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Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients’ circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψm) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψm. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψm. It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψm. The specificity of these changes was here controlled by membrane depolarization with the Ψm disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06–0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs.

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1&micro, M). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-&alpha, content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1&beta, (IL-1&beta, ), and tumor necrosis factor-&alpha, (TNF-&alpha, ) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 &mu, M) significantly inhibited the release of NO, TNF-&alpha, and expression of iNOS, IL-1&beta, and TNF-&alpha, in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

One of the approaches to address cancer treatment is to develop new drugs not only to obtain compounds with less side effects, but also to have a broader set of alternatives to tackle the resistant forms of this pathology. In this regard, growing evidence supports the use of bacteria-derived peptides such as bacteriocins, which have emerged as promising anti-cancer molecules. In addition to test the activity of these molecules on cancer cells in culture, their in vivo antitumorigenic properties must be validated in animal models. Although the standard approach for such assays employs experiments in nude mice, at the initial stages of testing, the use of high-throughput animal models would permit rapid proof-of-concept experiments, screening a high number of compounds, and thus increasing the possibilities of finding new anti-cancer molecules. A validated and promising alternative animal model are zebrafish larvae harboring xenografts of human cancer cells. Here, we addressed the anti-cancer properties of the antibacterial peptide microcin E492 (MccE492), a bacteriocin produced by Klebsiella pneumoniae, showing that this peptide has a marked cytotoxic effect on human colorectal cancer cells in vitro. Furthermore, we developed a zebrafish xenograft model using these cells to test the antitumor effect of MccE492 in vivo, demonstrating that intratumor injection of this peptide significantly reduced the tumor cell mass. Our results provide, for the first time, evidence of the in vivo antitumoral properties of a bacteriocin tested in an animal model. This evidence strongly supports the potential of this bacteriocin for the development of novel anti-cancer therapies.

Aim: To develop a real-time PCR-based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real-time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml−1 and 10 spores ml−1 were determined. Compared to spread plate method, this real-time PCR-based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false-positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g−1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real-time PCR-based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.

CD34, a glycophosphoprotein present in lymphohaematopoietic stem and progenitor cells, as well as in other cell types, exists in both transmembrane and intracytoplasmic forms. Transmembrane CD34 expression, which is high in the earliest haematopoietic precursors, decreases as cells mature. However, to our knowledge, there is no information on whether a decrease in transmembrane CD34 can also predict a release of the molecule from the cell membrane into the extracellular fluid. To investigate the above possibility, we studied conditions (incubation time, cell density and proliferative status) in human myeloid cells (lines KG-1a, KG-1 and cord blood-derived cells) that may cause a decrease in surface CD34 and the generation of a soluble form of the molecule. The latter, as demonstrated by Western blot analysis, adds more complexity to the proposed structural features and functional properties of CD34 in myeloid cells.

Respiratory syncytial virus (RSV) and adenovirus (Advs) serotype 3 (Adv3) and 7h (Adv7h) are associated with mild to severe respiratory infection and are indistinguishable during the acute phases of the illnesses. However, outcome and long-term prognosis are different with both infections. RSV infection is associated with later development of asthma, and Adv, mainly Adv7h, with severe lung damage, bronchiectasis, and hyperlucent lung. We hypothesized that this difference could be partly due to different immune responses induced by these viruses. To test this hypothesis we quantified TCD4+, TCD8+, and BCD19+ expressing the interleukin-2 receptor-alpha chain (CD25) and interferon-gamma (IFN-gamma), interleukin (IL)-10, and IL-4 in the supernatant of peripheral blood mononuclear cells (PBMC) from school children infected in vitro with and without RSV, Adv7h, and Adv3 and after phytohemagglutinin (PHA) stimulation in the presence or absence of these viruses at a multiplicity of infection (MOI) of 1. PBMC from every child produced more IL-10 (p

