Characterization of the B-Cell Epitopes of Echinococcus granulosus Histones H4 and H2A Recognized by Sera From Patients With Liver Cysts.

Cystic echinococcosis (CE) is a zoonotic disease worldwide distributed, caused by the cestode Echinococcus granulosus sensu lato (E. granulosus), with an incidence rate of 50/100,000 person/year and a high prevalence in humans of 5-10%. Serology has variable sensitivity and specificity and low predictive values. Antigens used are from the hydatid fluid and recombinant antigens have not demonstrated superiority over hydatid fluid. A cell line called EGPE was obtained from E. granulosus sensu lato G1 strain from bovine liver. Serum from CE patients recognizes protein extracts from EGPE cells with higher sensitivity than protein extracts from hydatid fluid. In the present study, EGPE cell protein extracts and supernatants from cell colonies were eluted from a protein G affinity column performed with sera from 11 CE patients. LC-MS/MS proteomic analysis of the eluted proteins identified four E. granulosus histones: one histone H4 in the cell extract and supernatant, one histone H2A only in the cell extract, and two histones H2A only in the supernatant. This differential distribution of histones could reflect different parasite viability stages regarding their role in gene transcription and silencing and could interact with host cells. Bioinformatics tools characterized the linear and conformational epitopes involved in antibody recognition. The three-dimensional structure of each histone was obtained by molecular modeling and validated by molecular dynamics simulation and PCR confirmed the presence of the epitopes in the parasite genome. The three histones H2A were very different and had a less conserved sequence than the histone H4. Comparison of the histones of E. granulosus with those of other organisms showed exclusive regions for E. granulosus. Since histones play a role in the host-parasite relationship they could be good candidates to improve the predictive value of serology in CE.

First report of cystic echinococcosis caused by Echinococcus granulosus sensu stricto/G1 in Felis catus from the Patagonian region of Argentina.

Two domestic cats from the Patagonia rural area in Argentina were found to be naturally infected with Echinococcus granulosus sensu stricto/G1 genotype; so far, the only species/genotype of E. granulosus sensu lato complex described to infect domestic cats. The felines developed abdominal disseminated larval disease; the diagnosis was performed by ultrasound, exploratory laparotomy, and molecular techniques. These results indicate that cystic echinococcosis must be considered for differential diagnosis of felines with abdominal distension and/or observation of vesicles through ultrasound, from endemic areas. Even though cats and dogs are carnivores, differences in digestive physiology and immunological characteristics between them could allow the development of larval or adult worm parasites. Domestic cats with cystic echinococcosis show to be environmentally infected with E. granulosus s. s./G1 eggs.

A protocol for umbilical cord tissue cryopreservation as a source of mesenchymal stem cells.

Mesenchymal stem cells (MSC) differentiate into different cell types and have immunomodulatory and paracrine effects. Cryopreservation of umbilical cord tissue as a source of MSC is very promising for regenerative medicine. We aim to evaluate a protocol for cryopreserving this tissue sectioned into small fragments with viable MSC. A total of 723 samples were frozen, thawed and cultured to obtain primary cultures of MSC. These were followed until 90-100% confluence and flow cytometric analysis were performed to confirm the mesenchymal phenotype. Samples in which protocol alterations at the collection of the samples were reported, were excluded for microbial contamination analysis leaving a total of 634 samples composed of 181 vaginal and 453 cesarean births. All cultures reach confluence with a media of 22.57 days and 97% in 28 or fewer days. Evaluated cultures showed low percentage of CD45+ and high of CD73 and CD90. Eight samples were subcultured 4 or 5 times and differentiated to chondrocytes and osteocytes to test differentiation potential with positive results. Umbilical cord tissue collections showed similar microbial profile and risk factors to those reported of umbilical cord blood collections, but with higher contamination frequencies. Cryopreserved tissue samples had viable cells that can be expanded without losing differentiation potential. Higher contamination frequencies compared to umbilical cord blood collection are not surprising, however, microbial load and survival of microorganisms to cryopreservation are expected to be lower.

