. burgdorferi spirochetes and then adoptively transferred into naive syngeneic mice mediated a protective immune response against tick-transmitted spirochetes. This protection appeared not to be due to killing of spirochetes in the feeding ticks, since ticks fed to repletion on B. burgdorferi-pulsed dendritic cell-sensitized mice still harbored live spirochetes. Western blot analysis of the sera collected from dendritic cell-sensitized mice demonstrated that the mice responded to a limited set of B. burgdorferi antigens, including OspA, -B, and -C compared to control groups that either had received unpulsed dendritic cells or were not treated. Finally, mice in the early stage of B. burgdorferi infection were able to develop anti-OspA antibody following injection with B. burgdorferi-pulsed dendritic cells. Our results demonstrate for the first time that adoptive transfer of B. burgdorferi-pulsed dendritic cells induces a protective immune response against tick-transmitted B. burgdorferi and stimulates the production of antibodies specific for a limited set of B. burgdorferi antigens in vivo.

Adoptive Transfer↗
PubMed1996

Leishmania major: differential resistance to infection in C57BL/6 (high interferon-alpha/beta) and congenic B6.C-H-28c (low interferon-alpha/beta) mice.

In murine cutaneous leishmaniasis caused by Leishmania major (Lm), resistance often associates with the outgrowth of Lm-specific Th1 cells. Parasites are eliminated by Th1-mediated activation of infected macrophages (M phi) which destroy Lm by producing toxic nitrogen and oxygen radicals. The cytokine IFN-alpha activates microbicidal functions of M phis and facilitates outgrowth of Th1 cells. Therefore, we compared the course of infection with Lm in resistant C57BL/6 mice, bearing the If-1h high expression allele for IFN-alpha/beta, with the congenic B6.C-H-28c mouse, bearing the If-1I low expression allele from the Lm-susceptible BALB/c strain. We observed that B6.C-H-28c animals developed up to 70% larger footpad lesions and harbored up to 1000-fold more parasites than C57BL/6 mice. Furthermore, peak Lm-specific IFN-gamma production in the B6.C-H-28c animals was lower and delayed by approximately 2 weeks, whereas IL-4 production was higher and persisted approximately 2 weeks longer. Since these results suggested that IFN-alpha/beta plays a protective role in mice infected with Lm, we determined whether infusing B6.C-H-28c mice with IFN-alpha would influence the course of infection with Lm. Unfortunately, the mice developed severe peritoneal hemorrhaging in response to injection with IFN-alpha. Therefore, we examined the ability of IFN-alpha to activate M phis to destroy Lm in vitro. We observed that rIFN-alpha could synergize with subactivating doses of LPS to activate both C57BL/6 and BALB/c peritoneal M phis to produce NO and to kill intracellular Lm. Taken as a whole, these results suggest that type I interferons may play a protective role in cutaneous leishmaniasis.

Animals↗
PubMed1996

Blockade of CD86 ameliorates Leishmania major infection by down-regulating the Th2 response.

The costimulatory molecules CD80 and CD86 affect the differentiation of Th1 and Th2 subsets in experimental allergic encephalomyelitis, an autoimmune disorder. It is reported that the CD86 costimulator significantly affects disease outcome in Leishmania major infection, a classic model of Th subset polarization. Treatment of both L. major-resistant (C57BL/6) and susceptible (BALB/c) strains of mice with anti-CD86 substantially decreased parasite burden. This was accompanied, in BALB/c mice, by a decrease in Th2 cytokines. In contrast, anti-CD80 treatment did not affect parasite burden or cytokine levels in either strain. These data illustrate that in L. major infection, anti-CD86 can abrogate Th2 differentiation in a Th2-dominated susceptible mouse and can ameliorate disease in a Th1-dominated resistant strain, although the mechanism involved in the latter is not clear. It is concluded that in L. major infection, Th2 subset differentiation is critically dependent on interaction with the CD86 costimulatory molecule.

Animals↗
PubMed1996

Effects of sand fly vector saliva on development of cutaneous lesions and the immune response to Leishmania braziliensis in BALB/c mice.

The saliva of the sand fly vector of leishmaniasis significantly enhanced infection with doses of >10(4) Leishmania braziliensis parasites in BALB/c mice. With a dose of 10(7) L. braziliensis parasites, 60 to 70% of the mice developed lifetime infections. Mice infected with L. braziliensis plus saliva produced two- to threefold more interleukin-4 than did mice infected with L. braziliensis alone. Treatment with a neutralizing anti-interleukin-4 antibody abrogated the effects of the saliva.

Animals↗
PubMed1995

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Animals↗
PubMed1995

Sand fly vector saliva selectively modulates macrophage functions that inhibit killing of Leishmania major and nitric oxide production.

The saliva of Phlebotomus papatasi, a sand fly vector for Leishmania major, contains a factor that exacerbates leishmaniasis and may be required for the establishment of infection with Leishmania in nature. We have examined the effect of sand fly saliva on various macrophage functions in vitro. Our data demonstrate that although saliva does not alter uptake of L. major by macrophages, it inhibits the ability of IFN-gamma to activate macrophages to kill the intracellular parasite. This inhibition of parasite killing is observed when both the promastigote and amastigote forms of the parasite are used for infection. Furthermore, this inhibition of parasite destruction correlates with reduction of nitric oxide (NO) production, suggesting that the ability of sand fly saliva to reduce nitrogen oxidation in response to IFN-gamma may be responsible for the inhibitory effect of saliva on intracellular killing of L. major. Finally, despite the fact that saliva inhibits NO production in IFN-gamma-activated macrophages, it does not prevent IFN-gamma from up-regulating class II MHC expression on macrophages. This suggests that the immunosuppressive effect of sand fly saliva on the macrophage is targeted to certain critical, but not all, functions of the cell.

