Bacteriological, cytological, and molecular investigation of Corynebacterium pseudotuberculosis, mycobacteria, and other bacteria in caseous lymphadenitis and healthy lymph nodes of slaughtered sheep.

Caseous lymphadenitis (CL) in sheep is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, commonly characterized by abscess formation in peripheral lymph nodes and disseminated infections. Nonetheless, other microorganisms, including with zoonotic relevance, can be isolated from CL-resembling lymph nodes. Currently, mycobacteria have been reported in visceral granulomatous lesions in small ruminants, a fact that poses a public health issue, particularly in slaughtered sheep intended for human consumption. Cytology using fine needle aspiration and microbiological culturing are suitable tests for routine diagnostic, whereas present drawbacks and molecular methods have been confirmatory. Data about the occurrence of mycobacteria in both lymph nodes with aspect of CL and apparently healthy visceral nodes of sheep slaughtered for human consumption are scarce. In this study, 197 visceral lymph nodes of sheep showed lymphadenitis and 202 healthy visceral lymph nodes of slaughtered sheep intended for human consumption were submitted to conventional bacteriological diagnosis, mycobacteria culturing, and cytological evaluation. Compatible Corynebacterium isolates were subjected to multiplex PCR targeting 16S rRNA, rpoB, and pld genes to detect C. pseudotuberculosis. Based on microbiological identification, C. pseudotuberculosis (86/197; 43.7%), streptococci γ-hemolytic (17/197; 8.6%), and Trueperella pyogenes (12/197; 6.1%) were prevalent in lymph nodes with abscesses, as opposed to staphylococci (53/202; 26.2%) in apparently healthy lymph nodes. No mycobacteria were isolated. Cytology identified 49.2% (97/197) Gram-positive pleomorphic organisms (coryneform aspect). Multiplex PCR confirmed genetic material of C. pseudotuberculosis in 74.4% (64/86) of the samples with C. pseudotuberculosis isolation and 66% (64/97) samples with cytological coryneform aspect (κ = 86.78%; 95% CI = 79.87-93.68%). These findings emphasize the prevalence of C. pseudotuberculosis in abscess formation among peripheral lymph nodes of sheep. Other bacteria were also identified in lymph nodes sampled that resembling C. pseudotuberculosis-induced infections that may difficult the diagnosis. Multiplex PCR revealed a valuable assay to detect C. pseudotuberculosis, in addition to routine methods applied to CL-diagnosis. No mycobacteria were identified in lymph nodes sampled, with and without apparent lesions. Nonetheless, due to public health impacts, this pathogen should be considered as a differential diagnosis of C. pseudotuberculosis-induced infections during inspection procedures of slaughtered sheep intended for human consumption.

Obesity impairs resistance to Leishmania major infection in C57BL/6 mice.

An association between increased susceptibility to infectious diseases and obesity has been described as a result of impaired immunity in obese individuals. It is not clear whether a similar linkage can be drawn between obesity and parasitic diseases. To evaluate the effect of obesity in the immune response to cutaneous Leishmania major infection, we studied the ability of C57BL/6 mice fed a hypercaloric diet (HSB) to control leishmaniasis. Mice with diet-induced obesity presented thicker lesions with higher parasite burden and a more intense inflammatory infiltrate in the infected ear after infection with L. major. There was no difference between control and obese mice in IFN-gamma or IL-4 production by auricular draining lymph node cells, but obese mice produced higher levels of IgG1 and IL-17. Peritoneal macrophages from obese mice were less efficient to kill L. major when infected in vitro than macrophages from control mice. In vitro stimulation of macrophages with IL-17 decreased their capacity to kill the parasite. Moreover, macrophages from obese mice presented higher arginase activity. To confirm the role of IL-17 in the context of obesity and infection, we studied lesion development in obese IL-17R-/- mice infected with L. major and found no difference in skin lesions and the leukocyte accumulation in the draining lymph node is redcuced in knockout mice compared between obese and lean animals. Our results indicate that diet-induced obesity impairs resistance to L. major in C57BL/6 mice and that IL-17 is involved in lesion development.

Neutrophils Which Migrate to Lymph Nodes Modulate CD4+ T Cell Response by a PD-L1 Dependent Mechanism.

