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)

Eupatilin Ameliorates Collagen Induced Arthritis

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.

FNAC of Lymph Node Disorders.

Introduction: Fine needle aspiration cytology (=FNAC) is most popular diagnostic aid in patients with lymphadenopathy. This paper proves it to be highly sensitive also. Materials and Methods: The study comprises 300 lymph node aspirates done at Shri Mahant Indiresh Hospital of SGRR Medical College during a period of two years from January 2009 to December 2010. Results: Cytology was unsatisfactory in 3.3% (n=3) cases; showed reactive non specific lymphadenitis in 41.7% (n=125) cases; tuberculosis in 31.7% (n=95) cases; primary lymphomas in 6.3% (n=19) cases and metastatic tumor in 10.7% (n=32) cases. Conclusion: FNAC is a very sensitive procedure in patients with lymphadenopathy; sensitivity of 96.7% reported in our study.

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.

O papel da expressão de Bcl-2 em material obtido por PAAF no diagnóstico de doenças linfoproliferativas B / The role of Bcl-2 expression in fine needle aspiration specimens for the diagnostic accuracy in lymphoproliferative diseases

INTRODUÇÃO: O diagnóstico das doenças linfoproliferativas (DLP) tradicionalmente baseia-se no estudo histológico dos linfonodos (LN) acrescido de imuno-histoquímica. A imunofenotipagem (IFT) pela citometria de fluxo (CF) é uma ferramenta sensível e rápida, que pode ser aplicada nas DLP, em material obtido por punção aspirativa por agulha fina (PAAF) de LN. O Bcl-2 é um proto-oncogene que se expressa em várias DLP, porém em níveis especialmente elevados no linfoma folicular (LF). OBJETIVOS: Diagnosticar DLP, através de morfologia e imunofenotipagem por CF, em amostras obtidas por PAAF de LN. MATERIAL e MÉTODO: Amostras de 25 pacientes com adenopatias e de duas tonsilas reacionais foram analisadas pela morfologia e IFT, utilizando um painel inicial de AcMo (CD3, CD4, CD8, CD19, anti-kappa; e anti-lambda;), ampliado conforme a necessidade (CD5, CD10, CD11c, CD23, CD79b, sIgM, FMC-7 e Bcl-2). Os resultados foram comparados com a histologia. RESULTADOS:Dos 25 casos, quatro foram classificados como reacionais e 21 como DLP-B, havendo concordância com resultados histológicos em todos os casos. A intensidade média de fluorescência (IMF) da Bcl-2 no LF (19,92) foi maior que em outras DLP-B (11,93) e que nos controles (3,49) (p = 0,032). CONCLUSÃO:A PAAF de LN combinada com a citomorfologia e a IFT por CF permite uma rápida diferenciação entre os processos reacionais e linfoproliferativos B. A elevada expressão da Bcl-2 nos LFs pela citometria mostra sua utilidade no diagnóstico do tipo mais freqüente das DLP-B. A obtenção de células por PAAF requer treinamento e recomendamos mais de uma punção.

BACKGROUND: The diagnosis of lymphoproliferative disorders (LPD) is routinely made through histological and immunohistochemical analysis of lymph nodes. Immunophenotyping by flow cytometry (FC) is a sensitive and fast tool, which may be applied in samples obtained through fine needle aspiration for the diagnosis of LPD. Bcl-2 is a proto-oncogene that appears in several LPD and it has a significantly high expression in follicular lymphomas. OBJECTIVES: to diagnose LPD in FNA samples through morphology and flow cytometry immunophenotyping. MATERIAL AND METHODS: Samples from 25 patients with lymphadenopathies and 2 reactive tonsils were studied through morphology and immunophenotyping. The antigens expressions were evaluated by using a screening panel of monoclonal antibodies (CD3, CD4, CD8, CD19, light chains kappa; and lambda), followed by CD5, CD10, CD11c, CD23, CD79b, sIgM, FMC-7 and Bcl-2 when required. The results were compared with histology. RESULTS:Four out of 25 samples were reactive processes and 21were B-LPD. In all cases there was consistency with histological results. The mean fluorescence intensity of Bcl-2 in Follicular Lymphoma (19.92) was higher compared with other lymphoproliferative diseases (11.93) and controls (3.49) (p = 0.032). CONCLUSION: Fine needle aspiration of lymph nodes associated with cytomorphology and flow cytometry immunophenotyping allows a fast differentiation between reactive processes and B lymphoproliferative cases. The high expression of Bcl-2 by cytometry shows its usefulness in the diagnosis of the most frequent type of B-LPD. Fine needle aspiration sampling requires training and more than one aspiration is recommended.

