Is cell surface calreticulin involved in phagocytosis by insect hemocytes?

The innate immune system of insects consists of humoral and cellular components involved in the recognition of and responses to intruding foreign micro- or macroorganisms. Several molecules have been identified so far that recognize molecular patterns present on microorganisms, such as lipopolysaccharides, peptidoglycans and lipoteichonic acid. These molecules, acting as opsonins, trigger immune responses such as phagocytosis, nodule formation, melanization and encapsulation. Here, we investigated the role of calreticulin (CRT) present on the surface of Pieris rapae hemocytes in phagocytosis. Comparative phagocytosis assays using yeast cells showed that hemocytes from different insects exhibit significant variation in their phagocytosing potential and relative CRT involvement. (C) 2003 Elsevier Science Ltd. All rights reserved.

Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity.

Visiting-economists through Hirschman`s eyes

Since the nineteenth century, Latin America and other poor areas of the world received periodical visits from missions of economic experts. This paper analyzes the picture that Hirschman draws of these money doctors, whose main task was to advise on the economic and financial reforms that were deemed necessary for economic development. Hirschman coins the expression `visiting-economist syndrome` to criticize the work done by these money doctors. I discuss whether Hirschman, as a money doctor himself, was able to acquire immunity from the disease he feared.

Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM

We determined the prophylactic effect of both the d-mannose-binding lectin ArtinM extracted from the seeds of Artocarpus integrifolia (jackfruit) and its recombinant counterpart during the course of experimental paracoccidioidomycosis induced in BALB/c mice. Four experimental protocols of prophylaxis were employed to evaluate the most protective regimen of ArtinM administration. It was demonstrated that the best effect was obtained by administration of two ArtinM doses on days 10 and 3 before the challenge with Paracoccidioides brasiliensis. By following this protocol, the lungs of mice that received native or recombinant ArtinM exhibited reduced fungal burden and granuloma incidence. In addition, the protocol augmented contents of IL-12, IFN-gamma, TNF-alpha and NO. On the other hand, the control group consisting of untreated infected mice had higher pulmonary levels of IL-4 and IL-10. In conclusion, prophylaxis with ArtinM significantly reproduces the effect of its therapeutic administration, i.e, it confers resistance to P. brasiliensis infection in mouse models by promoting IL-12 production and favours Th1-immunity.

TRADE-OFF BETWEEN IMMUNE STIMULATION AND EXPRESSION OF STORAGE PROTEIN GENES

Proteins stored in insect hemolymph may serve (is a source of amino acids and energy for metabolism, and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCH and Western blot.. After ensuring that. the immune system had, been activated by measuring the ensuing expression (, the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (pro PO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein. were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLP-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLP-III transcripts. Our findings are consistent with a down-regulation, of the express and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon. represents a strategy to redirect resources to combat injury or infection. (C) 2009 Wiley Periodicals, Inc.

Incorporation of antigenic GPI-proteins from Leishmania amazonensis to membrane mimetic systems: Influence of DPPC/cholesterol ratio

The reconstitution of membrane proteins into liposomes is a useful tool to prepare antigenic components that induce immunity. We have investigated the influence of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol molar ratio on the incorporation of a GPI-protein from Leishmania amazonensis on liposomes and Langmuir monolayers. The latter system is a well behaved and practical model, for understanding the effect of variables such as surface composition and lipid packing on protein incorporation. We have found that the DPPC/cholesterol molar ratio significantly alters the incorporation of the GPI-protein. In the absence of cholesterol, reconstitution is more difficult and proteoliposomes cannot be prepared, which we correlated with disruption of the DPPC layer. Our results provide important information that Could be employed in the development of a vaccine system for this disease or be used to produce other GPI-systems for biotechnological application. (c) 2009 Elsevier Inc. All rights reserved.

A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp paratuberculosis: clinical disease, faecal culture and immunological studies

Two longitudinal experiments involving Merino sheep challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma test, the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Infections were induced with either a bovine or ovine strain of Map in separate experiments with infections being more easily established, in terms of faecal bacterial shedding and clinical disease when the challenge inoculum was prepared from gut mucosal tissue than cultured bacteria. The patterns of response for shedding and clinical disease were similar. Cell-mediated immune responses were proportionally elevated by at least an order of magnitude in all sheep dosed with either a bovine or ovine strain of Map. Conversely, antibody responses were only elevated in a relatively small proportion of infected sheep. Neither of the clinically affected tissue challenged sheep developed an antibody response despite the presence of persistent shedding and the development and decline in cell-mediated immunity. The results indicated that for sheep the interferon-gamma test may be useful for determining if a flock has been exposed to ovine Johne's disease. (C) 2004 Elsevier B.V. All rights reserved.

Use of proteoliposome as a vaccine against Trypanosoma cruzi in mice

We have generated proteoliposomes carrying proteins of Topanosoma cruzi for use as immunogens in BALB/c mice. T cruzi trypomastigote and amastigote forms were sonicated and mixed with SDS, with 94% recovery of soluble proteins. To prepare proteoliposomes, we have used a protocol in which dipalmitoylphosphatidylcholine, dipalmitoyl-phosphatidylserine and cholesterol were incubated with the parasite proteins. BALB/c mice immunized with 20 mu g were able to generate antibodies which, in Western blotting, reacted with the proteins of T cruzi. We further investigated the ability of peritoneal cells from immunized mice to arrest the intracellular replication of trypomastigotes, in vitro. After 72h of culture, the number of intracellular parasites in immunized macrophages decreased significantly, as compared to controls. Despite the fact that exposure of mice to T cruzi proteins incorporated into proteoliposomes generate antibodies and activate macrophages, the immunized mice were not protected against T cruzi intraperitoneal challenge. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

Bovine mammary epithelial cells, initiators of innate immune responses to mastitis

Innate immunity plays a vital role in the protection of the bovine mammary gland against mastitis. Until recently, the migration of effector cells such as neutrophils and monocytes into the mammary gland was thought to provide the only defence against invading pathogens. However, mammary epithelial cells may also play an important role in the immune response, contributing to the innate defence of the mammary tissue through secretion of antimicrobial peptides and attraction of circulating immune effector cells. This paper reviews the innate immune pathways in mammary epithelial cells and examines their role in the initiation of an innate immune response to Gram-positive and Gram-negative bacteria.