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increase in the percentage of autoreactive B and T lymphocytes. Since dendritic cells (DCs) are essential for B cell and T cell function, we hypothesized that changes in DC biology may play a critical role in the pathogenesis of the disease. We analyzed the phenotype and distribution of two main DC subsets, conventional (cDC) and plasmacytoid (pDC), in lupus prone (NZW × NZB)F1 (BWF1) mice and age-matched NZW × BALB/c control mice. Our results show that both subsets of lupic DCs displayed an abnormal phenotype, characterized by an over-expression of the co-stimulatory molecules CD80, CD86, PD-L1 and PD-L2 compared with control mice. Accordingly, spleen CD4(+) T cells from lupic mice exhibit an activated phenotype characterized by a higher expression of PD-1, CD25, CD69 and increased secretion of IFN-γ and IL-10. Interestingly, lupic mice also present an increase in the percentage of cDC in peripheral blood and an increase in the percentage of pDCs in spleen and mesenteric lymph nodes (MLNs) compared with control and pre-lupic mice. Homing experiments demonstrate that lupic and pre-lupic DCs migrate preferentially to the spleen compared to DCs from control mice. This preferential recruitment and retention of DCs in the spleen is related to an altered expression of different chemokine and chemokine receptors on both, DCs and stromal cells from lupic mice. Our results suggest that this altered phenotype and migratory behavior shown by DCs from lupic mice may account for the abnormal T cell and B cell responses in lupus.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic β-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.

Depressed immune response is well documented in protein-calorie malnutrition (PCM). Also, central and peripheral noradrenaline (NA) activities have been reported to be increased in malnourished animals. Since increases in central and peripheral NA may inhibit immune function, it is possible that malnutrition-induced immunodepression could be mediated by noradrenergic hyperactivity. To address this hypothesis the effect of malnutrition on cell-mediated immune response, as well as on NA levels of the median eminence, spleen and thymus was studied in PCM rats. Decreased lymphoproliferative response and IL-1 production by mononuclear macrophages was observed in PCM. Besides, increased NA concentration was detected in thymuses of PCM rats, while unchanged levels of this neurotransmitter were observed in median eminence and spleen. These data suggest a positive correlation between malnutrition-induced immunodepression and sympathetic noradrenergic activity in thymus, an organ implicated in immune cell differentiation during early development.

Type 1 diabetes mellitus (T1D) is due to autoimmune destruction of pancreatic beta-cells. Previously, we have shown that intravenously administered bone marrow-derived multipotent mesenchymal stromal cells (MSCs) allows pancreatic islet recovery, improves insulin secretion and reverts hyperglycemia in low doses streptozotocin (STZ)-induced diabetic mice. Here we evaluate whether insulin prophylaxis and the administration of a second dose of cells affect the antidiabetic therapeutic effect of MSC transplantation. Insulitis and subsequent elimination of pancreatic beta-cells was promoted in C57BL/6 mice by the injection of 40 mg/kg/day STZ for five days. Twenty-four days later, diabetic mice were distributed into experimental groups according to if they received or not insulin and/or one or two doses of healthy donor-derived MSCs. Three and half months later: glycemia, pancreatic islets number, insulinemia, glycated hemoglobin level and glucose tolerance were determined in animals that did not received exogenous insulin for the last 1.5 months. Also, we characterized MSCs isolated from mice healthy or diabetic. The therapeutic effect of MSC transplantation was observed in diabetic mice that received or not insulin prophylaxis. Improvements were similar irrespective if they received one or two doses of cells. Compared to MSCs from healthy mice, MSCs from diabetic mice had the same proliferation and adipogenic potentials, but were less abundant, with altered immunophenotype and no osteogenic potential. Our preclinical results should be taken into account when designing phase II clinical trials aimed to evaluate MSC transplantation in patients with T1D. Cells should be isolated form healthy donor, insulin prophylaxis could be maintained and a second dose, after an elapse of two months, appears unnecessary in the medium-term.

