Canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gamma c) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or 'stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA+ (naive) phenotype to a CD45RA- (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand-receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage phiX174. The number of thymocytes in the XSCID thymus is approximately 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4-CD8- thymocytes in XSCID dogs is increased 3.5-fold and the CD4+CD8+ population is decreased 2.3-fold. These findings demonstrate that (1) a functional gamma c is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gamma c, and (3) B cell development can occur through a gamma c-independent pathway.     

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34+ cells in a canine X-linked severe combined immunodeficiency model

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).     

Ex vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency and the development of a thymic T cell lymphoma

We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (na?ve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease.     

Isolation and characterization of pediatric canine bone marrow CD34+ cells

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.     

B-cell function in canine X-linked severe combined immunodeficiency

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gammac) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and has a similar clinical phenotype to human XSCID. We have previously shown that the block in T-cell development is more profound in XSCID dogs than in genetically engineered gamma c-deficient mice. In this study we evaluated the B-cell function in XSCID dogs. In contrast to the marked decrease in peripheral B-cells in gamma c-deficient mice, XSCID dogs have increased proportions and numbers of peripheral B-cells as observed in XSCID boys. Canine XSCID B-cells do not proliferate following stimulation with the T-cell-dependent B-cell mitogen, pokeweed mitogen (PWM); however, they proliferate normally in response to the T-cell-independent B-cell mitogen, formalin-fixed, heat-killed Staphylococcus aureus. Canine XSCID B-cells are capable of producing IgM but are incapable of normal class-switching to IgG antibody production as demonstrated by in vitro stimulation with PWM and immunization with the T-cell-dependent antigen, bacteriophage PhiX174. Similar results have been reported for XSCID boys. Thus, it appears that gamma c-dependent cytokines have differing roles in human and canine B-cell development than in the mouse making the XSCID dog a valuable model for studying the role of these cytokines in B-cell development and function.     

CD34 glycoprotein identifies putative stem cells located in the isthmic region of canine hair follicles

It is widely documented that a pool of multipotent stem cells located in humans and mice hair follicle outer root sheath (bulge region) is involved in the restoration of the whole follicular unit during each anagen phase. To the authors' knowledge, data regarding the location and characterization of hair follicle stem compartment in dogs have not been reported in the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor cell antigen CD34 as a marker of putative stem cells located in a bulge-like region of canine hair follicles. The presence of CD34 mRNA and glycoprotein was assessed on formalin-fixed, paraffin-embedded canine skin samples by in situ hybridization technique and by standard immunohistochemistry, respectively. A strong expression of CD34 mRNA and glycoprotein was observed in a well-defined area of the hair follicle isthmic region and appeared uniformly concentrated at the level of the basal layer of the outer root sheath. These findings provide compelling support to the hypothesis that in dogs, a subpopulation of basal keratinocytes located in the hair follicle isthmic region and characterized by the selective expression of CD34 is potentially associated with the stem cell compartment of this skin appendage.     

Phenotypical characterization of T and B cell areas in lymphoid tissues of dogs with spontaneous distemper

CD3, CD4, CD5, and CD8 antigen expression of T cells and IgG expression of B cells and canine distemper virus (CDV) antigen distribution were immunohistochemically examined in lymphoid tissues (lymph node, spleen, thymus, and tonsil) of control dogs and animals with spontaneous canine distemper. In addition, CNS tissue of all animals was studied for neuropathological changes and CDV antigen distribution. Based on the degree of depletion distemper dogs were classified into two groups. Group I represented animals with moderate to marked lymphoid depletion, while group II dogs displayed mild or no depletion. CDV antigen was mainly found in lymphocytes and macrophages of group I dogs, whereas CDV expression was most prominent in dendritic cells of group II animals. In group I dogs, a marked loss of CD3, CD4, CD5, CD8, and IgG expression was noticed, hereby loss of CD4+ cells was more prominent than depletion of CD8+ cells. In the lymphoid tissues of group II animals, a significant increase in the number of T and B cells was observed compared to group I dogs. The number of CD3+, CD4+, and CD8+ cells in group II dogs was similar to the findings in controls, however, CD5 and IgG expression was mildly reduced in T and B cell areas, respectively. Additionally, in groups I and II dogs, CD3+ and CD5- T cells were detected in T cell areas. Whether this cell population represents a cell type with autoimmune reactive potential remains to be determined. Surprisingly in group II animals, viral antigen was found predominantly in dendritic cells indicating a change in the cell tropism of CDV during chronic infection and a possible mechanism of viral persistence. The two patterns of lymphoid depletions correlated to two different types of canine distemper encephalitis (CDE). Group I dogs displayed acute non-inflammatory CDE, whereas group II dogs suffered from chronic inflammatory demyelinating CDE, indicating a pathogenic relationship between lymphocytic depletion and inflammatory brain lesions in distemper.     