It is well known that neutrophils are rapidly recruited to a site of injury or infection and perform a critical role in pathogen clearance and inflammation. However, they are also able to interact with and regulate innate and adaptive immune cells and some stimuli induce the migration of neutrophils to lymph nodes (LNs). Previously, we demonstrated that the immune complex (IC) generated by injecting OVA into the footpad of OVA/CFA immunized mice induced the migration of OVA+ neutrophils to draining LNs (dLNs). Here we investigate the effects of these neutrophils which reach dLNs on CD4+ T cell response. Our findings here strongly support a dual role for neutrophils in dLNs regarding CD4+ T cell response modulation. On the one hand, the CD4+ T cell population expands after the influx of OVA+ neutrophils to dLNs. These CD4+ T cells enlarge their proliferative response, activation markers and IL-17 and IFN-γ cytokine production. On the other hand, these neutrophils also restrict CD4+ T cell expansion. The neutrophils in the dLNs upregulate PD-L1 molecules and are capable of suppressing CD4+ T cell proliferation. These results indicate that neutrophils migration to dLNs have an important role in the homeostasis of adaptive immunity. This report describes for the first time that the influx of neutrophils to dLNs dependent on IC presence improves CD4+ T cell response, at the same time controlling CD4+ T cell proliferation through a PD-L1 dependent mechanism.

Characterization and expression of DEC205 in the cDC1 and cDC2 subsets of porcine dendritic cells from spleen, tonsil, and submaxillary and mesenteric lymph nodes.

Conventional dendritic cells (cDCs) are divided into the following different subtypes: cDC1, which promotes a Th1 response, and cDC2, which stimulates a Th2 and Th17 response. These cells have not been characterized in porcine lymphoid tissues. DEC205 is a receptor that increases antigen presentation and allows DCs to cross-present antigens. The objectives of this work were to characterize cDCs subsets in the tonsil, submaxillary and mesenteric lymph nodes and spleen lymphoid tissues and to determine their expression of DEC205 by flow cytometry. The cDC1 (MHCIIhighCADM1highCD172a-/low) and cDC2 (MHCIIhighCADM1highCD172a+) phenotypes were confirmed by the expression of characteristic cDC1 and cDC2 transcripts (FLT3, XCR1 and FCER1α). Among all lymphoid tissues, the spleen had the highest frequency of total cDCs. The cDC1:cDC2 ratio showed that all lymph tissues had higher levels of cDC1 than levels of cDC2. DEC205+ cDCs were found in all analyzed tissues, albeit with different frequencies. Our research will facilitate the study on the function of these cells and the investigation of the strategies for DEC205 targeting and functional studies.

Linfoma multicêntrico em cão, uma abordagem clinica e laboratorial / Multicenter lymphoma in dogs, a clinical and laboratory approach: a case report

O linfoma é uma forma de apresentação do distúrbio linfoproliferativo, em que o tumor se origina em um órgão hematopoiético sólido, como o linfonodo. É considerada uma neoplasia de maior incidência em cães e gatos. O trabalho tem como objetivo apresentar um relato de caso sobre um linfoma multicêntrico, com abordagem clínica e laboratorial. Um cão sem raça definida, foi atendido no Hospital Veterinário da Universidade Estadual do CEARÁ (UHV-UECE), apresentando hematúria e uma discreta linfadenomegalia no exame físico. Foram solicitados hemograma completo (HC), análise de bioquímica sérica, sumário de urina e exame ultrassonográfico (US). As alterações no hemograma foram anemia normocítica normocrômica e trombocitopenia. A análise dos exames de bioquímica sérica revelou um aumento na enzima aspartato amino-transferase (AST). No sumário de urina (SU), observaram-se alterações das características físicas e da sedimentoscopia. O exame ultrassonográfico mostrou alterações de ecogenicidade na bexiga, próstata, testículo e baço. Houve piora do quadro clínico do animal, com perda de peso e aumento evidente dos linfonodos. Um exame citológico foi realizado com aspirado de linfonodos, que apresentou configuração celular compatível com linfoma; sendo também encontradas células neoplásicas no esfregaço de sangue periférico, no hemograma. Diante dos achados e do agravamento do quadro clínico, o animal veio a óbito.