Nodal status, C-ERBB-2 expression combined with lymphovascular invasion creates a more powerful tool for predicting outcome in breast cancer

To evaluate the prognostic role of peritumoral vascular invasion [PVI] and its association with axillary nodal status and c-erbB-2 expression. Seventy five patients with stage I and II breast carcinoma who underwent conservative breast surgery or modified radical mastectomy were assessed clinically and pathologically for tumor size, grade, axillary lymph node status and peritumoral vascular invasion [PVI]. The immunophenotype of the tumor was determined as: the expression of oestrogen [ER] and progesterone [PgR] receptors, and c-erbB2. Thirty eight patients [50.7%] showed PVI. It was found that extensive PVI was significantly more likely to be associated with nodal positivity, higher tumor grade and c-erbB-2 over-expression. - 52/75 [69.3%] patients showed positive nodal status. The analysis showed that nodal positivity was significantly associated with tumor size, higher grade, presence of PVI and c-erbB-2 overexpression. - PVI and nodal positivity showed no significant association with receptor status. These data suggest that assessment of PVI together with axillary nodal status and c-erbB-2 expression creates a more powerful tool for predicting outcome in patients with breast cancer

B Cells in Murine Cervical Lymph Nodes are Conventional B-2 Cells

We investigated the characteristic features of cervical lymph node B cells to determine whether their behavior differs from that of B cells located elsewhere, because cervical lymph nodes may be exposed to continual antigenic stimulation from the naso- and/or oropharynx. B cells were isolated from cervical lymph nodes, spleen and peritoneal fluid of mice, cultured in medium, and exposed to various stimuli. The expression of various surface molecules characteristic of lymphoid B cells was assayed by flow cytometry, and immunoglobulin secreted into the culture supernatants was evaluated by enzyme-linked immunosorbent assay. B220+ cells were cultured in medium alone or with lipopolysaccharide, and their entrance into S phase in response to stimuli was measured by proliferative assays. Phenotypic characteristics of cervical lymph node B cells included CD5low, CD23high, CD43low, B7.1low, B7.2low, and Syndecan-1low. Unstimulated lymphoid B cells did not secrete immunoglobulin, but, upon stimulation, secretion of IgM was increased more than secretion of IgA and IgG. B cells actively entered S phase after 48 hr stimulation. These results show that B cells in cervical lymph nodes are conventional B2 cells, like splenic B cells.

Murine model of BCG lung infection: dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes.

C57Bl/6 female mice were infected with an intrapulmonary dose of 2.5 x 10(4) BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1, 2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4-6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3+ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1+) per lung increased by about 50%, 5-6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNgamma producing non-T cells outnumbered T cells producing IFNgamma. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNgamma producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNgamma response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNgamma, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine.

Administration of interferon-gama to pregnant rats reverses the depressed adjuvant-induced arthritis of their chronically Trypanosoma cruzi-infected offspring

We demonstrated that administration of interferon gamma (IFN-gama) to the inbred "l" strain of pregnant rats conferred partial resistance on their offspring to challenge with Trypanosoma cruzi. We now examine if this intervention also modifies the reportedly immunodepressed cellular responses which occur during chronic infection. Offspring were born to mothers undergoing one of the following procedures during gestation: subcutaneous injections of recombinant rat IFN-gama, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating (IFN-Mo); infection with 106 trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gama treatment as given to the former group (TcIFN-Mo); the same protocol except that physiological saline was injected instead of IFN-gama (Tc-Mo); injection of physiological saline only (control-Mo). All offspring groups (N = 8-10/group) were infected at weaning and were assessed 90 days later for their adjuvant-induced arthritic response or levels of major T cell subsets in spleen and lymph nodes. TcIFN-Mo and IFN-Mo offspring showed a reestablished arthritic response, which remained within the range seen in controls. Immunolabeling studies on parallel groups of 90-day-infected offspring showed that the inverse CD4/CD8 cell ratio that is usually seen in lymphoid organs from these chronically infected rats (median 0.61) appeared to have recovered in the TcIFN-Mo and IFN-Mo groups (median 1.66 and 1.78, respectively) and was not different from uninfected controls (1.96). These studies indicate that early stimulation with IFN-gama is able to reverse the immunosuppressive state that is usually present during the chronic period of the experimental infection.

