Early Development and Putative Primordial Germ Cells Characterization in Dogs

Contents Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.

Use of extensible internal device in the femur of young dogs

An extensible internal device (EID) was developed to preserve growth plate during the treatment of fracture complications or segmental bone loss from tumour resection in children. Since this type of extensible, trans-physeal, internal fixation device has only been used in a few paediatric cases; the aim of this study was to evaluate an in vivo canine study, a surgical application of this device, and its interference with longitudinal growth of the non-fractured distal femur. Ton clinically healthy two- to three-month-old poodles weighing 1.5-2.3 kg were used. Following a medial approach to the right distal femur, one extremity of the EID, similar to a T-plate, was fixed in the femoral condyle with two cortical screws placed below the growth plate. The other extremity, consisting of an adaptable brim with two screw holes and a plate guide, was fixed in the third distal of the femoral diaphysis with two cortical screws. The EID was removed 180 days after application. All of the dogs demonstrated full weight-bearing after surgery. The values of thigh and stifle circumferences, and stifle joint motion range did not show any difference between operated and control hindlimbs. The plate slid in the device according to longitudinal bone growth, in all but one dog. In this dog, a 10.5% shortening of the femoral shaft was observed due to a lack of EID sliding. The other dogs had the some longitudinal lengths in both femurs. The EID permits longitudinal bone growth without blocking the distal femur growth plate if appropriately placed.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

Animal models for genetic neuromuscular diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for delta-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy(2J)/dy(2J) mouse, both presenting significant reduction of alpha 2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large(myd)) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of alpha-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) is a dimorphic fungal pathogen that causes paracoccidioidomycosis the most severe deep mycosis from South America Although cell mediated immunity is considered the most efficient protective mechanism against Pb infection mechanisms of innate immunity are poorly defined Herein we investigated the interaction of the complement system with high and low virulence isolates of Pb We demonstrated that Pb18 a high virulence Pb Isolate when incubated with normal human serum (NHS) induces consumption of hemolytic complement and when immobilized promotes binding of C4b C3b and C5b-C9 Both low virulence (Pb265) and high virulence (Pb18) isolates consumed C4 C3 and mannose-binding learn (MBL) of MBL-sufficient but not of MBL-deficient serum as revealed by deposition of residual C4 C3 and MBL on immune complexes and mannan However higher complement components consumption was observed with Pb265 as compared with Pb18 The suggested relationship between low virulence and significant complement activation properties of Pb isolates was confirmed by the demonstration that virulence attenuation of Pb 18 results in acquisition of the ability to activate complement Conversely reactivation of attenuated Pb18 results in loss of the ability to activate complement Our results demonstrate for the first time that Pb yeasts activate the complement system by the lectin pathway and there is an Inverse correlation between complement activating ability and Pb virulence These differences could exert an influence on Innate immunity and severity of the disease developed by infected hosts (C) 2010 Elsevier Ltd All rights reserved

Matrix metalloproteinases with gelatinolytic activity induced by Paracoccidioides brasiliensis infection

P>Matrix metalloproteinases (MMPs) modulate extracellular matrix turnover, inflammation and immunity. We studied MMP-9 and MMP-2 in experimental paracoccidioidomycosis. At 15 and 120 days after infection (DAI) with virulent Paracoccidioides brasiliensis, MMP-9 was positive by immunohistochemistry in multinucleated giant cells, in mononuclear cells with macrophage and lymphocyte morphologies and also in fungal cells in the lesions of susceptible and resistant mice. Using gelatin zymography, pro- and active MMP-9 and active MMP-2 were detected in all infected mice, but not in controls. Gelatinolytic activity was not observed in P. brasiliensis extracts. Semiquantitative analysis of gelatinolytic activities revealed weak or absent MMP-2 and strong MMP-9 activity in both mouse strains at 15 DAI, declining at 120 DAI. Avirulent P. brasiliensis-infected mice had residual lesions with MMP-9-positive pseudoxantomatous macrophages, but no gelatinase activity at 120 DAI. Our findings demonstrate the induction of MMPs, particularly MMP-9, in experimental paracoccidioidomycosis, suggesting a possible influence in the pattern of granulomas and in fungal dissemination.

Nitric oxide participation in granulomatous response induced by Paracoccidioides brasiliensis infection in mice

The role of nitric oxide (NO) in granulomas of Paracoccidioides brasiliensis-infected inducible NO synthase-deficient C57BL/6 mice (iNOS KO) and their wild-type counterparts and its association with osteopontin (OPN) and matrix metalloproteinases (MMPs) was studied. At 15 days after infection (DAI), iNOS KO mice showed compact and necrotic granulomas with OPN+ macrophages and multinucleated giant cells, whereas wild-type mice developed loose granulomas with many fungi and OPN+ cells distributed throughout the tissue. In addition, high OPN levels and fungal load were observed in iNOS KO mice. Both experimental groups had MMP-9 activity. At 120 DAI, iNOS KO had smaller granulomas with OPN+ cells, lower OPN levels, lower fungal load and decreased MMP-9 activity compared with wild-type mice. These findings suggest that NO has an important role in granuloma modulation, by controlling OPN and MMP production, as well as by inducing loose granulomas formation and fungal dissemination, resulting, at later phases, in progression of paracoccidioidomycosis.