In animal models it has been shown that mesenchymal stromal cells (MSC) contribute to skin regeneration and accelerate wound healing. We evaluated whether allogeneic MSC administration resulted in an improvement in the skin of two patients with recessive dystrophic epidermolysis bullosa (RDEB; OMIM 226600). Patients had absent type VII collagen immunohistofluorescence and since birth had suffered severe blistering and wounds that heal with scarring. Vehicle or 0.5 x 10(6) MSC were infused intradermally in intact and chronic ulcerated sites. One week after intervention, in MSC-treated skin type VII collagen was detected along the basement membrane zone and the dermal-epidermal junction was continuous. Re-epithelialization of chronic ulcerated skin was observed only near MSC administration sites. In both patients the observed clinical benefit lasted for 4 months. Thus intradermal administration of allogeneic MSC associates with type VII collagen replenishment at the dermal-epidermal junction, prevents blistering and improves wound healing in unconditioned patients with RDEB.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-γ ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3+CD8+ T cells were specific for the single HLA-B*3501-restricted epitope Gn465–473 years after the acute infection. Remarkably, Gn465–473–specific cells readily secreted IFN-γ, granzyme B and TNF-α but not IL-2 upon stimulation and showed a ‘revertant’ CD45RA+CD27−CD28−CCR7−CD127− effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines., Author Summary In man, hantavirus cardiopulmonary syndrome (HCPS) caused by Andes Virus (ANDV) is endemic in the Southern cone of Chile and Argentina but cases of HCPS are being increasingly reported all over South America since 1995. HCPS is characterized by fulminant pulmonary edema which progresses to shock and death in about 36% of patients with HCPS. Nevertheless, to date, neither antiviral treatments nor vaccines inducing neutralizing antibodies (NAb) have proven effective against HCPS-causing hantaviruses. We set out for the first study on human cellular immunity towards ANDV in 78 convalescent survivors of ANDV infection. We found that Gn-specific responses were predominant as compared to N- and Gc-specific responses, even up to 13 years after the infection. Surprisingly, most of the Gn-specific responses were restricted to two neighboring epitopes within the Gn carboxyterminus. Interestingly, among HLA-B*3501+ patients, Gn465−473-specific CD8+ T-cells showed highly differentiated but resting phenotype and functions. It remains to be seen in future studies whether the immunodominace of Gn-specific T-cells is crucial for protective immunity. Most intriguingly, titers of neutralizing antibodies increased in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest viral persistence or latency in part of ANDV-convalescent patients. However, it remains a major task for future studies to proof the concept of latent/persistent human ANDV infection by the determination of viral antigen in convalescent patients.

Viral infections have been associated with cellular immune responses and production of Th-1 cytokines. Respiratory syncytial virus (RSV), however, induces virus-specific IgE, which might be a consequence of a Th-2-like activation. To test this hypothesis we quantified interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the supernatant of peripheral blood mononuclear cells cultured for 24 and 48 h in the presence or absence of phytohemaglutinin and pokeweed mitogen and the lymphocyte phenotypes to analyze subsets and their activation markers, from 15 hospitalized infants during an acute lower respiratory infection caused by RSV and 17 healthy control infants from 1 to 15 mo of age. Compared with the control infants, those infected with RSV had an increase in the number of B-cells (p0.02) and decreases in both CD8+ T-cells (p0.01) and activated CD8+/CD25+ suppressor/ cytotoxic T-cells (p0.007). In RSV-infected infants, IFN-gamma production was subtotally suppressed, whereas IL-4 production was decreased to a lesser degree, giving significantly (p0.001) increased IL-4/IFN-gamma ratio compared with that in the control infants. These findings suggest a predominant Th-z-like response in RSV-infected infants, which could explain some aspects of the immunopathogenesis of RSV infection and the RSV-specific and nonspecific IgE antibody responses observed.

