Expression of Siglec-1, -3, -5 and -10 in porcine cDC1 and cDC2 subsets from blood, spleen and lymph nodes and functional capabilities of these cells.

Dendritic cells are professional antigen-presenting cells that play a critical role in the development of immune responses. DCs express a variety of Siglecs on their surface, which play a regulatory role modulating their activation through interaction with sialylated structures expressed by cells or pathogens. Here, we characterized the phenotype of porcine conventional dendritic cells subsets from blood, spleen and lymph nodes, emphasizing the analysis of the expression of Siglecs. Siglec-1 was detected in type 1 cDC and, at lower levels, in type 2 cDC in the spleen, being low to negative in blood and lymph node cDC. Siglec-3 and Siglec-5 were expressed in cDC1 at lower levels than in cDC2. Porcine cDCs did not express Siglec-10. cDC2 showed a higher capacity to phagocytose microspheres and to process DQ™-OVA than cDC1, but none of these functions was affected by engagement of Siglec-3 and -5 with antibodies on blood cDC.

Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena.

Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals. PRRSV-N-protein+ cells were linked to a marked drop of CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1+ cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.

Analysis of the expression of porcine CD200R1 and CD200R1L by using newly developed monoclonal antibodies.

CD200R1 and CD200R1-like are paired receptors which modulate activation of immune cells. Here, we describe the characterisation of their porcine homologues. Analysis of database porcine sequences shows an exceptionally high homology between the extracellular Ig-like domains of these receptors, being the rest more dissimilar. We have obtained two mAbs, PCT1 and PCT3, against a CD200R1-Fc recombinant protein, that bind on CHO cells expressing GFP-tagged CD200R1. The specificity of these mAbs was analysed on CD200R1 L, and also on a CD200R1 splicing variant that lacks the V-type Ig domain. PCT1 bound to both CD200R1 and CD200R1L, but not to the splicing variant, what suggests that recognises an epitope in the V-type Ig domain. PCT3 reacted with both CD200R1 variants, but not CD200R1L, probably binding to an epitope in the N-terminal sequence of CD200R1. Analysis of porcine cells with these mAbs showed expression of CD200R1/CD200R1L on B cells, monocytes and alveolar macrophages.

Characterisation of porcine bone marrow progenitor cells identified by the anti-c-kit (CD117) monoclonal antibody 2B8/BM.

c-kit (CD117) plays an important role in the early stages of haematopoiesis. Previous studies of porcine haematopoietic stem cells have relied for their identification on the use of the c-kit ligand stem cell factor. Here, we describe a new mAb, 2B8/BM, that recognizes a 155-kDa protein expressed on a small subset (2-8%) of bone marrow haematopoietic cells. 2B8/BM(+) cells have a blast appearance, and are mostly negative for lineage-specific markers or express low levels of CD172a or SLA-II. In in vitro colony-forming unit assays these cells were able to give rise to erythroid and myeloid colonies. Altogether these data suggested that the 2B8/BM antigen might be the porcine orthologue of the human c-kit. This specificity was confirmed by the binding of mAb 2B8/BM to CHO cells transfected with a plasmid encoding the porcine c-kit ectodomain. This antibody can facilitate the isolation and enrichment of porcine stem cells to be used in procedures aimed to induce xenograft tolerance or to test their potential to repair damaged tissues and organs.

Phenotypic and functional characterization of porcine granulocyte developmental stages using two new markers.

Here, we describe two new surface antigens, named 6D10 and 2B2, whose expression is restricted to porcine granulocytes. 6D10 is only detected in neutrophils and its expression decreases from promyelocytes to mature cells. By contrast, 2B2 antigen is selectively expressed in mature neutrophils, eosinophils and basophils. The expression of these antigens along granulocyte maturation allows the discrimination of several developmental stages of granulocytes based on phenotypic, morphological and functional characteristics previously established. Moreover, these new markers are useful tools to easily characterize the different granulocytes lineages (neutrophils, eosinophils and basophils). By using multiparameter flow cytometric analysis, we have performed a phenotypic and functional characterization of the granulocyte subsets identified by the combination of 6D10 and 2B2 antigens.

Restoration of endocrine function and fertility with a tubo-ovarian autotransplant as the anatomical-functional unit in rabbits using a vascular microsurgical technique.