Lymphoma is a form of presentation of the lymphoproliferative disorder which the tumor originates from a solid hematopoietic organ such as the lymph node. It is considered one of the neoplasia of higher incidence in dogs and cats. This paper aims to present a case report on a multicenter lymphoma with a clinical and laboratory approach. A dog without defined breed, at the Hospital Veterinário da Universidade Estadual do CEARÁ (UHV-UECE) presenting hematuria and a discrete lymphadenomegaly on physical examination. Complete blood counts (HC), serum biochemical analyzes, urine summary and ultrasound examination were requested. Changes in the hemogram were normocyclic normocytic anemia and thrombocytopenia. Biochemical analysis revealed an increase in asparate amino transferase (AST). The urine summary (SU) showed changes in physical characteristics and sedimentation. Ultrasound examination (US) showed changes in the bladder, prostate, testis echogenicity and the spleen’s size. There was worsening of the animal's size over time with progression of weight loss and evident enlargement of the lymph nodes. A cytology was performed through a lymph node aspirate which had a lymphoma compatible cell configuration and neoplastic cells were also found in the hemogram peripheral blood smear. The animal died with deterioration of the clinical picture.

Linfoma multicêntrico em cão, uma abordagem clinica e laboratorial / Multicenter lymphoma in dogs, a clinical and laboratory approach: a case report

O linfoma é uma forma de apresentação do distúrbio linfoproliferativo, em que o tumor se origina em um órgão hematopoiético sólido, como o linfonodo. É considerada uma neoplasia de maior incidência em cães e gatos. O trabalho tem como objetivo apresentar um relato de caso sobre um linfoma multicêntrico, com abordagem clínica e laboratorial. Um cão sem raça definida, foi atendido no Hospital Veterinário da Universidade Estadual do CEARÁ (UHV-UECE), apresentando hematúria e uma discreta linfadenomegalia no exame físico. Foram solicitados hemograma completo (HC), análise de bioquímica sérica, sumário de urina e exame ultrassonográfico (US). As alterações no hemograma foram anemia normocítica normocrômica e trombocitopenia. A análise dos exames de bioquímica sérica revelou um aumento na enzima aspartato amino-transferase (AST). No sumário de urina (SU), observaram-se alterações das características físicas e da sedimentoscopia. O exame ultrassonográfico mostrou alterações de ecogenicidade na bexiga, próstata, testículo e baço. Houve piora do quadro clínico do animal, com perda de peso e aumento evidente dos linfonodos. Um exame citológico foi realizado com aspirado de linfonodos, que apresentou configuração celular compatível com linfoma; sendo também encontradas células neoplásicas no esfregaço de sangue periférico, no hemograma. Diante dos achados e do agravamento do quadro clínico, o animal veio a óbito.(AU)

Lymphoma is a form of presentation of the lymphoproliferative disorder which the tumor originates from a solid hematopoietic organ such as the lymph node. It is considered one of the neoplasia of higher incidence in dogs and cats. This paper aims to present a case report on a multicenter lymphoma with a clinical and laboratory approach. A dog without defined breed, at the Hospital Veterinário da Universidade Estadual do CEARÁ (UHV-UECE) presenting hematuria and a discrete lymphadenomegaly on physical examination. Complete blood counts (HC), serum biochemical analyzes, urine summary and ultrasound examination were requested. Changes in the hemogram were normocyclic normocytic anemia and thrombocytopenia. Biochemical analysis revealed an increase in asparate amino transferase (AST). The urine summary (SU) showed changes in physical characteristics and sedimentation. Ultrasound examination (US) showed changes in the bladder, prostate, testis echogenicity and the spleens size. There was worsening of the animal's size over time with progression of weight loss and evident enlargement of the lymph nodes. A cytology was performed through a lymph node aspirate which had a lymphoma compatible cell configuration and neoplastic cells were also found in the hemogram peripheral blood smear. The animal died with deterioration of the clinical picture.(AU)

Quantification of Treg cells in peripheral blood and lymph nodes of dogs with multicentric lymphoma / Quantificação de células Treg no sangue periférico e linfonodos de cães com linfoma multicêntrico

