Biomedical subjects

Peter C Melby

Publications and source records attributed to Peter C Melby.

11 recordsLinked to original sources

PubMed2005

Selective enhancement of the type 1 cytokine response by expression of a canine interleukin (IL)-12 fused heterodimeric DNA.

Interleukin-12 (IL-12) is a heterodimeric cytokine that is a principal mediator of the innate immune response and modulator of acquired cell-mediated immunity. Administration of exogenous IL-12 can direct the host adaptive T cell response toward a type 1 phenotype. The co-administration of IL-12 with vaccine antigens has been shown to augment the vaccine-induced T(H)1 response and protection against intracellular pathogens. We show here that a canine IL-12 DNA, constructed by fusing the p35 and p40 subunit cDNAs with an interspacing linker, generated stable IL-12 transcripts when placed under control of a strong constitutive promoter. The protein expressed from this fused cDNA was fully functional in promoting a type 1 (IFN-gamma) and suppressing a type 2 (IL-4) cytokine response following both in vitro transfection of a canine cell line and in vivo delivery to dogs. This DNA construct may be useful as an adjuvant for vaccines that target tumors or intracellular pathogens of the dog.

Animals↗

PubMed2005

Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs.

Vaccination of dogs, the domestic reservoir of Leishmania chagasi, could not only decrease the burden of canine visceral leishmaniasis (VL), but could also indirectly reduce the incidence of human VL. Intramuscular vaccination of foxhounds with a Leishmania multicomponent (10 antigen) DNA vaccine resulted in antigen-induced lymphoproliferative and IFN-gamma (but not IL-4) responses. This response was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants. The multicomponent DNA vaccine also induced a delayed type hypersensitivity (DTH) response to viable L. donovani promastigotes and led to a reduction of parasite burden in an in vitro intracellular infection model, and in the draining lymph node of dogs early after cutaneous challenge. Thus, the multicomponent DNA vaccine was effective in priming dogs for a parasite-specific type 1 cellular immune response, which was able to restrict parasite growth.

Adjuvants, Immunologic↗

PubMed2003

Multinutrient undernutrition dysregulates the resident macrophage proinflammatory cytokine network, nuclear factor-kappaB activation, and nitric oxide production.

We have described previously a murine model of multinutrient undernutrition that reproduced the features of moderate human malnutrition and led to increased early dissemination of Leishmania donovani. Peritoneal cells from these malnourished mice produced decreased NO after stimulation with IFN-gamma/LPS. We hypothesized that malnutrition may cause a deficit in NF-kappaB activation, a principal transcription pathway for inducible NO synthase and proinflammatory cytokines. Macrophages from malnourished mice, stimulated with IFN-gamma/LPS, showed increased IL-6 production and decreased IL-10 and TNF-alpha production. Neutralization of TNF-alpha in macrophage cultures from the control mice mimicked the effect of malnutrition on NO and IL-10 production, whereas supplemental TNF-alpha added to cultures of macrophages from malnourished mice increased NO secretion. NF-kappaB nuclear binding activity in macrophages from the malnourished mice was reduced early after stimulation, but increased to supranormal values by 16- or 24-h poststimulation. Blocking NO production in the macrophages from the control mice reproduced the effect of malnutrition on the late activation of NF-kappaB, whereas supplemental NO decreased the late NF-kappaB activation in the malnourished mice. Thus, in macrophages from the malnourished mice, initial deficits in NF-kappaB activity probably lead to decreased TNF-alpha, which results in decreased NO; however, IL-6 is regulated independently from NF-kappaB and TNF-alpha. The late activation of NF-kappaB in the macrophages from malnourished mice is due to absence of negative feedback from NO.

Animals↗

PubMed2003

Fractalkine (CX3CL1) stimulated by nuclear factor kappaB (NF-kappaB)-dependent inflammatory signals induces aortic smooth muscle cell proliferation through an autocrine pathway.