Overcoming original antigenic sin to generate effective immune responses in antigen primed host

Original antigenic sin is failure to mount effective immunity to virus variants in a previously virus infected host. We have previously shown that prior immunity to a virus capsid protein inhibits induction from naive CD8 T cells of an IFN-g response to a MHC class I restricted epitope linked to the capsid protein, following immunisation with a capsid expressing the class I restricted epitope. The inhibition is independent of pre-existing antibody to the viral capsid, and the inhibition is observed in animal lacking B cells. CD8 restricted viral capsid specific T cell responses are also not required, but the inhibition is not observed in IL10 knockout mice. We now demonstrate that capsid antigen primed CD4+ T cells secrete IL10 in response to capsid antigen presented by DC, and deviate CD8 cells specific for the linked MHC Class I restricted epitope from IFN-g production to IL-5 production. Neutralizing IL10, either in vitro or in vivo, restores induction following immunisation of an antigen specific IFN-g response to an MHC Class I restricted epitope. This finding demonstrates a strategy for overcoming bias towards a Tc2 response to MHC Class I epitopes upon immunisation of a host already primed to antigen, facilitating immunotherapy for chronic viral infection or cancer

Defective NF-kappaB Activation in Response to LPS in Type 1 Diabetes Dendritic Cells

Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.

Mannose-binding lectin gene polymorphism and resistance to therapy in women with recurrent vulvovaginal candidiasis

Precis Women with recurrent vulvovaginal candidiasis (RVC) due to a polymorphism in codon 54 of the MBL2 gene respond better to fluconazole maintenance therapy than do women with other underlying causes. Objective To explain differences in response rates to maintenance therapy with fluconazole in women suffering from RVC by evaluating associations with a polymorphism in the gene coding for mannose-binding lectin (MBL). Design Follow-up study, neted case-control group. Setting Women attending vulvoginitis clinic for RVC. Population Women participating in a multicentric study in Belgium with a degressive dose of fluconazole for RVC (the ReCiDiF trial) were divided into good responders, intermediate responders and nonresponders according to the number of relapses they experienced during therapy. From 109 of these women with adequate follow-up data, vaginal lavage with 2 ml of saline were performed at the moment of a proven acute attack at inclusion in the study, before maintenance treatment was started. A buccal swab was obtained from 55 age-matched women without a history of Candida infections, serving as a control group. Methods Extracted DNA from buccal or vaginal cells was tested for codon 54 MBL2 gene polymorphism by polymerase chain reaction and endonuclease digestion. Main outcome measures Frequency of MBL2 condon 54 allele B in women with optimal or poor response to maintenance therapy in composition with controls. Results Women (n = 109) suffering from RVC were more likely to carry the variant MBL2 codon 54 allele B than control women (20 versus 6.6%, OR 3.4 [95% CI 1.3-8.2], P = 0.01). B alleles were present in 25% of the 36 women not suffering from any recurrence during the maintenance therapy with decreasing doses of fluconazole (OR 4.9 [95% CI 1.9-12.5], P = 0.0007 versus controls), in 20% of the 43 women with sporadic recurrences (OR 3.6 [95% CI 1.4-9.2], P = 0.007 versus controls) and in 15% of the 30 women who had to interrupt the treatment regimen due to frequent relapses (P = 0.097 versus controls). Conclusions The MBL2 codon 54 gene polymorphism is more frequent in Belgian women suffering from RVC than in controls. The presence of the B allele is associated with a superior response to fluconazole maintenance therapy as compared with RVC patients without this polymorphism. We conclude that RVC due to deficient MBL production is more easily helped with antifungal medication than is RVC due to some other mechanism.

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our under-standing of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field. (c) 2006 Elsevier B.V. All rights reserved.

Neutrophil depletion increases susceptibility to systemic and vaginal candidiasis in mice, and reveals differences between brain and kidney in mechanisms of host resistance

Infections caused by the yeast Candida albicans represent an increasing threat to debilitated and immunosuppressed patients, and neutropenia is an important risk factor. Monoclonal antibody depletion of neutrophils in mice was used to study the role of these cells in host resistance. Ablation of neutrophils increased susceptibility to both systemic and vaginal challenge. The fungal burden in the kidney increased threefold on day 1, and 100-fold on day 4, and infection was associated with extensive tissue destruction. However, a striking feature of the disseminated disease in neutrophil-depleted animals was the altered pattern of organ involvement. The brain, which is one of the primary target organs in normal mice, was little affected. There was a threefold increase in the number of organisms recovered from the brains of neutrophil-depleted mice on day 4 after infection, but detectable abscesses were rare. In contrast, the heart, which in normal mice shows only minor lesions, developed severe tissue damage following neutrophil depletion. Mice deficient in C5 demonstrated both qualitative and quantitative increases in the severity of infection after neutrophil depletion when compared with C5-sufficient strains. The results are interpreted as reflecting organ-specific differences in the mechanisms of host resistance.