Detection of Echinococcus granulosus sensu lato infection by using extracts derived from a protoscoleces G1 cell line.

Cystic echinococcosis (CE) can be diagnosed by means of several serological approaches, but their results vary among laboratories due to the molecular characteristics of the reference antigens used. Thus, this study aimed to address both the relevance of an EGPE cell line previously obtained from Echinococcus granulosus protoscoleces G1 and the complexity of the immune response by using two different in vitro growth stages as separate sources of parasite antigens. The serum reactivity was investigated by western blotting (WB) in 21 CE patients from an endemic area in a matched case-control design and also in seven experimentally infected sheep and five healthy control sheep. EGPE-antigen-human serum sensitivity by WB was higher than that of hydatid fluid (HF) WB, ELISA and DD5 (P < .05, Chi-square test). EGPE protein extract was immunogenic in mice and hyperimmune plasma reacted with HF proteins, and AgB2 expression was detected by molecular analysis. Proteins of 37 to 60 kDa were recognized by 95.24% of the CE patients' sera but, with poor specificity. Statistically significant differences were found between serum protein extract recognition at 7 and 20 days of cell growth. The EGPE cell line is a laboratory source of antigens for improvement of CE serological diagnosis.

B cells inhibit the antitumor immunity against an established murine fibrosarcoma.

Despite the classic role of B cells in favoring the immune response, an inhibitory action of B lymphocytes in tumor immunity has emerged in certain studies. In methylcolanthrene-induced murine fibrosarcoma (MCC), the loss of immunogenicity and the establishment of tolerance are paralleled by systemic immune suppression and the appearance of B+IL-10+ cells in tumor-draining lymph nodes. The present study aimed to assess the role of the B+IL-10+ cell population in the immune evasion and tolerance induced by MCC through the depletion of B cells in mice at various times of tumor progression: Prior to or subsequent to tumor implantation. Tumor growth and immunological parameters were evaluated. B cell depletion prior to tumor inoculum enhanced tumor growth, initiating the onset of the tumor-induced systemic immune response; however, an increase in the T regulatory cells (Tregs) at the tumor-draining lymph node could account for tumor exacerbation. B cell depletion once the tumor was established resulted in decreased tumor growth and a delayed onset of tolerance. Additionally, B cell absence exacerbated T cell dependent-tumor rejection, reduced Tregs and increased cytotoxic CD8+ T cells. In vitro analysis showed a direct effect of B cells upon T cell proliferation. In conclusion, B cell depletion exerts opposite effects when performed prior to or subsequent to tumor implantation. In this initially immunogenic tumor, B cell absence would delay the establishment of immunological tolerance probably by unmasking a pre-existing antitumor response. The present findings elucidate the convenience of modulating B cells in the development of future and more effective immunotherapies against cancer.

Regulatory cells induced by acute toxoplasmosis prevent the development of allergic lung inflammation.

The increased prevalence of allergies in developed countries has been attributed to a reduction of some infections. Supporting epidemiological studies, we previously showed that both acute and chronic Toxoplasma gondii infection can diminish allergic airway inflammation in BALB/c mice. The mechanisms involved when sensitization occurs during acute phase would be related to the strong Th1 response induced by the parasite. Here, we further investigated the mechanisms involved in T. gondii allergy protection in mice sensitized during acute T. gondii infection. Adoptive transference assays and ex vivo co-cultures experiments showed that not only thoracic lymph node cells from infected and sensitized mice but also from non-sensitized infected animals diminished both allergic lung inflammation and the proliferation of effector T cells from allergic mice. This ability was found to be contact-independent and correlated with high levels of CD4(+)FoxP3(+) cells. IL-10 would not be involved in allergy suppression since IL-10-deficient mice behaved similar to wild type mice. Our results extend earlier work and show that, in addition to immune deviation, acute T. gondii infection can suppress allergic airway inflammation through immune suppression.

Regulatory B cells present in lymph nodes draining a murine tumor.