Lymphoma is a malignant tumor characterized by cell proliferation of lymphoid origin and corresponds to 90% of all hematopoietic neoplasms of dogs. Regulatory T cells (Tregs) have been the target of many investigations in oncology due to their potential of down-regulating immune responses, as well as ensuring the maintenance of active mechanisms of tumor suppression. The aims of the present study were to compare the percentage of Tregs in peripheral blood between dogs with multicentric lymphoma and healthy animals, together with the percentage of Tregs in peripheral blood and lymph nodes of dogs with multicentric lymphoma. Twenty-six animals were enrolled in the study: 10 healthy dogs comprised the control group (CG) and 16 dogs with multicentric lymphoma comprised the Lymphoma Group (LG). We observed that dogs in the LG showed a significantly higher Tregs expression in peripheral blood compared to the CG. No significant difference was observed between Tregs expression in lymph nodes and peripheral blood of the LG, however. With these results, it is possible to conclude that multicentric lymphoma is a neoplasm with high Tregs expression, which poses this as a condition of interest when investigating treatments that can suppress Regulatory T cells.(AU)

O linfoma é uma neoplasia maligna caracterizada pela proliferação neoplásica de células originadas de tecido linfoide e corresponde a cerca de 90% das neoplasias hematopoiéticas em cães. Células T reguladoras (Tregs) têm sido alvo de diversas investigações na área da oncologia devido ao potencial de regulação negativa da resposta do sistema imune e à manutenção ativa do mecanismo de imunossupressão tumoral. O objetivo do presente estudo foi a comparação da porcentagem de Tregs no sangue periférico entre cães com linfoma multicêntrico e animais saudáveis e a porcentagem de Tregs no sangue periférico e nos linfonodos de cães com linfoma multicêntrico. Foram utilizados 26 animais: 10 cães saudáveis, como grupo controle (CG), e 16 cães com linfoma multicêntrico, como grupo linfoma (LG). Observou-se maior expressão de Tregs no sangue periférico de cães do LG em comparação ao CG. Entretanto, não foi observada diferença significativa entre as expressões de Treg nos linfonodos e no sangue periférico do LG. Com esses resultados, foi possível concluir que o linfoma multicêntrico apresenta alta expressão de Tregs, tornando-se condição interessante para o estudo de tratamentos capazes de suprimir as células T reguladoras.(AU)

Quantification of Treg cells in peripheral blood and lymph nodes of dogs with multicentric lymphoma / Quantificação de células Treg no sangue periférico e linfonodos de cães com linfoma multicêntrico

Lymphoma is a malignant tumor characterized by cell proliferation of lymphoid origin and corresponds to 90% of all hematopoietic neoplasms of dogs. Regulatory T cells (Tregs) have been the target of many investigations in oncology due to their potential of down-regulating immune responses, as well as ensuring the maintenance of active mechanisms of tumor suppression. The aims of the present study were to compare the percentage of Tregs in peripheral blood between dogs with multicentric lymphoma and healthy animals, together with the percentage of Tregs in peripheral blood and lymph nodes of dogs with multicentric lymphoma. Twenty-six animals were enrolled in the study: 10 healthy dogs comprised the control group (CG) and 16 dogs with multicentric lymphoma comprised the Lymphoma Group (LG). We observed that dogs in the LG showed a significantly higher Tregs expression in peripheral blood compared to the CG. No significant difference was observed between Tregs expression in lymph nodes and peripheral blood of the LG, however. With these results, it is possible to conclude that multicentric lymphoma is a neoplasm with high Tregs expression, which poses this as a condition of interest when investigating treatments that can suppress Regulatory T cells.(AU)

O linfoma é uma neoplasia maligna caracterizada pela proliferação neoplásica de células originadas de tecido linfoide e corresponde a cerca de 90% das neoplasias hematopoiéticas em cães. Células T reguladoras (Tregs) têm sido alvo de diversas investigações na área da oncologia devido ao potencial de regulação negativa da resposta do sistema imune e à manutenção ativa do mecanismo de imunossupressão tumoral. O objetivo do presente estudo foi a comparação da porcentagem de Tregs no sangue periférico entre cães com linfoma multicêntrico e animais saudáveis e a porcentagem de Tregs no sangue periférico e nos linfonodos de cães com linfoma multicêntrico. Foram utilizados 26 animais: 10 cães saudáveis, como grupo controle (CG), e 16 cães com linfoma multicêntrico, como grupo linfoma (LG). Observou-se maior expressão de Tregs no sangue periférico de cães do LG em comparação ao CG. Entretanto, não foi observada diferença significativa entre as expressões de Treg nos linfonodos e no sangue periférico do LG. Com esses resultados, foi possível concluir que o linfoma multicêntrico apresenta alta expressão de Tregs, tornando-se condição interessante para o estudo de tratamentos capazes de suprimir as células T reguladoras.(AU)

Migratory capacity and function of dendritic cells from mesenteric afferent lymph nodes after feeding a single dose of vitamin A.

Lamina propria dendritic cells (DCs) have a permanent turnover with constitutive migration to mesenteric lymph nodes and replenishment by progenitors. Luminal bacteria and dietary constituents provide key signals that endow DCs their unique properties in vivo. Taking into account that the intestinal immune system is greatly influenced by retinoids, we evaluated in B6 mice 3, 8, 16 and 24 h after feeding a single dose of vitamin A phenotype and function of cells present in mesenteric afferent lymph nodes as well as signals involved in migration. We studied the frequency of CD11c+MHC-II+CD103+CD86+ and RALDH+ DCs by flow cytometry, we determined CCL-21 and D6 levels in tissue homogenates by Western blot, and we co-cultured cells isolated from afferent lymphatics with sorted CD4+ lymphocytes to assess Foxp-3 induction and homing receptor expression. Sixteen hours after vitamin A administration, DCs isolated from afferent lymphatics were able to induce homing receptors and Foxp3 expression in CD4+ lymphocytes. Our results show that a single dose of vitamin A generated a stream of signals and amplified the tolerogenic activity of DCs migrating to lymphoid tissue.

Isolation and high-dimensional phenotyping of gastrointestinal immune cells.

The gastrointestinal immune system plays a pivotal role in the host relationship with food antigens, the homeostatic microbiome and enteric pathogens. Here, we describe how to collect and process liver and intestinal samples to efficiently isolate and analyse resident immune cells. Furthermore, we describe a step-by-step methodology showing how to high-dimensionally immunophenotype resident leucocytes using cytometry by time-of-flight, providing a well-characterized antibody platform that allows the identification of every leucocyte subset simultaneously. This protocol also includes instructions to purify and cultivate primary murine hepatocytes, a powerful tool to assess basic cell biology and toxicology assays. Gut and liver samples from the same mouse can be collected, processed and stained in less than 6 hr. This protocol enables the recovery of several populations of purified and viable immune cells from solid and fibrous organs, preventing unwanted loss of adherent cells during isolation.

M1 polarization and the effect of PGE2 on TNF-α production by lymph node cells from dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is caused by the intracellular parasite Leishmania infantum. Increased levels of arginase, nitric oxide (NO2 ) and prostaglandin E2 (PGE2 ) can play a regulatory role regarding the immune response in CVL cases. This study aimed to evaluate the arginase activity in adherent macrophages cultured from the lymph nodes of healthy and naturally infected dogs and to examine the NO2 and PGE2 levels in the supernatant of these cultures. In addition, the regulatory effect of PGE2 on the production of tumour necrosis factor (TNF-α) and interleukin-10 (IL-10) in supernatants from the total lymph node was observed in leucocyte cultures. The arginase activity was lower in the adherent macrophages cultured from the lymph nodes of naturally infected dogs and there were higher concentrations of NO2 and PGE2 in the supernatants of these cultures. Higher TNF-α and IL-10 concentrations were observed in supernatants from total lymph node leucocytes cultures, from infected dogs, and the presence of indomethacin only decreased TNF-α in the supernatant of these cultures. We conclude that the low arginase activity in macrophages suggested that M1 polarization and PGE2 were participating in the immune response and were increasing TNF-α in CVL.

Dehydroepiandrosterone (DHEA) restrains intestinal inflammation by rendering leukocytes hyporesponsive and balancing colitogenic inflammatory responses.