Infertility has been considered a global public health problem in many countries worldwide. Our objective was to restore endocrine function and fertility in tubal-oophorectomized rabbits using an orthotopic tubal-ovarian vascularized autotransplant model as the anatomical-functional unit while employing a microvascular surgical technique. Twenty New Zealand white (NZW) sexually mature female rabbits and four male NZW rabbits of proven fertility were divided into two study groups. In group I (n = 10), a left salpingo-oophorectomy was performed. Group II (n = 10) was subjected to a bilateral salpingo-oophorectomy, plus a right orthotopic tubal-ovarian autotransplant. Our testing variables were vascular and tubal-anastomoses permeability, estradiol (E2) and progesterone (P4) serum levels, pregnancy, number of offspring, histopathological study of the uteri, fallopian tubes, and ovaries. One hundred percent immediate permeability of the tubal anastomoses was achieved, while late permeability was found to be 64%. Immediate permeability of vascular anastomoses was 90%, and late permeability was recorded at 80%. E2 serum levels in both groups at different times showed no statistically significant differences. In the case of P4, a small difference was found during pregnancy, especially greater in the control group (P < .05). In the autotransplanted group, four rabbits became pregnant (44%). Endocrine function and fertility were restored in the rabbits with the tubal-ovarian transplant as the anatomical-functional unit. The use of isotransplants and allotransplants should be considered a therapeutic alternative in the infertile woman with irreparable bilateral tubal damage, ovarian dysgenesis, surgical absence of ovaries and fallopian tubes, or when the conventional IVF/TE in these cases has been unsuccessful.

Restoration of endocrine function and ovulation after a heterotopic ovarian transplant in the inguinal region of rabbits using a vascular microsurgical technique.

The aim of this study was to define an experimental model in rabbits for subcutaneous heterotopic ovarian autotransplants and allotransplants in the inguinal region using a microvascular technique to restore endocrine function and ovulation. Forty sexually mature New Zealand white receptor rabbits and 20 donating Californian rabbits were divided into two experimental models: model A; autogenic model-control group 1 (n = 10), right ovariectomy; group II (n = 10), heterotopic ovarian autotransplant with peritoneal pouch plus left ovariectomy; model B: allogenic model-donator group III (n = 10), right ovariectomy with peritoneal tissue; receptor group (n = 10), ovarian heterotopic allotransplant with peritoneal pouch and bilateral ovariectomy, without immunosuppression; group IV donator (n = 10), receptor (n = 10) using the same procedure as in group III, administering cyclosporine 4 mg/kg/d intramuscularly and prednisone 1 mg/kg/d PO for 28 days. Ovarian function was assessed in the transplanted ovary after stimulation with human chorionic gonadotropin (100 IU). Exfoliative vaginal cytology was done, serum estradiol (E2) and progesterone (P(4)) were measure, and a histological study of ovaries and uteri was done. Late vascular permeability was 73.3%. Serum E2 and P4 levels during the poststimulation period were extremely low exclusively in group III (P < .05). In all viable grafts, the histological study showed follicular development and presence of luteal bodies. In the uteri, the endometrium was proliferative and vaginal cytology showed the karyopicnotic index was >20%. Endocrine function and ovulation were restored in the heterotopic transplanted ovary. Allogenic heterotopic ovarian transplants are indicated in women with gonadal dysgenesia or premature surgical menopause.

Intrahepatic DNA vaccination: unexpected increased resistance against murine cysticercosis induced by non-specific enhanced immunity.

Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model with which to test the efficacy of new vaccine candidates and delivery systems against pig cysticercosis. A high level of protection against murine cysticercosis was previously observed by intramuscular or intradermal DNA immunization with the use of the sequence of the recombinant KETc7 antigen cloned in pcDNA3 (pTc-sp7). To determine the effect of KETc7 differential expression in DNA vaccination, KETc7 was cloned in pGEM 11Zf(+) under the control of the tissue-specific regulatory promoter phosphoenolpyruvate carboxykinase (pPc-sp7). A high level of protection was induced by intrahepatic immunization with pPc-sp7, pTc-sp7 and the empty vector in the absence of any specific immunity. The empty vector pGEM 11Zf(+), the plasmid with the highest content of CpG sequences, provided to the most efficient protection. This protection was related to an increased number of splenocytes, enhanced nonspecific splenocyte proliferation, and intensified intrahepatic INF-gamma production. Overall, intrahepatic plasmid CpG-DNA immunization provokes an exacerbated nonspecific immune response that can effectively control Taenia crassiceps cysticercosis.