Fractalkine (also known as CX3CL1), a CX3C chemokine, activates and attracts monocytes/macrophages to the site of injury/inflammation. It binds to CX3C receptor 1 (CX3CR1), a pertussis toxin-sensitive G-protein-coupled receptor. In smooth muscle cells (SMCs), fractalkine is induced by proinflammatory cytokines [tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)], which may mediate monocyte adhesion to SMCs. However, the mechanisms underlying its induction are unknown. In addition, it is unlear whether SMCs express CX3CR1. TNF-alpha activated nuclear factor kappaB (NF-kappaB) and induced fractalkine and CX3CR1 expression in a time-dependent manner in rat aortic SMCs. Transient transfections with dominant-negative (dn) inhibitory kappaB (IkappaB)-alpha, dnIkappaB-beta, dnIkappaB kinase (IKK)-gamma, kinase-dead (kd) NF-kappaB-inducing kinase (NIK) and kdIKK-beta, or pretreatment with wortmannin, Akt inhibitor, pyrrolidinecarbodithioc acid ammonium salt ('PDTC') or MG-132, significantly attenuated TNF-alpha-induced fractalkine and CX3CR1 expression. Furthermore, expression of dn TNF-alpha-receptor-associated factor 2 (TRAF2), but not dnTRAF6, inhibited TNF-alpha signal transduction. Pretreatment with pertussis toxin or neutralizing anti-CX3CR1 antibodies attenuated TNF-alpha-induced fractalkine expression, indicating that fractalkine autoregulation plays a role in TNF-alpha-induced sustained fractalkine expression. Fractalkine induced its own expression, via pertussis toxin-sensitive G-proteins, phosphoinositide 3-kinase (PI 3-kinase), phosphoinositide-dependent kinase 1 (PDK1), Akt, NIK, IKK and NF-kappaB activation, and induced SMC cell-cell adhesion and cellular proliferation. Taken together, our results demonstrate that TNF-alpha induces the expression of fractalkine and CX3CR1 in rat aortic SMCs and that this induction is mediated by NF-kappaB activation. We also show that fractalkine induces its own expression, which is mediated by the PI 3-kinase/PDK1/Akt/NIK/IKK/NF-kappaB signalling pathway. More importantly, fractalkine increased cell-cell adhesion and aortic SMC proliferation, indicating a role in initiation and progression of atherosclerotic vascular disease.

Androstadienes↗

PubMed2003

The site of cutaneous infection influences the immunological response and clinical outcome of hamsters infected with Leishmania panamensis.

We determined that the site of inoculation (foot or snout) influences the clinical evolution and immune responses of hamsters infected with Leishmania (Viannia) panamensis. Hamsters infected in the snout showed (i) a more rapid and severe lesion evolution at multiple time points (P < 0.05), (ii) a more extensive inflammatory infiltrate and tissue necrosis, (iii) a higher tissue parasite burden, (iv) a higher antibody titre (P < 0.01), but lower antigen-specific spleen cell proliferative response (P = 0.02), and (v) a slower response to anti-leishmanial drug treatment (P < 0.002). In both inoculation groups there was co-expression of type 1 (IFN-gamma and IL-12) and some type 2 (IL-10 and TGF-beta, but not IL-4) cytokines in the cutaneous lesions and spleen. Early in the course of infection, hamsters infected in the snout showed higher expression of splenic IL-10 (P = 0.04) and intra-lesional IFN-gamma (P = 0.02) than foot infections. No expression of IL-12p40 or IL-4 was detected. During the chronic phase, snout lesions expressed more IFN-gamma (P = 0.001), IL-12p40 (P = 0.01), IL-10 (P = 0.009) and TGF-beta (P = 0.001), and the level of expression of each of these cytokines correlated with lesion size (P < or = 0.01). These results suggest that the site of infection influences the clinical outcome in experimental cutaneous leishmaniasis, and that the expression of macrophage-deactivating type 2 cytokines and/or an exaggerated type 1 proinflammatory cytokine response may contribute to lesion severity.

Animals↗

PubMed2003

Short report: a disseminated infection of Leishmania mexicana in an eastern woodrat, Neotoma floridana, collected in Texas.

An eastern woodrat (Neotoma floridana) collected in January 2001 near Bedias, Grimes County, Texas, had extensive lesions of both ears and swollen feet. Impression smears and histologic sections demonstrated the presence of Leishmania in both ears and the one foot that was screened. Polymerase chain reaction screening using species-specific primers detected parasites in both ears and all four feet and indicated the parasites were L. mexicana. The detection of L. mexicana in N. floridana represents a new host record in a new ecologic region and may help explain a human infection acquired outside the previously-known range of the disease. Given the geographic distribution of N. floridana and the two other species of Neotoma found naturally infected, enzootic foci of Leishmania could be present over much of the southern United States.