nuclear proliferation antigen in chronic lymphoproliferative disease

To determine whether the proliferation rates of tumour cells, in chronic lymphoproliferative disorders may reflect disease activity and relate to prognosis, we studied the expression of ki-67% [a nuclear proliferation marker] by alkaline phosphatase anti-alkaline phosphatase technique [APAAP], in peripheral blood and bone marrow mononuclear cells [separated on Ficoll-Hypaque] and in lymph-node biopsies, from patients with chronic lymphatic leukemia [CLL], chronic lymphatic leukemia/prolymphocytic leukemia [CLL/PLL], prolymphocytic leukemia [PLL] and non-Hodgkin's lymphoma with leukemic phase. The proliferation rate was determined for these patients at presentation and again two months after therapy [to detect any change with therapy]. We found that the highest rate of proliferation in each group was parallel to the degree of malignancy i.e. PLL showed higher proliferation than CLL/PLL, and CLL/PLL showed higher proliferation than CLL. In the NHL group the highest proliferation rate was found in the high-grade NHL. followed by intermediate grade NHL then the low grade NHL Lymph node biopsies also showed the same relation between proliferation rates and degree of malignancy. Bone marrow cells did not show a particular pattern probably due to interference from the erythroid element and contamination by peripheral blood. Ki-67% was compared to other proliferation markers serum B2 micro globulin and lactate dehydrogenase, It was found to be an independent marker of proliferation it is unaffected by hepatic, renal and gastrointestinal elements and thus its specificity for the tumour proliferation

Touch imprints cytology of lymph nodes

Touch imprints [Tl] is an inexpensive and a rapid diagnostic technique that can be utilized during surgery. In this study touch imprints of 54 cases of lymphadenopathy were studied and reports were delivered on the same day. The results were correlated with histopathology later on. In 38 cases tuberculosis was diagnosed, 5 were Non Hodgkin's lymphoma, 2 Hodgkin's lymphoma, 7 metastatic tumour and 2 had reactive hyperplasia. From this study it was concluded that touch imprints give early diagnosis with more than 90% accuracy, so it can be used for intraoperative diagnosis in radical surgery

Fine needle aspiration cytology in the diagnosis of lymphadenopathy

The results of fine needle aspiration cytology [FNAC] were compared with the clinical and histopathological results in 125 patients with lymphadenopathy with the final diagnosis of benign lymphadenopathy in 54 cases and malignant lymphadenopathy in 71 cases. The material was sufficient for cytological analysis in 92% of the patients. Correct cytological diagnosis was achieved in 79.6% of benign lymphadenopathy and in 85.9% of malignant lymphadenopathy with an overall accuracy of 83.2%, FNAC is effective, safe and simple technique for discovering the etiology of lymphadenopathy. Rebiopsy is recommended in lesions with no diagnostic cells in the aspirate and when the diagnosis is doubtful, surgical excision for histopathology is mandatory

Bacterial translocation and wasting in stressed mice

The effects of strees immunity and on the bacterial translocation from intestine to mesenteric lymph nodes, liver, and spleen were studied in a group of newborn CD1 mice. Animals were separated into three experimental groups. Mice from group I were stressed by intraperitoneal (IP) injections of heatkilled staphylococci for 4 weeks. Mice from group II were IP injected with saline solution only. The remaining mice, group III, were not injected. The clinical condition, presence of bacteria in abdominal organs, mitochondrial activity in splenic cells, lymphocyte proliferative response to Concanavalin-A and in vitro antibody production were evaluated in each mouse. Results showed that prolonged IP stressor challenge causes severe weight loss and immunodeficiency. The splenic lymphocytes from stressed mice exhibited a significant depression of both proliferative response to Concanavalin-A stimulation and anti-erythrocytes antibody synthesis. Instead, cultured in basal conditions, the splenic cells from stressed mice have an increased capacity to reduce the tetrazolium salts. Bacterial dissemination from intestine to mesenteric lymphoid nodes was also confirmed in the same group of mice. In contrast, mice in groups II and III presented no weight loss and immunodeficiency. Results suggest that chronic biological stress induced in newborn mice could facilitate the translocation of Gramnegative bacteria. Probable pathogenic mechanisms are commented upon and a correlation is proposed between the bacterial dissemination and the wasting development

Clasificación de la enfermedad de Hodgkin. Posibilidades de la biopsia por aspiración con aguja delgada en ganglio linfático / The classification of Hodgkin disease. posibilities of fine needle aspiration in lymph node