Osteopontin involvement in granuloma formation and in the severity of Paracoccidioides brasiliensis infection

The participation of osteopontin (OPN) in Paracoccidioides brasiliensis infected mice, its association to granulomatogenesis, severity of infection, pattern of lesions, nitric oxide (NO) levels and fungal load were evaluated in this investigation. Immunohistochemistry analysis showed marked OPN staining in extracellular matrix and in macrophages and multinucleated giant cells at the center of lesions, suggesting a possible role of OPN in the distribution of these cells within the granulomas. At 15 days post-infection with a virulent P. brasiliensis isolate, OPN(+) cells were more numerous and intensely immunostained in the loose granulomas of susceptible mice than in those of resistant mice. In addition, high fungal loads and low NO levels were observed in susceptible mice. At 120 days after infection, resistant mice had increased total OPN levels (ELISA) and OPN positivity in compact granulomas, higher NO levels and lower fungal loads than susceptible mice. Residual lesions associated with low OPN levels, high NO and control of fungal dissemination were observed in both mouse strains at 120 days post-infection with the slightly virulent fungal isolate. Therefore, OPN could be associated with higher severity of the disease in an early phase of infection and with a degree of control of the progressive infection.

MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection

The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.

Alveolar macrophages from susceptible mice are more competent than those of resistant mice to control initial Paracoccidioides brasiliensis infection

Alveolar macrophages ( AM) are the first host cells to interact with Paracoccidioides brasiliensis (Pb), a primary human pathogen that causes severe pulmonary infections in Latin America. To better understand innate immunity in pulmonary paracoccidioidomycosis, we decided to study the fungicidal and secretory abilities of AM from resistant (A/J) and susceptible (B10.A) mice to infection. Untreated, IFN-gamma and IL-12 primed AM from B10. A and A/J mice were challenged with P. brasiliensis yeasts and cocultured for 72 h. B10. A macrophages presented an efficient fungicidal ability, were easily activated by both cytokines, produced high levels of nitric oxide ( NO), IL-12, and MCP-1 associated with low amounts of IL-10 and GM-CSF. In contrast, A/J AM showed impaired cytokine activation and fungal killing, secreted high levels of IL- 10 and GM-CSF but low concentrations of NO, IL- 12, and MCP-1. The fungicidal ability of B10. A but not of A/J macrophages was diminished by aminoguanidine treatment, although only the neutralization of TGF-beta restored the fungicidal activity of A/J cells. This pattern of macrophage activation resulted in high expression of MHC class II antigens by A/J cells, while B10. A macrophages expressed elevated levels of CD40. Unexpectedly, our results demonstrated that susceptibility to a fungal pathogen can be associated with an efficient innate immunity, while a deficient innate response can ultimately favor the development of a resistant pattern to infection. Moreover, our data suggest that different pathogen recognition receptors are used by resistant and susceptible hosts to interact with P. brasiliensis yeasts, resulting in divergent antigen presentation, acquired immunity, and disease outcomes.

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4(+) T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Characterization of Paracoccidioides brasiliensis COX9, COX12, and COX16 respiratory genes

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that is the causative agent of paracoccidioidomyicosis (PCM), a human systemic granulomatous mycosis found in Latin America. Dimorphic transition from mycelium to yeast is required for establishing pathogenicity. Dimorphism is marked by changes in mitochondrial physiology, including modulation of respiration rate. In this work, we present the identification of three P. brasiliensis nuclear genes PbCOX9, PbCOX12, and PbCOX16 that code for structural sub-units and a putative assembly facilitator (PbCOX16) of the mitochondrial cytochrome c oxidase (COX), the terminal enzyme complex of the respiratory chain. We measured their expression pattern during the dimorphic transition from mycelium to yeast and back by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). Our results show that messages from these genes increase during the mycelium to yeast transition and decrease during the opposite conversion. This result supports active mitochondrial participation in the transition. Heterologous complementation of the corresponding Saccharomyces cerevisiae null mutant with the PbCOX9 gene was successfully obtained. (C) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis

Paracoccidioidomycosis is a systemic granulomatous disease manifested in the acute/subacute or chronic forms. The anergic cases of the acute/subacute form are most severe, leading to death threatening conditions. Drug treatment is required to control the disease but the response in anergic patients is generally poor. A 15-mer peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4(+) helper-1 immune response in mice of different haplotypes and protects against intratracheal challenge with virulent P. brasiliensis. Presently, P10 immunization and chemotherapy were associated in an attempt to improve antifungal treatment in Balb/c mice made anergic by adding dexamethasone to the drinking water. The combined drug/peptide treatment significantly reduced the lung CFUs in infected anergic mice, largely preserved lung alveolar structure and prevented fungal dissemination to liver and spleen. Results recommend that a P10-based vaccine should be associated to chemotherapy for improved treatment of paracoccidioidomycosis aiming especially at anergic cases. (C) 2008 Elsevier Masson SAS. All rights reserved.