Animals↗

PubMed2002

Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.

It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.

Animals↗

PubMed2002

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation.

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.

Animals↗

PubMed2002

Recent developments in leishmaniasis.

PURPOSE OF REVIEW: The leishmaniases, caused by protozoan parasites of the genus Leishmania, are a significant health problem in many regions of the world. This review highlights the recent advances in the study of leishmaniasis related to parasite biology, disease pathogenesis, clinical evaluation and treatment, and prevention. RECENT FINDINGS: Genetic heterogeneity and clonal diversity is common among Leishmania strains. Gene knockout, overexpression, and re-introduction studies have identified a number of genes that play a role in parasite virulence. Surprisingly, the importance of the surface lipophosphoglycan in parasite virulence appears to differ among Leishmania spp. Studies in experimental animal models have further defined the roles of CD4 and CD8 T cells, IL-4, IL-10, and IL-12 in the control, maintenance, or progression of disease. The effect of Leishmania on dendritic cells and macrophage effector function has also been an important area of investigation. A number of new vaccine candidates have been identified through experimental animal studies. Clinical studies of leishmaniasis have focused on the host determinants of disease (most notably HIV co-infection), serological and DNA-based diagnostic assays, and treatment. Antimony-resistant cases of cutaneous and visceral leishmaniasis have become more common; liposomal amphotericin and oral miltefosine are promising alternative therapies. SUMMARY: Significant advances have been made in the areas of pathogenesis, host defence, and treatment of leishmaniasis. A number of new vaccine candidates and potential targets of drug therapy have been identified, but progress from preclinical studies to clinical trials has been slow. Translational research, built upon the solid foundation of existing and ongoing basic investigation, is a high priority.

Animals↗

PubMed2002

Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp.

In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender on Leishmania infection have not included parasites of the subgenus Viannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection by Leishmania (Viannia) spp., but cutaneous infection of hamsters with L. (V.) panamensis or L. (V.) guyanensis resulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P = 0.04), IL-10 (P = 0.04), and transforming growth factor beta (TGF-beta) (P < 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-beta mRNA and lesion size (Spearman's correlation coefficient = 0.873; P < 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.

Age Factors↗

PubMed2002

Vaccination against cutaneous leishmaniasis: current status.

The different cutaneous leishmaniases are distinct in their etiology, epidemiology, transmission, and geographical distribution. In most instances cutaneous leishmaniasis is limited to one or a few skin ulcers that develop at the site where the parasites were deposited during the bite of the sandfly vector. Lesions typically heal spontaneously after several months but some lesions can be large and follow a chronic, more severe course. Protective immunity is usually acquired following cutaneous infection with Leishmania spp., so prevention of disease through prophylactic immunization appears to be feasible. Since vaccination with live, virulent parasites is associated with an unacceptable rate of adverse events, attention has turned to the use of killed or attenuated parasite vaccines and defined subunit vaccines. Whole parasite vaccines have the advantage of delivering multiple antigenic epitopes that may be necessary for initiation of a broad-based immune response. Persistent or repeated immune-stimulation by parasite antigens and/or sustained expression of interleukin-12 appear to be critical elements in the development of durable immunity. A number of purified or recombinant antigens, when co-administered with a vaccine adjuvant, appear promising as vaccine candidates against cutaneous leishmaniasis. The sustained expression of recombinant Leishmania antigens by vaccination with DNA is an attractive approach because it mimics the persistent antigenic stimulation of subclinical infection. Effective vaccine-induced immunity must generate an antigen-specific memory T cell population that, upon exposure to the infecting parasite, rapidly produces a type 1 effector T cell response that leads to interferon-gamma-mediated activation of infected macrophages to kill the intracellular parasites. This parasite-directed recall response must be prompt and of sufficient magnitude to overcome the subversive effect that the intracellular infection has on macrophage effector function. It is unlikely that vaccination against cutaneous leishmaniasis would induce sterile immunity, but a small number of parasites are likely to persist subclinically.

Adjuvants, Immunologic↗