Animals↗
PubMed1995

T cell and non-T cell compartments can independently determine resistance to Leishmania major.

In experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm), the cellular determinants governing development of protective or exacerbative T cells are not well understood. We, therefore, attempted to determine the influence of T cell and non-T cell compartments on disease outcome. To this end, T cell chimeric mice were constructed using adult thymectomized lethally irradiated, bone marrow-reconstituted (ATXBM) animals of genetically resistant, C57BL/6, or susceptible, BALB/c, backgrounds. These hosts were engrafted with naive T cell populations from H-2-congenic susceptible, BALB.B6-H-2b, or resistant, C57BL/6.C-H-2d, animals, respectively. Chimeric mice were then infected with Lm, and disease outcome was monitored. BALB/c T cell chimeric mice, BALB/c ATXBM hosts given naive C57BL/6.C-H-2d T cells, resolved their infections as indicated by reductions in both lesion size and parasite numbers. Furthermore, the mice developed typical Th1 (interferon[IFN]-gamma hiinterleukin[IL]-4lo) cytokine patterns. In contrast, both sham chimeric, BALB/c ATXBM hosts given naive BALB/c T cells, and control irradiated euthymic mice succumbed to infection, producing Th2 profiles (IFN-gamma loIL-4hiIL-10hi). C57BL/6 T cell chimeras, C57BL/6 ATXBM hosts given naive BALB.B6-H-2b T cells, resolved their infections as did C57BL/6 sham chimeras and euthymic controls. Interestingly, whereas C57BL/6 control animals produced Th1 cytokines, chimeric animals progressed from Th0 (IFN-gamma hiIL-4hiIL-10hi) to Th2 (IFN-gamma loIL-4hiIL-10hi) cytokine profiles as cure ensued. Both reconstitution and chimeric status of all mice were confirmed by flow cytometry. In addition, T cell receptor V beta usage of Lm-specific blasts was determined. In all cases, V beta use was multiclonal, involving primarily V beta 2, 4, 6, 8.1, 8.2, 8.3, 10, and 14, with relative V beta frequencies differing between H-2b and H-2d animals. Most importantly, however, these differences did not segregate between cure and noncure outcomes. These findings indicate that: (a) genetic traits determining cure in Lm infection can direct disease outcome from both T cell and non-T cell compartments; (b) the presence of the curing genotype in only one compartment is sufficient to confer cure; (c) curing genotype T cells autonomously assume a Th1 cytokine profile-mediating cure; (d) noncuring genotype T cells can mediate cure in a curing environment, despite the onset of Th2 cytokine production; and lastly, (e) antigen specificity of responding T cells, as assessed by V beta T cell receptor diversity, is not a critical determinant of disease outcome.

Animals↗
PubMed1995

Class II major histocompatibility complex-deficient mice initially control an infection with Leishmania major but succumb to the disease.

Class II major histocompatibility complex (MHC)-deficient (H-2b) mice do not express I-A or I-E molecules and, as a result, do not develop CD4 cells. Thus, they represent the ideal model for examining the importance of CD4 cells and MHC class II molecules in resistance to infection with Leishmania major and the capacity of MHC class I-restricted T cells to mediate resistance to L. major. Class II MHC-deficient mice and control (C57BL/6, normal and nude) mice were infected with L. major. Although MHC class II-deficient mice were able to control infection more effectively than nude mice, cutaneous lesions on the mice eventually progressed, and parasite replication became uncontrolled. These results suggest that CD4 cells and MHC class II molecules are essential for resistance to L. major and that in the absence of these cells and molecules, such mice can transiently control infection with L. major but are unable to resolve such infections.

Animals↗
PubMed1994

Leishmania major-parasitized macrophages augment Th2-type T cell activation.

We studied the ability of resident peritoneal M phi, parasitized in vitro with Leishmania major, to present Ag to three Th2-type T cell clones that are specific for non-leishmanial Ags. Results indicated that L. major-infected M phi enhanced the proliferation and IL-4 secretion of Th2 T cells in response to stimulation with their cognate Ag. Augmentation of Th2 T cell proliferation was seen in Ag-driven responses but not in mitogenic Con A stimulation. Furthermore, live L. major promastigotes and amastigotes, but not killed parasites, augmented the Th2 T cell response. To delineate the augmentative effect of L. major-infected M phi for Th2 T cell activation, we analyzed the cytokines produced by infected M phi in the presence or absence of Th2 T cell clones. The supernatants contained IL-1 but not IL-6 or IL-10. Interestingly, addition of a neutralizing anti-IL-1 alpha mAb to the cultures reduced the augmentative effect for Th2 proliferation. Moreover, additional experiments showed that IL-1 alpha could substitute for L. major and enhance T cell activation in cultures consisting of Th2 cells, normal M phi, and Ag. Thus, our study shows that L. major-infected M phi present Ags in a manner that augments Th2 T cell proliferation and IL-4 synthesis, and that the phenomenon is at least in part mediated by IL-1 secreted by the infected M phi. For purposes of comparison, two non-leishmanial Ag specific Th1-type T cell clones were stimulated with L. major-infected M phi. Our data, as already reported by others, showed that infected M phi inhibited the response of Th1 T cells to their cognate Ag. Taken together, this report shows that leishmanial-infected M phi favor the activation of Th2 T cells over Th1 cells and that Th1 and Th2 cells require distinct activation signals from M phi.

Animals↗