Dehydroepiandrosterone (DHEA) is a hormone that plays an important role in the modulation of inflammatory responses. However, the precise mechanisms that link the actions of this androgen with protection or susceptibility to inflammatory bowel diseases (IBD) remain uknown. Here we showed that low dose DHEA inhibited proliferation of spleen cells and IFN-у production. The hormone was not toxic to myeloid lineage cells, although it caused necrosis of spleen cells at the intermediate and highest doses in vitro (50 and 100µM). The treatment of C57BL/6 mice with DHEA during colitis induction by dextran sodium sulfate (DSS) led to a reduction in weight loss and clinical signs of disease. There were decreased peripheral blood monocytes on day 6 of DSS exposure and treatment, besides increase in circulating neutrophils in the tissue repair phase. DHEA also led to reduced lamina propria cellularity and restoration of normal colon length. These results were accompanied by decreased expression of IL-6 and TGF-ß mRNA, while IL-13 was augmented in the colon on day 6, which was probably related to attenuation of inflammation. There was retention of CD4(+) cells in the spleen after use of DHEA, along with augmented frequency of CD4(+)IL-4(+) cells, decreased CD4(+)IFN-É£(+) in spleen and constrained CD4(+)IL-17(+) population in the mesenteric lymph nodes. Moreover, splenocytes of mice treated with DHEA became hyporesponsive, as observed by reduced proliferation after re-stimulation ex-vivo. In conclusion, DHEA modifyies leukocyte activity and balances the exacerbated immune responses which drive local and systemic damages in IBD.

Distinct courses of infection with Leishmania (L.) amazonensis are observed in BALB/c, BALB/c nude and C57BL/6 mice.

Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

Desiccating stress-induced disruption of ocular surface immune tolerance drives dry eye disease.

Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surface's mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF-κB)- and time-dependent disruption of the ocular surface's immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF-κB inhibitors reduced corneal epithelial damage and interleukin (IL)-1ß and IL-6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF-κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF-κB activation could have therapeutic potential in dry eye.

Dynamics of murine B lymphocytes is modulated by in vivo treatment with steroid ouabain.

Ouabain (OUA) is a steroid hormone capable of inhibiting the protein Na+K+ATPase present in the plasma membrane of cells. Ouabain was initially extracted from the roots of African trees such as Acocanthera ouabaio and Strophantus gratus seeds and later described as an endogenous component found in higher mammals. The adrenal gland is the main site of synthesis of ouabain and it is released in stressful situations, conditions similar to those where there is secretion of corticosteroids. Immunological functions have been shown to be regulated by ouabain. In order to understand the effects of ouabain on B lymphocyte populations in different lymphoid organs, mice received intraperitoneal injections of ouabain for 3 consecutive days. Twenty-four hours after the last injection, cells were analyzed by flow cytometry. In the spleen, ouabain modulated especially follicular B cells, inducing a significant decrease in the percentage and absolute numbers of those cells. Ouabain also reduced the absolute number of marginal zone B lymphocytes. No difference in the percentage or absolute number of B lymphocytes in the spleen forty-eight hours after the last injection was observed. An increase in the number of B cells was seen in mesenteric lymph nodes and this retention appears to be directly related to increased expression of CXCR5 chemokine receptor and reduction of CD62L, which also explains the observed reduction of B cells in the spleen. Our results indicate that ouabain regulates the dynamics of B lymphocytes in peripheral organs but production of total IgM and IgG in the serum of animals treated in vivo with ouabain was not affected.

Detection of Leishmania infantum in peripheral blood smear and lymph node of a domestic feline / Detecção de Leishmania infantum em esfregaço de sangue periférico e linfonodo de um felino doméstico

Background: Cats can become infected and develop feline visceral leishmaniasis (LVF) and manifest variable clinical signs. The diagnosis of the disease in this species has been made more frequently by means of serological and molecular techniques. Different from what happens with dogs, the use of parasitological analysis of lymph node, in clinical practice, for agent detection in felines are infrequent. Therefore, the aim of this study was to disclose the diagnosis of visceral leishmaniasis in a domestic cat from the blood smear analysis and aspiration cytology of lymph node. Case: An adult mixed breed queen, from the city of Campo Grande, Mato Grosso do Sul state, Brazil was referred for clinical care due to the presence of a nodule, with approximately 4 cm, near the inguinal breast. Other parameters, on physical examination, are within the normal range for the specie. For the investigation, additional tests were requested. Eosinophilia was found in a complete blood count (CBC). The association between macroscopic characteristics and cytological findings allowed the diagnosis of inguinal lymph node hyperplastic. Furthermore, amastigotes of Leishmania sp. were visualized both in the peripheral blood sample and in the analyzed material mass. The diagnosis of visceral leishmaniasis and identification of specie of Leishmania was performed by PCR technique (Polymerase Chain [...]