In cancer, B cells have been classically associated with antibody secretion, antigen presentation and T cell activation. However, a possible role for B lymphocytes in impairing antitumor response and collaborating with tumor growth has been brought into focus. Recent reports have described the capacity of B cells to negatively affect immune responses in autoimmune diseases. The highly immunogenic mouse tumor MCC loses its immunogenicity and induces systemic immune suppression and tolerance as it grows. We have previously demonstrated that MCC growth induces a distinct and progressive increase in B cell number and proportion in the tumor draining lymph nodes (TDLN), as well as a less prominent increase in T regulatory cells. The aim of this research was to study B cell characteristics and function in the lymph node draining MCC tumor and to analyze whether these cells may be playing a role in suppressing antitumor response and favoring tumor progression. Results indicate that B cells from TDLN expressed increased CD86 and MHCII co-stimulatory molecules indicating activated phenotype, as well as intracellular IL-10, FASL and Granzyme B, molecules with regulatory immunosuppressive properties. Additionally, B cells showed high inhibitory upon T cell proliferation ex vivo, and a mild capacity to secrete antibodies. Our conclusion is that even when evidence of B cell-mediated activity of the immune response is present, B cells from TDLN exhibit regulatory phenotype and inhibitory activity, probably contributing to the state of immunological tolerance characteristic of the advanced tumor condition.

Regulatory B cells present in lymph nodes draining a murine tumor

In cancer, B cells have been classically associated with antibody secretion, antigen presentation and T cell activation. However, a possible role for B lymphocytes in impairing antitumor response and collaborating with tumor growth has been brought into focus. Recent reports have described the capacity of B cells to negatively affect immune responses in autoimmune diseases. The highly immunogenic mouse tumor MCC loses its immunogenicity and induces systemic immune suppression and tolerance as it grows. We have previously demonstrated that MCC growth induces a distinct and progressive increase in B cell number and proportion in the tumor draining lymph nodes (TDLN), as well as a less prominent increase in T regulatory cells. The aim of this research was to study B cell characteristics and function in the lymph node draining MCC tumor and to analyze whether these cells may be playing a role in suppressing antitumor response and favoring tumor progression. Results indicate that B cells from TDLN expressed increased CD86 and MHCII co-stimulatory molecules indicating activated phenotype, as well as intracellular IL-10, FASL and Granzyme B, molecules with regulatory immunosuppressive properties. Additionally, B cells showed high inhibitory upon T cell proliferation ex vivo, and a mild capacity to secrete antibodies. Our conclusion is that even when evidence of B cell-mediated activity of the immune response is present, B cells from TDLN exhibit regulatory phenotype and inhibitory activity, probably contributing to the state of immunological tolerance characteristic of the advanced tumor condition.

Dehydroepiandrosterone and metyrapone partially restore the adaptive humoral and cellular immune response in endotoxin immunosuppressed mice.

Prior exposure to endotoxins renders the host temporarily refractory to subsequent endotoxin challenge (endotoxin tolerance). Clinically, this state has also been pointed out as the initial cause of the non-specific humoral and cellular immunosuppression described in these patients. We recently demonstrated the restoration of immune response with mifepristone (RU486), a receptor antagonist of glucocorticoids. Here we report the treatment with other modulators of glucocorticoids, i.e. dehydroepiandrosterone (DHEA), a hormone with anti-glucocorticoid properties, or metyrapone (MET) an inhibitor of corticosterone synthesis. These drugs were able to partially, but significantly, restore the humoral immune response in immunosuppressed mice. A significant recovery of proliferative responsiveness was also observed when splenocytes were obtained from DHEA- or MET-treated immunosuppressed mice. In addition, these treatments restored the hypersensitivity response in immunosuppressed mice. Finally, although neither DHEA nor MET improved the reduced CD4 lymphocyte count in spleen from immunosuppressed mice, both treatments promoted spleen architecture reorganization, partially restoring the distinct cellular components and their localization in the spleen. The results from this study indicate that DHEA and MET could play an important role in the restoration of both adaptive humoral and cellular immune response in LPS-immunosuppressed mice, reinforcing the concept of a central involvement of endogenous glucocorticoids on this phenomenon.