Identification and characterisation of side population cells in the canine pituitary gland

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.     

High Mobility Group Box 1-Protein expression in canine haematopoietic cells and influence on canine peripheral blood mononuclear cell proliferative activity

High Mobility Group Box 1-Protein (HMGB1) is a nuclear chromosomal protein occurring ubiquitary in mammalian tissues. HMGB1 demonstrates cytokine function and induces inflammation when actively released by haematopoietic cells or passively released during cell necrosis. This study aimed at the determination of HMGB1 expression in different cell types and at the evaluation of the role of HMGB1 in PBMC proliferation. Therefore we investigated the HMGB1 mRNA expression level in different canine haematopoietic cell types and the influence of exogenous rhHMGB1 on canine PBMC proliferation. Differentiated haematopoietic blood cells showed lower relative HMGB1 expression levels compared to CD34+ haematopoietic stem cells. Relative HMGB1 expression seemed also to decrease during differentiation of CD34+ stem cells into dendritic cells. Furthermore, peripheral blood CD14+ monocytes and granulocytes showed a lower relative HMGB1 expression in comparison to CD3+ T-lymphocytes. When exogenous rhHMGB1 at low concentrations was added to single PBMC cultures an increase of proliferation was obvious. However, in higher concentrations HMGB1 lost its stimulative effect. In conclusion, HMGB1 is broadly expressed in canine haematopoietic cells with highest levels in haematopoietic stem cells. HMGB1 induced directly PBMC proliferation.     

Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs.

A recent epizootic of swine infertility and respiratory syndrome (SIRS) in a Minnesota swine herd was investigated. Examination of a sow, neonatal piglets, and stillborn fetuses obtained during the epizootic from the affected herd revealed interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in the piglets and focal vasculitis in the brain of the sow. Fetuses did not have microscopic lesions. No cause for the infertility and respiratory syndrome was determined. Therefore, attempts were made to experimentally reproduce the disease. Eleven 3-day-old gnotobiotic piglets exposed intranasally to tissue homogenates of piglets from the epizootic became inappetent and febrile by 2-4 days postexposure and had interstitial pneumonitis and encephalitis similar to that seen in the field outbreak. After 2 blind passages in gnotobiotic piglets, tissue homogenates were cultured on continuous cell line CL2621, and a cytopathic virus (ATCC VR-2332), provisionally named SIRS virus, was isolated. Gnotobiotic piglets exposed intranasally to the SIRS virus developed clinical signs and microscopic lesions that were the same as those in piglets exposed to the tissue homogenates, and the virus was reisolated from their lungs. This is the first isolate of SIRS virus in the United States that fulfills Koch's postulates in producing the respiratory form of the disease in gnotobiotic piglets and the first report of isolation and propagation of the virus on a continuous cell line (CL2621). The virus is designated as American Type Culture Collection VR-2332.     

Hematologic changes after total body irradiation and autologous transplantation of hematopoietic peripheral blood progenitor cells in dogs with lymphoma

Dogs with and without lymphoma have undergone hematopoietic cell transplantation in a research setting for decades. North Carolina State University is currently treating dogs with B- and T-cell lymphoma in a clinical setting with autologous peripheral blood progenitor cell transplants, using peripheral blood CD34+ progenitor cells harvested using an apheresis machine. Complete blood counts were performed daily for 15 to 19 days posttransplantation to monitor peripheral blood cell nadirs and subsequent CD34+ cell engraftment. This study documents the hematologic toxicities of total body irradiation in 10 dogs and the subsequent recovery of the affected cell lines after peripheral blood progenitor cell transplant, indicating successful CD34+ engraftment. All peripheral blood cell lines, excluding red blood cells, experienced grade 4 toxicities. All dogs had ≥ 500 neutrophils/μl by day 12, while thrombocytopenia persisted for many weeks. All dogs were clinically normal at discharge.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

关于牛CD34cDNA的克隆及应用

CD34是白细胞抗原，可在不同类型的细胞包括造血细胞中进行表达，抗人，鼠和犬CD34鼠白的单克隆抗体已用于对淋巴造血干细胞的鉴别，本试验克隆出编码牛CD34的cDNA，并测定其核苷酸顺序，推测其蛋白的氨基酸顺序与人，鼠和犬的CD34蛋白的氨基酸顺序的同源性分别为61.1%,56.0%,和66.1%。并以cDNA作为探针进行Northern杂交，探测CD34RNA在胎牛脑，脾，心和肺中的表达。     

Canine lymphoproliferative disease characterized by lymphocytosis: immunophenotypic markers of prognosis

BACKGROUND: Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse. HYPOTHESIS: Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis. ANIMALS: Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005. METHODS: Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study. RESULTS: Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P < .0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis >30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with <30,000 lymphocytes/muL (1098 days, P < .0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.     

Acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus

Gnotobiotic puppies were inoculated intracerebrally with a strain of canine parainfluenza virus (CPI-78-238). Four of eight dogs developed histological evidence of acute encephalitis. Clinical signs of encephalitis were seen in two of these four dogs; one had signs and lesions of interstitial pneumonia. Of six inoculated dogs observed for six months after infection, five developed internal hydrocephalus. Virus was reisolated from two dogs with acute encephalitis but not from dogs with hydrocephalus. Hemagglutination-inhibition antibodies persisted throughout the observation period of six months at high levels in the serum and cerebrospinal fluid of hydrocephalic dogs.     

Generation and characterization of bone marrow-derived cultured canine mast cells

Disorders of mast cells, particularly mast cell tumors (MCTs), are common in dogs. There now is evidence that many of these disorders exhibit breed predilections, suggesting an underlying heritable component. In comparison to humans and mice, little is known regarding the biology of canine mast cells. To facilitate the study of mast cell biology in other species, bone marrow-derived cultured mast cells (BMCMCs) often are used because these represent a ready source of large numbers of cells. We have developed a protocol to successfully generate canine BMCMCs from purified CD34(+) cells. After 5-7 weeks of culture with recombinant canine stem cell factor (rcSCF), greater than 90% of the cell population consisted of mast cells as evidenced by staining with Wright's-Giemsa, as well as production of chymase, tryptase, IL-8 and MCP-1. These cells expressed cell surface markers typical of mast cells including Kit, Fc epsilonRI, CD44, CD45 and CD18/CD11b. The canine BMCMCs were dependent on rcSCF for survival and proliferation, and migrated in response to rcSCF gradients. Cross-linking of cell surface-bound IgE induced the release of histamine and TNFalpha. Histamine release could also be stimulated by ConA, compound 48/80, and calcium ionophore. In summary, canine BMCMCs possess phenotypic and functional properties similar to mast cells found in vivo. These cells represent a novel, valuable resource for investigating normal canine mast cell biology as well as for identifying factors that lead to mast cell dysregulation in the dog.     

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.     

Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs

Despite accumulating knowledge of porcine macrophages and dendritic cells (DCs) from in vitro studies, information regarding monocytes/macrophages and DCs in lymphoid tissues of enteric pathogen-infected neonatal animals in vivo is limited. In this study we evaluated the influence of commensal bacterial [two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and L. reuteri] colonization and rotavirus infection on distribution and frequencies of monocytes/macrophages and conventional DCs (cDCs) in ileum, spleen and blood. Gnotobiotic pigs were inoculated with LAB and virulent Wa strain human rotavirus (HRV) (LAB+HRV+), HRV only (LAB-HRV+), LAB only (LAB+HRV-) or mock (LAB-HRV-). The cDCs were characterized as SWC3(+)CD11R1(+), whereas monocytes/macrophages were identified as SWC3(+)CD11R1(-) by flow cytometry in the gnotobiotic pigs at 10 days of age. Infection with HRV alone activated/recruited significantly more monocytes/macrophages to the intestine than LAB colonization and 56% versus 28% of these cells expressed CD14. Colonization with LAB alone also significantly increased the frequencies of monocytes/macrophages and cDCs and the CD14 expression on monocytes/macrophages in ileum and spleen compared to the controls. LAB colonization plus HRV infection significantly reduced macrophage and cDC frequencies in spleen compared to LAB colonization or HRV infection alone, suggesting that LAB colonization down-regulated HRV- infection-induced monocyte/macrophage activation/recruitment at the systemic lymphoid tissue. These results illustrated the distribution of porcine monocytes/macrophages and cDCs and the frequencies of CD14 expression on these cells in intestinal and systemic lymphoid tissues in the early stage of immune responses to intestinal colonization by LAB versus infection by an enteric pathogen HRV and will facilitate further in vivo studies on functional characterization of these immune cells in neonates.     

Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis

While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN in Leishmania (Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti-Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8+ T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.     

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis

A method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin-eosin and Wright' stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin-eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy.