Inverse correlation of CD3 & vascular endothelial growth factor in incisional oral squamous cell carcinoma biopsies predicts nodal metastasis & poor survival of patients

Background & objectives: Oral squamous cell carcinoma (OSCC) is widely prevalent in the Indian subcontinent mainly due to habit-associated aetiologies. Immune regulation and angiogenesis are the part of tumourigenesis that play a crucial role in metastasis and survival. However, the concurrent expression of vascular endothelial growth factor (VEGF) and CD3 (immune regulator receptor on T-lymphocyte) in the same OSCC tissue samples has not been reported in the Indian population. The present study evaluated the expression of CD3+ T-cells and VEGF in OSCC tissue samples and studied the clinicopathological correlation and survival analysis in an Indian population. Methods: This was a retrospective study conducted on 30 formalin-fixed and paraffin embedded sections which were histologically diagnosed as OSCC cases comprising of 15 metastatic OSCC and 15 non- metastatic OSCC with available clinical data and survival status. Results: Reduced expression of CD3+ T-cells and increased VEGF expression were observed in metastatic OSCC samples. The correlation of expression of CD3+ T-cells and VEGF with clinicopathological parameters showed a significant association between these markers with age, nodal status, site of the lesion and survival. Interpretation & conclusions: Reduced expression of CD3+ T-cells in OSCC was found to be associated with a significantly poor survival. VEGF was found to be over expressed in metastatic OSCC as compared to that in non-metastatic OSCC. The study findings suggest that the evaluation of CD3 and VEGF in incisional OSCC biopsies can be considered for predicting the survival outcome and metastasis

Glofitamab for diffuse large B cell lymphoma / 中国药房

Diffuse large B cell lymphoma （DLBCL） is a malignant tumor derived from mature B cells. Currently， chemotherapy is still the main clinical treatment. However， some patients experience recurrence or refractory conditions after treatment. On June 15， 2023， the FDA approved the marketing of glofitamab， a CD3/CD20 bispecific monoclonal antibody， to provide the new treatment plan for patients with recurrent or refractory DLBCL after receiving 2-line or above systemic treatment. This article reviews pharmacological effects， clinical studies， safety， usage and dosage of glofitamab. Glofitamab mainly plays a therapeutic role in DLBCL by promoting the activation and proliferation of T cells，activating T cells to release tumor cell-killing proteins， and mediating the lysis of B cells. Clinical studies have shown that glofitamab has a better complete and objective response rate for recurrent or refractory DLBCL. Common adverse reactions caused by glofitamab include mild/moderate cytokine release syndrome， musculoskeletal pain， rash， fatigue， and so on，without significant drug interactions.

Determinación del intervalo de referencia para subpoblaciones linfocitarias CD3+, CD4+ y CD8+ y el índice CD4+/CD8+ en la población chilena adulta

Resumen Introducción: En varios estudios se ha documentado la influencia de factores étnicos en la distribución de las subpoblaciones linfocitarias; sin embargo, los intervalos de referencia utilizados en Chile fueron obtenidos de un estudio realizado en Países Bajos el año 1997. Objetivo: Determinar el intervalo de referencia para subpoblaciones linfocitarias CD3+, CD4+ y CD8+, además del índice CD4+/CD8+ en la población chilena. Metodología: Se analizó un total de 200 muestras de sangre total obtenida de hombres y mujeres adultos sanos, utilizando el método establecido por el CLSI estandarizado en el protocolo EP28-A3c desde la etapa pre analítica en adelante. Resultados: Los rangos de referencia para CD3+, CD4+ y CD8+ fueron 54,7-81,6% (789-2732 céls/μL), 28,1-57,7% (447-1703 céls/μL) y 15,1-38,8% (226-996 céls/μL), respectivamente. El índice CD4+/CD8+ fue de 0,84-3,77. Discusión: Los valores de referencia de las subpoblaciones linfocitarias en la población chilena sana son diferentes de los que se usan actualmente en Chile. Estas observaciones muestran datos locales que pudieran tener implicaciones para el tratamiento de la infección por VIH, y los rangos de referencia encontrados en este estudio pudieran ser usados para entender la situación local de algunos pacientes. Conclusiones: Otros estudios deberán ser realizados para confirmar estas observaciones dada la falta de datos previos y debido a que este es el primer estudio en población chilena.

