A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells.

OBJECTIVE: Surfactant protein D (SP-D), which is secreted mainly in the lung, is an oligometric C type lectin that promotes phagocytosis by binding to carbohydrates on microbial surfaces. SP-D can also bind SIRPα, leading to a decrease in cytokine production by monocytes/macrophages. In the present study, we examined the possibility that SP-D suppresses macrophage-mediated xenogeneic cytotoxicity, by creating a membrane-type SP-D. METHODS: The cDNA for the carbohydrate recognition domain (CRD) of human SP-D was switched to that of a membrane-type protein, collectin placenta 1 (CL-P1), with a Flag-tag. The cDNA of CD47 was prepared as a control. The suppressive function of the membrane-type protein of the hybrid molecule, CL-SP-D, to monocytes/macrophages was then studied and the results compared with that for CD47. RESULTS: The expression of Flag-tagged CL-SP-D on the transfected SECs and the SIRPα on monocyte-like cells, THP-1 cells, was confirmed by FACS using anti-Flag Ab and anti-CD172a, respectively. The molecular size of the hybrid protein was next assessed by western blot. While significant cytotoxicity against SEC was induced in differentiated THP-1 cells, CL-SP-D significantly reduced THP-1-mediated cytotoxicity. In addition, phosphorylated SHP-1 was clearly detected in SEC/CL-SP-D in western blots. Moreover, IL-10 production was upregulated and IL-1ß production was suppressed in the case of THP-1 and SEC/CL-SP-D, compared with naïve SEC. Next, the cytotoxicity caused by the in vitro generated macrophage was assessed under the same conditions as were used for THP-1. CL-SP-D also showed the significant down-regulation on the macrophage. In addition, changes in IL-10 production by the macrophage confirmed the results. CONCLUSIONS: These findings indicate that the membrane-type SP-D serve as an effective therapeutic strategy for inhibiting macrophage-mediated xenograft rejection in xenotransplantation.

[Therapeutic intervention alternatives in cancer, using attenuated live bacterial vectors: Salmonella enterica as a carrier of heterologous molecules]. / Alternativas de intervención terapéutica en cáncer usando vectores bacterianos vivos atenuados: Salmonella enterica como acarreador de moléculas heterólogas.

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

Human regulatory T cells control xenogeneic graft-versus-host disease induced by autologous T cells in RAG2-/-gammac-/- immunodeficient mice.

PURPOSE: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell-dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2-/-gammac-/- mice by i.v. administration of human peripheral blood mononuclear cells (PBMC). EXPERIMENTAL DESIGN: Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals. RESULTS: Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8+ T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-gamma, suggesting the de novo development of TR1 cells. CONCLUSIONS: These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.

Immunotherapy for gynaecological malignancies.

Gynaecological malignancies, excluding breast cancer, cause approximately 25,000 deaths yearly among women in the US. Therefore, novel approaches for the prevention or treatment of these diseases are urgently required. In the case of cervical cancer, human papillomavirus (HPV) xenoantigens are readily recognised by the immune system, and their targeting has shown great promise in preclinical models of therapeutic vaccination and in clinical studies of preventative vaccination. A growing body of evidence indicates that ovarian cancer is also immunogenic and can thus be targeted through immunotherapy. This review outlines the principles and problems of immunotherapy for cervical and ovarian cancer, including the authors' personal assessment.

DNA vaccines: an active immunization strategy for prostate cancer.

Immunotherapy is currently being investigated as a treatment for patients with asymptomatic, recurrent prostate cancer manifested only by a rising prostate-specific antigen (PSA) level. Several different approaches to active immunization against antigens found on cancer cells have been explored. Immunization with DNA overcomes many of the obstacles noted in previous studies. Injection of plasmid DNA encoding a xenogeneic differentiation antigen (prostate-specific membrane antigen [PSMA]) is a potent means to induce antibody and T-cell responses to these otherwise poorly immunogenic self proteins. Use of the xenogeneic DNA (ie, human PSMA DNA injected into mouse) has been shown to be an absolute requirement to overcome immunologic tolerance. We are currently conducting a phase I trial of human and mouse PSMA DNA vaccines in patients with recurrent prostate cancer, based on preclinical experiments described below.

Peach profilin: cloning, heterologous expression and cross-reactivity with Bet v 2.