Regulatory B cells present in lymph nodes draining a murine tumor. / Regulatory B cells present in lymph nodes draining a murine tumor.

In cancer, B cells have been classically associated with antibody secretion, antigen presentation and T cell activation. However, a possible role for B lymphocytes in impairing antitumor response and collaborating with tumor growth has been brought into focus. Recent reports have described the capacity of B cells to negatively affect immune responses in autoimmune diseases. The highly immunogenic mouse tumor MCC loses its immunogenicity and induces systemic immune suppression and tolerance as it grows. We have previously demonstrated that MCC growth induces a distinct and progressive increase in B cell number and proportion in the tumor draining lymph nodes (TDLN), as well as a less prominent increase in T regulatory cells. The aim of this research was to study B cell characteristics and function in the lymph node draining MCC tumor and to analyze whether these cells may be playing a role in suppressing antitumor response and favoring tumor progression. Results indicate that B cells from TDLN expressed increased CD86 and MHCII co-stimulatory molecules indicating activated phenotype, as well as intracellular IL-10, FASL and Granzyme B, molecules with regulatory immunosuppressive properties. Additionally, B cells showed high inhibitory upon T cell proliferation ex vivo, and a mild capacity to secrete antibodies. Our conclusion is that even when evidence of B cell-mediated activity of the immune response is present, B cells from TDLN exhibit regulatory phenotype and inhibitory activity, probably contributing to the state of immunological tolerance characteristic of the advanced tumor condition.

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen.

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP+rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP+rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-γ than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8(+) T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity.

B-cell lymphopoiesis is regulated by cathepsin L.

Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSL(nkt/nkt) mice that lack CTSL activity, we have previously demonstrated that the absence of CTSL activity affects the homeostasis of the T-cell pool by decreasing CD4+ cell thymic production and increasing CD8+ thymocyte production. Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSL (nkt/nkt) mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSL (nkt/nkt) mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSL (nkt/nkt) mice. Besides, BM B-cell emigration to the spleen was increased in CTSL (nkt/nkt) mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSL (nkt/nkt) mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.

[Increase of regulatory T cells in the lymph node of cathepsin L mutant mice]. / Aumento de linfocitos T regulatorios en los ganglios linfoides de ratones mutantes para catepsina L.

Regulatory CD4+CD25+Foxp3+ T cells (Treg) have been implicated in different pathologies including cancer, infections and autoimmune diseases and in the rejection of allogeneic organ transplantation. Thus, modulation of Treg activity has a great potential in the treatment of these pathologies. Herein, we evaluated the influence of cathepsin L (CTSL) on Treg homeostasis. CTSL mutant mice (CTSLnkt/nkt) showed a decrease in the absolute number of thymic Treg cells. In contrast, the absolute number of lymph node Treg cells and their frequency within CD4+ cells were increased. The absence of CTSL activity in CD4+ T cells -and not in their environment- increased the proliferation rate of lymph node CD4+ T cells. Treg and T CD4+ conventional (CD4+CD25-Foxp3-) cells from mutant mice showed similar increases in their proliferative levels as compared with control mice, suggesting that although proliferation contributes to the increases in their number, the augmentation in the frequency of Treg cells is not only associated to increases in proliferation. Furthermore, the Treg apoptosis rate was not decreased in the lymph node of CTSLnkt/nkt mice. Taking into account that the daily CD4+ thymic production is diminished in mutant mice, our results suggest that peripheral Treg increases are probably not the result of increased thymic output and raise the possibility that a conversion to Treg phenotype would be favored in the CD4+ T cells peripheral pool of CTSL mutant mice.

Plant Hsp90 proteins interact with B-cells and stimulate their proliferation.