El diagnóstico de enfermedad de Hodgkin (EH) en aspirados con aguja delgada (BAAD) de ganglios linfáticos, se basea exclusivamente en la identificación de células clásicas de Reed-Sternberg (R-S), y sus variantes y en la relación de éstas con el fondo reactivo. Por las características del material no existen criterios estructurales evaluables. Se han identificado casos de lesiones benignas y malignas que presentan como constituyente neoplásico, células que son morfológicamente indistinguibles de las de R-S o sus variantes. Este grupo de lesiones se evalúan mejor cuando se realiza inmunohistoquímica con anticuerpos contra CD 15, CD 30, CD 45, Pan T y Pan B. En años recientes, han sido publicados criterios citológicos que permiten clasificar a la EH en más de 70 por ciento de los casos. Con el objetivo de evaluar la reproducibilidad de dichos parámetros citológicos, identificamos en un período de cinco años, nueve aspirados diagnosticados como EH, en donde el material era adecuando. Todos los casos tenían corroboración histológica y a todos se les realizó estudio de inmunohistoquímica con anticuerpos contra CD 15, CD 30, CD 45, Pan T, Pan B. Unicamente en cinco de los nueve casos se pudo precisar el tipo de EH, cuatro como celularidad mixta y uno como predominio linfocítico. Tres aspirados no tenían suficientes células para realizar una aproximación porcentual objetiva siguiendo los criterios de Das y Cols. Uno de los casos mostró, además de las células de R-S, numerosas células linfoides atípicas que sugirieron el diagnóstico de linfoma anaplástica de células grandes, que fue corroborado posteriormente con estudio de inmunohistoquímica. La clasificación de EH en BAAD de ganglios linfáticos es poco reproducible. La correlación en la clasificación citológica e histológica fue de 33 por ciento en el presente estudio. Aunque el diagnóstico inicial de enfermedad de Hodgkin se puede realizar en el material obtenido por BAAD, siempre que sea posible, es recomendable obtener tejido linfoide para clasificar la enfermedad y realizar inmunohistoquímica en caso de ser necesario

Evaluation of fine needle aspiration cytology and imprint cytology in some cases of lymphadenopathy

This study included 115 patients with lymphadenopathy. They were subjected to clinical examination and fine needle aspiration cytology of one of the enlarged lymph nodes. This was followed by imprint cytology and histologic examination of this lymph node after its excision. Clinical examination was correct in 74% of the cases. The overall accuracy of fine needle aspiration was 86%. It was accurate in all cases of reactive hyperplasia, 85% of TB. Lymphadenitis, 88% in Hodgkin's lymphoma, 78% in non-Hodgkin's lymphoma, and 84% of metastatic lymphadenopathy. On the other hand, the overall accuracy of imprint cytology was higher than that of fine needle aspiration, being 93%. It diagnosed all cases of reactive hyperplasia and non-Hodgkin's lymphoma, 90% in TB lymphadenitis, 88% in Hodgkin's lymphoma and 89.5% in metastatic lymphadenopathy. These techniques proved to be reliable, rapid, and inexpensive procedures in diagnosis of lymphadenopathy. They can differentiate well between inflammatory and neoplastic lesions, but in cases of lymphoma, cytologic diagnosis should be followed by histologic diagnosis for accurate classification and grading

Fine needle aspiration biopsy in diagnosis of tuberculosis

Fine needle aspiration biopsy [FNA] is alternative to excision biopsy in the diagnosis of tuberculosis. Biopsy materials from 45 cases were studied by both Z-N stain and cultures. Z-N stain was positive in 27 cases out of 45 [60%], cultures were positive in 35 cases out of 45[78%]

Identification T-cell reactive antigens in Nippostrongylus brasiliensis using a cell immunoblotting technique

Antigens of the nematode Nippostrongylus brasiliensis were fractionated by SDS-PAGE under reducing conditions, transferred electrophoretically onto nitrocelluose and converted to antigen-bound nitrocellulose particles for use in vitro proliferation assays. Mesenteric lymph node cells from unfected rats were analyzed for reactivity against the fractionated antigens, revealing a range of different molecular weight antigens. Ian addition, when supernatants from these cultures were assyed for IL3, further reactive antigens wee detected. The results demonstrated that these approaches are useful for the identification of T-cell reactive components of a complex mixture of parasite antigens in helminth infections, where the cellular nature of protection is not well defined

Utilidad de la impronta de ganglio linfático en el diagnóstico del linfoma / Lymph node imprints in the diagnosis of lymphoma

Para conocer la utilidad de la impronta de ganglio linfático, se revisó el archivo de citomorfología del laboratorio de Estudios Espaciales del Servicio de Hematología en el período de febrero de 1983 a diciembre de 1986 y se correlacionó con los reportes de histopatología de la Unidad de Patolog del Hospital General, S.S. Se revisaron 44 improntas de pacientes con diagnóstico en 31 casos (72%) y no habiendo correlación diagnóstico en 13 casos (28%). Los resultados de este estudio justifican la introducción de la impronta de ganglio dentro de los exámenes de procedimientos diagnóstico en pacientes con probable linfoma. Su uso sicesivo y la adquisición de mayor experiencia del observador hará que este porcentaje se mejore sin que reemplace el diagnóstico histopatológico .