BACKGROUND: Peach is among the main foods causing allergic reactions in the Mediterranean adult population. Only a single peach allergen, named Pru p 3, has been characterized. However, a potential role of profilin has also been suggested in grass pollen-associated allergy to peach. METHODS: Complementary DNA clones for two different peach profilin isoforms were obtained by reverse transcriptase polymerase chain reaction using non-degenerated primers. Expression of recombinant peach profilin was performed in Escherichia coli, and confirmed using rabbit polyclonal antibodies to sunflower pollen profilin. Twenty-nine individual sera from patients with peach allergy proved by double-blind, placebo-controlled food challenges (DBPCFC), either with (n = 15) or without (n = 14) specific IgE to Bet v 2, were used in immunodetection assays to test recombinant peach profilin reactivity. RESULTS: Each peach profilin cDNA included an open reading frame coding for a 131 amino acid protein. The peach profilin isoforms, designated Pru p 4.01 and Pru p 4.02, showed 80% of amino acid sequence identity, and were very similar (>70% identity) to allergenic profilins from plant foods and pollens. Recombinant Pru p 4.01 was expressed in E. coli as a nonfusion protein, displaying the expected molecular size and reacting with anti-profilin antibodies. rPru p 4.01 was recognized by all sera (15 of 15) with specific IgE to Bet v 2, whereas no sera (zero of 14) without IgE to this birch allergen reacted with rPru p 4.01. CONCLUSIONS: Peach profilin Pru p 4 is very closed to other allergenic profilins from plant foods and pollens. A complete correlation between reactivity to rPru p 4 and rBet v 2 has been found in sera from peach allergic patients.

Characterization of the immune response to valve bioprostheses and its role in primary tissue failure.

BACKGROUND: The role of an immune response in the failure of bioprosthetic heart valves is poorly understood and disregarded by many. To elucidate the nature of the immune response to glutaraldehyde-treated tissue and the possible role of graft-specific antibody in graft mineralization, we performed immune-calcification studies in the rabbit and correlated those results with the analysis of specific antibodies. METHODS: Aortic wall buttons (6 mm) were punched from porcine aortic wall tissue fixed with 0.2% glutaraldehyde and detoxified with urazole and then subsequently perforated under sterile conditions. The perforated buttons were then incubated with either immune serum prepared by immunization of New Zealand White rabbits (n = 5) with Freund's incomplete adjuvant emulsions of tissue homogenates of similarly treated aortic wall tissue, or incubated with the corresponding control preimmune sera obtained before immunization of the same animals. The tissue was then implanted subdermally on the back of unrelated New Zealand White rabbits (n = 8) for a period of 3 weeks. After the buttons were explanted, tissue calcium levels were determined by atomic absorption spectroscopy. RESULTS: Tissue calcium was increased in all five immune serum-treated replicates (range, 61.8% to 431.2%; mean, 225.9%+/-73.2%) when compared with control samples treated with preimmune sera. Overall, the mean calcium level was significantly increased (p < 0.0001) when tissue was treated with immune sera (66.0+/-10.0 microg/mg versus 22.6+/-4.8 microg/mg in control tissue). Graft specificity of immune sera was confirmed by Western blot analysis. CONCLUSIONS: These results strongly suggest a role of circulating graft-specific antibody in the disease of bioprosthetic graft calcification.

Pig xenogeneic antigen modification with green coffee bean alpha-galactosidase.