Effective protection against experimental Taenia solium tapeworm infection in hamsters by primo-infection and by vaccination with recombinant or synthetic heterologous antigens.

The disease caused by Taenia solium is progressively being recognized as a growing global threat for public human health and pig husbandry that requires the development of effective control measures. A central participant in the taeniasis/cysticercosis transmission network is the human carrier of the adult tapeworm because of its great potential in spreading the infection. Herein, evidence is presented that a primary infection of golden hamsters with orally administered T. solium cysticerci improved the host's resistance against a secondary infection. Likewise, previous vaccination increased the hamster's resistance. Similar high levels of protection (> 78%) were induced by systemic or oral vaccination with the S3Pvac anticysticercosis synthetic peptide vaccine or the highly immunogenic recombinant chimera based on the protective peptide KETc1 bound to Brucella spp. lumazine synthase (BLS-KETc1). Increased resistance after primo-infection and vaccination possibly results from changes in the immune conditions prevailing in the host's intestine. The contribution to protection from the KETc1 and BLS epitopes in a chimeric vaccine is under study. Preventive vaccination of definitive hosts of T. solium against the tapeworm, the most relevant step in the taeniasis/cysticercosis transmission, may greatly impact the dynamics of endemic disease and has not been studied or tried previously.

Analysis of functional heterogeneity of porcine memory CD4+ T cells.

Previous studies have identified swine helper memory T cells as CD4+CD8alpha+SLADR+. We have recently described a new porcine surface antigen (2E3) selectively expressed on CD4+ T cells that allows to divide these cells into naive (2E3+) and effector/memory (2E3-). However, although the majority of CD4+2E3- cells are CD8alpha+SLADR+, a minor proportion do not express SLADR and/or CD8alpha. Here, we have analyzed the functional capacity of these CD4+2E3- subsets to proliferate to a recall antigen. Both SLADR- and CD8alpha- cells proliferated in response to lysozyme, but at lower levels compared to the whole population CD4+2E3-. Besides, after activation with PMA plus ionomycin, CD4+2E3-SLADR- T cells produced IFNgamma and TNFalpha, although they did also in lower proportion than the whole CD4+2E3- population. Most of the IFNgamma-TNFalpha+, IFNgamma+TNFalpha+, IFNgamma+TNFalpha- cells were CD8alpha+ and CD45RA-, while IFNgamma-TNFalpha- cells showed a less differentiate phenotype.

Differential expression of chemokine receptors and CD95 in porcine CD4(+) T cell subsets.

Among other differences, naïve and memory T cells show distinct migratory patterns and susceptibility to CD95-mediated cell death. We have recently characterised in the pig two subsets of CD4(+) T cells, based on the expression of the 2E3 marker, that display phenotypic and functional features of naïve (CD4(+)2E3(+)) and effector/memory (CD4(+)2E3(-)) T cells. In this study, we have analysed the expression of several chemokine receptors, as well as the distribution of CD95 antigen (APO-1/Fas) in these CD4(+) T cell subsets. CD4(+)2E3(-) T cells express high levels of CXCR3 and CCR4 transcripts but not of CCR7. On the contrary, CCR7 is clearly detected in CD4(+)2E3(+) T cells, whereas CXCR3 and CCR4 are negative or present at trace levels. These subsets also differ in the expression of CD95 antigen, being CD95 positive cells significantly more abundant in the CD4(+)2E3(-) cell subset. These findings, although based on a small number of animals, fit well with those reported for naïve and memory CD4(+) T cells in humans.

Molecular characterization of porcine Siglec-10 and analysis of its expression in blood and tissues.

Siglecs are sialic acid binding Ig-like proteins involved in the control of leukocyte responses. In this study we describe the characterization of a porcine orthologue of Siglec-10. A cDNA clone was obtained from a porcine library which encodes a protein with sequence homology to human Siglec-10. This cDNA codes for a type I transmembrane protein containing four Ig-like domains, a transmembrane region, and a cytoplasmic tail with three tyrosine-based motifs, including a membrane-proximal Grb2-binding motif, and two ITIM motifs. When expressed on transfected cells, porcine Siglec-10 was able to bind red blood cells in a sialic acid-dependent manner. Monoclonal antibodies were developed against this protein and used to examine its cell and tissue distribution in the pig. Siglec-10 was found to be expressed on blood B cells and B cell areas of the spleen and lymph nodes. A weak expression was also detected on monocytes.

Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues.

A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2-6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51-60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.

Phenotypic characterization of porcine IFN-gamma-producing lymphocytes by flow cytometry.

We have developed a three-colour flow cytometric assay for phenotypic characterization of porcine IFN-gamma-producing lymphocytes. Analyses of activated swine peripheral blood mononuclear cells (PBMC) showed a significant difference in the proportion of IFN-gamma producing cells between young and adult animals (13.2+/-5.8% versus 34.2+/-5.7%). The majority of IFN-gamma producing cells were alphabeta T lymphocytes, although there was also an important proportion of gammadelta T cells particularly in young animals. Within the alphabeta T lymphocytes, the double positive CD4(+)CD8(lo) subset, that contains memory T cells, produced high levels of IFN-gamma, whereas the CD8(hi) T cells ranged from low to high levels of IFN-gamma. Also, consistent with a higher production by memory T cells, the CD45RA(-) subset of both CD4(+) and CD8(+) cells contained higher numbers of IFN-gamma producing cells than the CD45RA(+) subset. Finally, no production of IFN-gamma by either B cells (CD21(+)) or monocytes (SWC3(+)) was detected. This assay may be useful for the assessment of cell-mediated immunity in vaccine trials and may contribute to our understanding of the role of IFN-gamma in protective immunity against important viral diseases of the pig.

Insulin resistance in offspring of hypertensive subjects.

OBJECTIVE: To assess whether apparently healthy subjects with a family history of systemic hypertension have a higher risk of presenting the insulin resistance syndrome. SUBJECTS: Three hundred and eighty-six subjects aged 20-65 years. SETTING: A middle socio-economic class urban community from Mexico City. METHOD: All subjects and, when necessary, their first-degree relatives, answered a questionnaire and underwent a physical examination with measurement of height, weight and blood pressure. Serum insulin, glucose, cholesterol and triglycerides were measured during fasting and 2 h after an oral load of 75 g glucose. RESULTS: A family history of systemic hypertension was present for 167 (43%) of the subjects, of whom 123 (31%) were obese. Subjects with a family history of hypertension had higher systolic blood pressures than did those without such a history (120 +/- 15 versus 115 +/- 10 mmHg). In the logistic regression model, the body mass index and age showed statistically significant effects on the fasting glucose:insulin ratio and on serum insulin levels after an oral load of glucose. When men and women were analysed separately, only in men were higher systolic and mean blood pressures and lower glucose:insulin ratios observed. In the logistic regression analysis the body mass index was a significant predictor of the glucose:insulin ratio and serum insulin levels after an oral load of glucose, especially in men. CONCLUSION: Apparently healthy male offspring of hypertensive parents have higher blood pressure levels and lower insulin sensitivities than do offspring of normotensive parents. Insulin resistance was related to obesity, but not to a family history of hypertension, as had previously been reported by other research groups.

2E3, a new marker that selectively identifies porcine CD4+ naive T cells.

We describe a novel antigen recognized by mAb 2E3 selectively expressed in the periphery by a subset of porcine CD4+ T cells. Both, CD4+CD8alpha- and CD4+CD8alphalow T cell subpopulations express this antigen. CD4+2E3+ T cells show phenotypical and functional characteristics of nai;ve cells. The majority of them are CD29low, CD45RAhigh, CD49dlow, CD11alow, CD18low, and SLA-II-. After mitogen activation CD4+2E3+ T cells express high levels of IL-2 mRNA, but only traces of IFN-gamma or IL-4 mRNA. Indeed a minor percentage of cells stained positive for IFN-gamma when assessed by flow cytometry. Moreover, CD4+2E3+ T cells did not proliferate in response to the recall antigen lysozyme, although they did efficiently to the mitogen ConA. By contrast, CD4+2E3- T cells show phenotypical and functional characteristics of primed cells. They express markers associated to a memory phenotype, respond to the recall antigen lysozyme, and produce high amounts of IFN-gamma and IL-4.

Phenotypic characterization of monocyte subpopulations in the pig.