Depressed immune function is well documented in protein calorie malnutrition (PCM). Also, central noradrenergic hyperactivity has recently been reported in malnourished animals. Increase in central noradrenaline activity could be responsible for cell-mediated immune depression. The present study is designed to address this hypothesis by testing whether neurotoxic lesion of central noradrenergic systems by 6-hydroxydopamine (6-OHDA) could improve lymphoproliferative response to mitogens and interleukin (IL)-l production in PCM rats. A significant enhancement of lymphoproliferative response to concanavalin A (ConA) and in IL-1 production was observed in spleen mononuclear cells of PCM rats injected intracerebroventricularly with 120 μg of 6-OHDA, as compared with solvent injected and untreated PCM animals. A significant decrease in brain noradrenaline levels was produced in the drug-injected animals. These results suggest that central noradrenergic hyperactivity is one of the mechanisms involved in the immunodepression produced by malnutrition.

Twenty-five to 40% of diabetic patients develop diabetic nephropathy, a clinical syndrome that comprises renal failure and increased risk of cardiovascular disease. It represents the major cause of chronic kidney disease and is associated with premature morbimortality of diabetic patients. Multipotent mesenchymal stromal cells (MSC) contribute to the regeneration of several organs, including acutely injured kidney. We sought to evaluate if MSC protect kidney function and structure when endovenously administered to mice with severe diabetes. A month after nonimmunologic diabetes induction by streptozotocin injection, C57BL/6 mice presented hyperglycemia, glycosuria, hypoinsulinemia, massive beta-pancreatic islet destruction, low albuminuria, but not renal histopathologic changes (DM mice). At this stage, one group of animals received the vehicle (untreated) and other group received 2 doses of 0.5 x 10(6) MSC/each (MSC-treated). Untreated DM mice gradually increased urinary albumin excretion and 4 months after diabetes onset, they reached values 15 times higher than normal animals. In contrast, MSC-treated DM mice maintained basal levels of albuminuria. Untreated DM mice had marked glomerular and tubular histopathologic changes (sclerosis, mesangial expansion, tubular dilatation, proteins cylinders, podocytes lost). However, MSC-treated mice showed only slight tubular dilatation. Observed renoprotection was not associated with an improvement in endocrine pancreas function in this animal model, because MSC-treated DM mice remained hyperglycemic and hypoinsulinemic, and maintained few remnant beta-pancreatic islets throughout the study period. To study MSC biodistribution, cells were isolated from isogenic mice that constitutively express GFP (MSC(GFP)) and endovenously administered to DM mice. Although at very low levels, donor cells were found in kidney of DM mice 3 month after transplantation. Presented preclinical results support MSC administration as a cell therapy strategy to prevent chronic renal diseases secondary to diabetes.

It has been established that early events in lymphocyte activation involve a rise in intracellular Ca++ as well as changes in the flux of other ions. Although a Ca++ channel has been postulated to participate in the early Ca++ rise, its presence in lymphocytes remains controversial. Also although yet undetected, electrophysiological data suggest the presence of a Ca++ activated K+ channel on human peripheral blood lymphocytes (HPBL). Here we report on the effect of specific channel blockers as an approach to the identification of these channels on HPBL. At 40 nM nifedipine, an inhibitor of voltage-gated Ca++ channels, fully inhibits the PHA-promoted activation of HPBL. This effect is concentration dependent with a half maximum effect at approximately 10 nM and is demonstrable whether the drug is added at the same time as or up to 18 h after the addition of the mitogen. This inhibition of activation is not seen if the lymphocytes are activated using IL-2 instead of PHA. Charybdotoxin a toxin which blocks a Ca++ activated K+ channel of muscle cells also blocks to almost 100 per cent the PHA-induced activation of HPBL. This inhibition can be demonstrated regardless of whether the blocker is added together with or up to 4 h after PHA. As opposed to nifedipine charybdotoxin shows no effect if added 18 h after the initiation of the activation process. When nifedipine and charybdotoxin were tested on mice splenocytes we found that nifedipine fully inhibits the LPS-promoted activation of these cells while charybdotoxin has no effect on their activation.(ABSTRACT TRUNCATED AT 250 WORDS)