Green coffee bean alpha-galactosidase can cleave the terminal alpha-galactose (alphaGal) on oligosaccharides that form the major antigen on pig endothelial cells recognized by primate-specific antibodies. Studies have been made of the conditions under which it is functional (e.g. temperature, pH) and of its biochemical and immunologic effects. Pig-to-rhesus monkey vein transplants were studied to identify the efficiency of the enzyme in delaying hyperacute rejection. When a graft became occluded, biopsies were taken for light microscopy (hematoxylin and eosin), scanning electron microscopy (SEM) and immunostaining with Griffonia simplicifolia IB4 lectin (GSIB4), and for IgM, IgG and C3. alpha-Galactosidase was stable for 72-96 h and was effective at 4 degrees C and pH 6.9 (conditions of human liver graft storage), although better function was obtained at 20 degrees C and pH 6.5. Using the porcine PK15 cell assay, the cytotoxicity of human serum was reduced after treatment of the pig cells with the enzyme. In vitro studies demonstrated that porcine veins treated with alpha-galactosidase lost endothelial expression of the Gal epitope within 30 min. SEM, however, demonstrated endothelial damage beginning within 2 h, probably caused by the alpha-galactosidase, as no damage was found in phosphate-buffered saline-treated veins, where the Gal epitope was preserved for >3 h. No change was found in either group on light microscopy. In vivo studies demonstrated that patency of the alpha-galactosidase-treated veins (mean 2.5 h) was longer than that of untreated veins (0.23 h) (P < 0.01). Biopsies showed no GSIB4 lectin staining for alpha-Gal epitopes and much less IgM and C3 deposition in the treated group. Light microscopy and SEM demonstrated more severe endothelial damage, hemorrhage, and fibrin formation in the untreated group. Galactosidase is effective in removing the terminal alphaGal and delays the onset of hyperacute rejection of pig veins transplanted into monkeys. However, its effect is temporary and, on its own, its use is unlikely to prolong survival of pig organs transplanted into primates sufficiently to be of clinical value.

[Immunomodulating action of heteropolysaccharides isolated from camomile flowers]. / Immunomoduliruiushchee deistvie geteropolisakharidov, vcydelennykh iz sotsvetii romashki.

Administration of heteropolysaccharides from the camomile flower clusters to rats which failed to perform a physical load (swimming) resulted in stimulation of development of the immune response to SRBCs. However, it did not influence development of the immune response to a bacterial lipopolysaccharide in the rats. A short-term exposure of the swimming animals to high doses of the heteropolysaccharides increased development of the immune response induced by their lipopolysaccharide. A long-term exposure of the swimming rats to low doses of the heteropolysaccharides increased development of the immune response to SRBCs and the lipopolysaccharide. The high doses of the heteropolysaccharides induced excretion of the helper factors by the spleen cells not adhesive to glass while the low doses of the heteropolysaccharides decreased sensitivity of the cells of the immune system to the influence of the suppressing factor excreted by the glass-adherent spleen cells from swimming rats.

[Effect of doxycycline on immune response in a multifactorial experiment]. / Izuchenie deistviia doksitsiklina na immunnyi otvet v mnogofaktornom éksperimente.

Multifactorial analysis was applied to studies on the effect of doxycycline on the primary immune response to sheep erythrocytes. The experimental factors were the following: the antibiotic dose, the antigen dose and the time of the onset of the antibiotic therapy with respect to the antigen action. Polynomial statistic models describing the delayed hypersensitivity and antibody titers within wide ranges of factor values were designed by the experimental data. It was shown that the prophylactic use of doxycycline prior to the antigenic stimulus markedly lowered the high-dose tolerance induced by high doses of the antigen.

[The correction of the functional activity of alveolar macrophages in inducing a primary immune response in influenza]. / Korrektsiia funktsional'noi aktivnosti al'veoliarnykh makrofagov v induktsii pervichnogo immunnogo otveta pri grippe.

The functional activity of alveolar macrophages obtained from mice, both healthy and infected with influenza virus A/Aichi 2/68 (H3N2), as manifested by their capacity to initiate the development of primary immune response to sheep red blood cells and Escherichia coli lipopolysaccharide after the transfer of these macrophages to intact syngeneic recipients was studied. The capacity of alveolar macrophages to perform antigen-presenting functions in the induction of humoral immune response was shown, and at the same time the development of experimental influenza infection was found to essentially decrease these properties. The injection of the immunomodulating agent diuciphon into experimental mice somewhat enhanced the immune response after the syngeneic transfer of alveolar macrophages from infected mice to intact recipients.

[Monoamines in the hypothalamic structures in the first few hours after immunization]. / Monoaminy v strukturakh gipotalamusa v pervye chasy posle immunizatsii.

This paper examines the time course of changes in the levels of dopamine, norepinephrine, serotonin and their metabolites in the hypothalamic structures closely related to neurohumoral regulation of immunogenesis. The findings suggest that the monoaminergic systems of the brain are widely involved in the body's response to antigen, which may lead to the conclusion that there are numerous modes of entering information from the immune to the nervous system.