BACKGROUND: The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro. METHODOLOGY: Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. CONCLUSIONS: Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of a non-pathogen-derived ligand for TLRs.

Lymphadenectomy exacerbates tumor growth while lymphadenectomy plus the adoptive transfer of autologous cytotoxic cells and low-dose cyclophosphamide induces regression of an established murine fibrosarcoma.

Tumor-draining lymph node (TDLN) ablation is routinely performed in the management of cancer; nevertheless, its usefulness is at present a matter of debate. TDLN are central sites where T cell priming to tumor antigens and onset of the antitumor immune response occur. However, tumor-induced immunosuppression has been demonstrated at TDLN, leading to downregulation of antitumor reaction and tolerance induction. Tolerance in turn is a main impairment for immunotherapy trials. We used a murine immunogenic fibrosarcoma that evolves to a tolerogenic state, to study the cellular and molecular mechanisms underlying tolerance induction at the level of TDLN and to design an appropriate immunotherapy. We determined that following a transient activation, the established tumor induces signs of immunosuppression at TDLN that coexist with local and systemic evidences of antitumor response. Therefore, we evaluated the feasibility of removing TDLN in order to eliminate a focus of immunosuppression and favor tumor rejection; but instead, a marked exacerbation of tumor growth was induced. Combining TDLN ablation with the in vivo depletion of regulatory cells by low-dose cyclophosphamide and the restoring of the TDLN-derived cells into the donor mouse by adoptive transference, resulted in lowered tumor growth, enhanced survival and a considerable degree of tumor regression. Our results demonstrate that important antitumor elements can be eliminated by lymphadenectomy and proved that the concurrent administration of low-dose chemotherapy along with the reinoculation of autologous cytotoxic cells provides protection. We suggest that this protocol may be useful, especially in the cases where lymphadenectomy is mandatory.

Increases in IgA(+) B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Anti-inflammatory pretreatment enables an efficient dendritic cell-based immunotherapy against established tumors.

Although animals can be immunized against the growth of some tumor implants, most of the attempts to use immunotherapy to cause the regression of animal and human tumors once they have become established have been disappointing even when strongly immunogenic tumors were used as target. In this paper, we demonstrate that the failure to achieve an efficient immunological treatment against an established strongly immunogenic murine fibrosarcoma was paralleled with the emergence of a state of immunological unresponsiveness (immunological eclipse) against tumor antigens observed when the tumor surpassed the critical size of 500 mm(3). In turn, the onset of the immunological eclipse was coincidental with the onset of a systemic inflammatory condition characterized by a high number of circulating and splenic polymorphonucleated neutrophils (PMN) displaying activation and Gr1(+)Mac1(+) phenotype and an increasing serum concentration of the pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 cytokines and C-reactive protein (CRP) and serum A amyloid (SAA) phase acute proteins. Treatment of tumor-bearing mice with a single low dose (0.75 mg/kg) of the synthetic corticoid dexamethasone (DX) significantly reduced all the systemic inflammatory parameters and simultaneously reversed the immunological eclipse, as evidenced by the restoration of specific T-cell-dependent concomitant immunity, ability of spleen cells to transfer anti-tumor activity and recovery of T-cell signal transduction molecules. Two other anti-inflammatory treatments by using indomethacin or dimeric TNF-alpha receptor, also partially reversed the immunological eclipse although the effect was not as striking as that observed with DX. The reversion of the immunological eclipse was not enough on its own to inhibit the primary growing tumor. However, when we used the two-step strategy of inoculating DX to reverse the eclipse and then dendritic cells loaded with tumor antigens (DC) as an immunization booster, a significant inhibition of the growth of both established tumors and remnant tumor cells after excision of large established tumors was observed, despite the fact that the vaccination alone (DC) had no effect or even enhanced tumor growth in certain circumstances. The two-step strategy of tumor immunotherapy that we present is based on the rationale that it is necessary to eliminate or ameliorate the immunological eclipse as a precondition to allow an otherwise ineffective anti-tumor immunological therapy to have a chance to be successful.