We have recently described the existence of two subsets of porcine monocytes based on the expression of CD163. In this study we compare the expression of a number of cell surface antigens in CD163+ and CD163- monocyte subsets using three-color flow cytometry. These monocyte subsets show differences with respect to the expression of MHC class II antigens (SLA-DR and DQ) and a variety of adhesion molecules (CD11a, wCD11c, wCD29, CD49d) that are expressed at higher levels on CD163+ monocytes, and of CD14 that is higher expressed by CD 163- cells. These differences on phenotype could reflect differences in the ability of these two subsets to migrate to tissues and may account for the higher allostimulatory capacity of CD163+ cells. In some aspects, the phenotype of CD163+ monocytes resembles that of mature macrophages. In vitro serum-induced maturation of monocytes into macrophages lead to the expression of SWC9 together with an increase in the expression of CD163 and a reduction in that of CD14. These results delineate a maturation pathway where CD14hiCD163-SWC9- monocytes develop into CD14loCD163+SWC9- monocytes and these cells into CD14loCD163+SWC9+ macrophages.

Nephropathy in low income diabetics: the Mexico City Diabetes Study.

Among the most serious complications associated with diabetes mellitus (DM) is nephropathy (DN). In Mexico, there is little information on the frequency and clinical characteristics of DN in the Mexican population. We present results of a population-based survey designed to estimate the prevalence of DN. The low income population consisted of 15,532 inhabitants. All 35- to 64-year-old males and non-pregnant women residing in the survey area were identified as eligible for the study (3505; 22.6%). A home interview was obtained in 2810 (80.2%). A physical exam with oral glucose tolerance test was obtained in 2282 (81.2% of those interviewed). DM was diagnosed in 304 (crude rate 13.3%). Mean age for men and women with DM was 51.6 +/- 8.4 and 52.2 +/- 7.5, respectively. Duration of DM in men was 9.2 +/- 8.1 and in women, 7.3 +/- 6.7 years. Hypertension was diagnosed in 19.8% of men and 18.1% of women. Diabetic retinopathy of any level was found in 55.4% of men and 45.7% of women. Mean glycohemoglobin in men was 9.6 +/- 2.1 and in women 9.5 +/- 2.2% (normal 4-8%). At baseline, proteinuria (1+ or more, by dipstick) was found in 24.7% of men and 9.6% of women, microalbuminuria (MA) in 84.4% of men and 63.8% of women. Quantitative albuminuria was abnormally high in 54.7% of men and 40.3% of women. In the 203 diabetics studied with 24 h urine collection for creatinine clearance, normal renal function was found in 69.1% of men and 47.5% of women, reduced renal function was found in 26% of men and 50% of women, renal insufficiency was diagnosed in 4.9% of men and 1.6% of women. One patient was on dialysis and in a subsequent follow up, we found that 2.3% of the patients had died of renal failure, six men (46-63 years) and a woman of 62 years. We conclude that DN is a very serious threat to this population. The high case fatality rate associated with this condition maintains a low prevalence. It is important to develop a program to diminish the frequency of this condition.

Neuroprotective effect of acidic fibroblast growth factor on seizure-associated brain damage.

The neuroprotective effect of acidic fibroblast growth factor (aFGF) has been analysed in a rat model of seizures-associated brain damage. We report that after treatment with a convulsivant dose of Kainic acid, systemically administered aFGF prevents neuronal degeneration in specific brain areas, mainly in the hippocampal formation. Our findings extend the potential pharmacological use of fibroblast growth factors and afford new data to understand the neurophysiology of these proteins.

Taenia crassiceps cysticercosis: protective effect and immune response elicited by DNA immunization.

The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci present in all stages of Taenia solium (KETc7), cloned into pcDNA3 plasmid with the signal peptide sequence of the beta-glycan receptor (pTc-sp7), has been shown to be effective in protecting mice against experimental infection of T. crassiceps. To explore further the possibilities of this form of immunization and the immune response induced, mice were injected intramuscularly (i.m.) or intradermally (i.d.) with 3 doses of pTc-sp7. Similar levels of resistance were found using either i.m. or i.d. immunization. Spleen cells from i.d. and i.m. DNA immunized mice induced a specific T-cell response to T. crassiceps antigens and to a synthetic peptide from the immunogen itself (GK-1). Proliferated cells were especially enriched in CD8+ CD4- T-lymphocytes. A clear increase in the percentage of CD3+ cells that produce gamma-interferon and interleukin-2 was detected when measuring the intracellular cytokine production, an indication of the pTc-sp7 capacity to induce an effective cellular response. These results provide encouraging information on the use of KETc7 in the prevention of cysticercosis as well as a first insight into the characterization of the immune response induced by pTc-sp7 that hints to the relevance of cellular immunity in protection.