Detection of Leishmania infantum in peripheral blood smear and lymph node of a domestic feline / Detecção de Leishmania infantum em esfregaço de sangue periférico e linfonodo de um felino doméstico

Background: Cats can become infected and develop feline visceral leishmaniasis (LVF) and manifest variable clinical signs. The diagnosis of the disease in this species has been made more frequently by means of serological and molecular techniques. Different from what happens with dogs, the use of parasitological analysis of lymph node, in clinical practice, for agent detection in felines are infrequent. Therefore, the aim of this study was to disclose the diagnosis of visceral leishmaniasis in a domestic cat from the blood smear analysis and aspiration cytology of lymph node. Case: An adult mixed breed queen, from the city of Campo Grande, Mato Grosso do Sul state, Brazil was referred for clinical care due to the presence of a nodule, with approximately 4 cm, near the inguinal breast. Other parameters, on physical examination, are within the normal range for the specie. For the investigation, additional tests were requested. Eosinophilia was found in a complete blood count (CBC). The association between macroscopic characteristics and cytological findings allowed the diagnosis of inguinal lymph node hyperplastic. Furthermore, amastigotes of Leishmania sp. were visualized both in the peripheral blood sample and in the analyzed material mass. The diagnosis of visceral leishmaniasis and identification of specie of Leishmania was performed by PCR technique (Polymerase Chain [...](AU)

Characterization of antigen-presenting cells from the porcine respiratory system.

Antigen-presenting cells (APCs) are strategically placed in all anatomic sites with high antigen exposure such as the respiratory system. The aim of this study was to evaluate phenotypic and functional properties of APCs from the lung (L-Cs), mediastinal lymph node (LN-Cs) and bronchoalveolar lavage cells (BAL-Cs). The APCs were first analyzed based on forward scatter and side scatter profiles and the selection of MHC-II(high)CD172a(+) cells (referred to as APCs); then the expression of CD1a, CD163, CD206, CD16 and CD11R3 was evaluated in the APCs. The results showed that CD1a, CD163 and CD206 were differentially expressed among L-Cs, LN-Cs and BAL-Cs, suggesting the phenotype MHC-II(high)CD172a(+)CD1a(low/-)CD163(low)CD206(-) for L-Cs and MHC-II(high)CD172a(+)CD1a(+)CD163(low/-)CD206(+) for LN-Cs. BAL-Cs were MHC-II(high)CD172a(+)CD1a(-)CD163(high)CD206(+/-). The functional characteristics of L-Cs and LN-Cs were different from those of BAL-Cs, confirming that L-Cs and LN-Cs resemble specialized APCs. In conclusion, we present the characterization of APCs from L-Cs, LN-Cs and BAL-Cs of the porcine respiratory system.

Neutrophils exhibit differential requirements for homing molecules in their lymphatic and blood trafficking into draining lymph nodes.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Postnatal development of lung T lymphocytes in a porcine model.

PURPOSE: Despite the high prevalence of respiratory diseases in the world and the extensive information available on the mucosal immune system, research on the development of the lung immune system in humans is limited by technical and ethical considerations; therefore, we studied the postnatal development of T lymphocytes in lung lobes in a porcine model. METHODS: Using less than 36-hour-old (NB), 1-week-weaned (5-week-old -AW-), 3-month-old (3M), and 4-year-old (4YR) healthy, nonvaccinated, specific pathogen free (SPF) Vietnamese miniature pigs, we studied the CD3+, CD4+, CD8+, TCR1 (gamma-delta T cells), and CD25+ (IL-2R-alpha) cell subpopulations in lung lobes parenchyma, bronchoalveolar lavage (BAL), peripheral blood mononuclear cells (PBMC), and cervical lymph nodes (LN) by flow cytometry. RESULTS: No differences among lung lobes were detected in any of the cell subpopulations tested. A low proportion of T cell subsets was detected in NB and 4YR groups in lung and BAL. Besides, the AW and 3M groups showed important changes in T cell subpopulations. CONCLUSIONS: These results suggest that in healthy animals the lung lobes behave as a homogeneous immune organ. T cells were detected in very low percentages at birth and in adult life, which may explain the high susceptibility to respiratory infections both early and later in life. Postweaning antigenic challenges and endocrine and sexual maturity at 3M had important effects on the development of the mucosal immune system. It was also evident that changes at mucosal sites were poorly correlated with PBMC and LN.