Abstract Background: Several studies have documented the influence of ethnic factors on the distribution of lymphocyte subpopulations; however, the reference intervals used in Chile were obtained from a study carried out in the Netherlands in 1997. Aim: To determine the reference interval for CD3+, CD4+ and CD8+ lymphocyte subpopulations, as well as the CD4+/CD8+ index in the Chilean population. Methods: A total of 200 whole blood samples obtained from healthy adult men and women were analyzed using the method established by CLSI standardized in protocol EP28-A3c from the pre-analytical stage onwards. Results: The reference ranges for CD3+, CD4+ and CD8+ were 54.7-81.6% (789-2732 cells/μL), 28.1-57.7% (447-1703 cells/μL) and 15.1-38.8% (226-996 cells/μL), respectively. The CD4+/ CD8+ index was 0.84-3.77. Discussion: The reference values for lymphocyte subpopulations in the healthy Chilean population are different from those currently used in Chile. These observations show local data that could have implications for the treatment of HIV infection and the reference ranges found in this study could be used to understand the local situation of some patients. Conclusions: Others studies must be done to confirm these observations due to lack of previous data and because this is the first study in Chilean population.

Analysis on dynamic changes of T lymphocyte subsets in recipients with stable graft status after lung transplantation / 器官移植

Objective To analyze the dynamic changes and the influencing factors of T lymphocyte subsets in recipients with stable graft status within 1 year after lung transplantation. Methods Clinical data of 41 recipients with stable graft status after allogeneic lung transplantation were analyzed. The absolute value and ratio of T lymphocyte subsets in peripheral blood from recipients were measured by flow cytometry before operation, 2 weeks and each month (within 1 year) after operation, respectively. The effects of age, gender, body mass index (BMI), surgical method, incidence of primary graft dysfunction (PGD) after operation, and primary disease upon the absolute values of T lymphocytes were evaluated. Results Within 1 year after lung transplantation, the absolute values of CD3+, CD3+CD4+, CD3+CD8+T lymphocytes and CD4+/CD8+ ratio were changed over time (all P < 0.001). Compared with preoperative values, there was no statistical significance in the absolute values of CD3+ and CD3+CD4+T lymphocytes at 12 months after operation (P=0.659, 0.109), whereas the absolute value of CD3+CD8+T lymphocytes was increased (P=0.02) and the CD4+/CD8+ ratio was decreased (P < 0.001). Age, gender, BMI, surgical method and incidence of PGD after operation exerted no significant effect on the dynamic changes of absolute values of CD3+CD4+ and CD3+CD8+T lymphocytes (all P > 0.05). Primary disease before lung transplantation exerted no effect on the changes of CD3+CD4+T lymphocytes, whereas the postoperative absolute value of CD3+CD8+T lymphocytes was higher in recipients with infectious lung diseases (P < 0.05). Conclusions The absolute values of CD3+, CD3+CD4+, CD3+CD8+T lymphocytes in recipients with stable graft status after lung transplantation are relatively low in the early stage after lung transplantation, then gradually restore, and stabilize at 6 months after operation. Dynamic changes are not associated with age, gender, BMI, surgical method and incidence of PGD after operation of recipients.

Expression of IL-2/IL-15Rβ on memory CD3 + CD8 + CD45RO + T cells in chronic hepatitis B patients undergoing antiviral treatment and its significance / 中华临床感染病杂志

Objective:To investigate the expression of IL-2/IL-15 receptor β subunit (IL-2/IL-15Rβ) on memory CD3 + CD8 + CD45RO + T cells in patients with chronic hepatitis B (CHB) receiving antiviral treatment and its significance. Methods:Sixty-eight patients with chronic active hepatitis B (CAHB) and 47 asymptomatic hepatitis B virus (HBV) carriers attending in the Department of Infectious Diseases, the First Affiliated Hospital of Wannan Medical College from March 2019 to December 2020 were enrolled in the study; and 30 health subjects were also enrolled as healthy control group. Among 60 CAHB patients there were 30 cases with positive HBeAg and 30 cases with negative HBeAg. All CAHB patients received nucleos(t)ide analogue therapy, the HBV-related markers, Alanine aminotransferase (ALT) and the expression of IL-2/IL-15Rβ on CD3 + CD8 + CD45RO + T cells were determined and compared between HBeAg-positive and negative patients, before and after treatment. Normal distribution measurement data among 3 groups were compared with One-way ANOVA; normal distribution measurement data between 2 groups were compared with paired samples t test; non-normal distribution measurement data between the two groups were compared with Mann-Whitney U test; Pearson’s correlation coefficient was performed for correlation analysis. P<0.05 was considered statistically significant. Results:The proportion of CD8 + CD45RO + T cells on PBMC CD3 + T cells in CAHB group [(8.6±3.7)%] was higher than that of asymptomatic HBV carriers group [(5.7±2.5)%] and healthy control group [(5.5±1.5)%] (all P<0.05). The expression percentage of IL-2/IL-15Rβ on PBMC CD3 + CD8 + CD45RO + T cells in CAHB group [(6.8±4.7)%] was higher than that of asymptomatic HBV carriers group [(4.7±2.8)%] and healthy control group [(4.3±2.2)%] (all P<0.05). The MFI of IL-2/IL-15Rβ on PBMC CD3 + CD8 + CD45RO + T cells in CAHB group (243±168) was higher than those of asymptomatic HBV carriers group (160±91) and healthy control group [160±63] (all P<0.05). The expression percentage and MFI of IL-2/IL-15Rβ on PBMC CD3 + CD8 + CD45RO + T cells were positively correlated with the percentage of CD3 + CD8 + CD45RO + T cells in CAHB patients ( r=0.33 and 0.28, all P<0.05). The proliferation percentage of PBMC CD3 + CD8 + CD45RO + T cells in CAHB group[ (43.7±16.0)%] was higher than that of asymptomatic HBV carriers group [(29.1±9.4)%] and healthy control group [(26.8±9.6)%] after stimulation with Anti-CD3+ super-2 (all P<0.05). After the expression of IL-2/IL-15Rβ was blocked, the proliferation percentage of CD3 + CD8 + CD45RO + T cells was decreased [(11.2±6.3)%] compared with the untreated CAHB group ( P<0.05). The percentages of PBMC CD3 + CD8 + CD45RO + T cells secreting IFN-γ, IL-2 and TNF-α in CAHB group were (13.8±5.4)%, (14.0±4.3)% and (12.3±4.6)% respectively, which were higher than those of asymptomatic HBV carriers [(8.4±2.6)%, (9.4±3.2)% and (6.8±3.3)%] and healthy control group [(6.9±2.7)%, (9.9±3.0)% and (7.7±3.8)%] after stimulation with Anti-CD3+ super-2 (all P<0.05). After the expression of IL-2/IL-15Rβ was blocked, the percentages of PBMC CD3 + CD8 + CD45RO + T cells secreting IFN-γ [(2.4±1.6)%], IL-2 [(4.1±1.9)%] and TNF-α [(4.1±1.8)%] were decreased compared with the untreated CAHB group (all P<0.05). HBeAg, ALT, the expression percentage and MFI of IL-2/IL-15Rβ on CD3 + CD8 + CD45RO + T cells were 521.4 (68.9, 1 339.0) COI, 292 (160, 528) U/L, (6.4±3.2)% and (239±136) in 30 HBeAg-positive CAHB patients before treatment, which were higher than those after treatment [3.5(1.5, 17.5)COI、20(14, 31) U/L, (4.1±2.4)% and (134±58)] ( Z=5.337 and 6.403, t=3.229 and 3.892, all P<0.05). HBsAg, ALT, the expression percentage and MFI of IL-2/IL-15Rβ on CD3 + CD8 + CD45RO + T cells were (5 310±2 851) COI, (328±207) U/L, (7.1±5.8)% and (252±110) in 30 HBeAg-negative CAHB patients before treatment, which were higher than those after 48 weeks of treatment [(3 811±2 495) COI, (33±14) U/L, (4.6±2.9)% and (154±73)] ( t=2.167, 5.595, 2.116 and 2.383, all P<0.05). Conclusion:The study suggests that up-regulated expression of IL-2/IL-15Rβ is associated with elevated frequency, proliferation and secretion function of memory CD3 + CD8 + CD45RO + T cells in CAHB patients.

Effect of Wenjing Huayu Zhitong Therapy in Treating Primary Dysmenorrhea and Its Regulatory Mechanisms on T Cell Subsets / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of Wenjing Huayu Zhitong therapy in treating primary dysmenorrhea (PD) with cold coagulation and blood stasis, and to explore its immune mechanisms on PD. Method:The 108 PD patients with cold coagulation and blood stasis syndrome were collected and randomly divided into traditional Chinese medicine (TCM) group, ibuprofen group and placebo group according to the random number table method, with 36 cases in each group. All patients received corresponding medicines three days before menstruation. The patients in TCM group were treated with TCM and ibuprofen sustained release capsule simulator. The patients in ibuprofen group were treated with ibuprofen sustained-release capsule and TCM simulator. The patients in placebo group were treated with TCM simulator and ibuprofen sustained-release capsule simulator. Treatment lasted for 6 consecutive days for three menstrual cycles, and follow-up was conducted for three menstrual cycles after drug withdrawal. The visual analogue score (VAS), total time of abdominal pain and TCM symptom scores in each menstrual cycle were recorded. The levels of CD3+, CD4+, CD8+ and the ratio of CD4+/CD8+ in peripheral blood before and after treatment were detected by flow cytometry. Result:After treatment for three menstrual cycles, both the TCM group and ibuprofen group were better than placebo group in reducing VAS score and reducing total abdominal pain time (P<0.01). The long-term follow-up effect after drug withdrawal in TCM group was significantly better than that in ibuprofen group (P<0.01). The total effective rate was 91.43% (32/35) in TCM group, 66.67% (10/33) in ibuprofen groups, and 30.30% (10/33) in placebo group . The efficacy of the TCM group was better than that of the ibuprofen group (χ2=-2.971, P<0.01), and the efficacy of the ibuprofen group was better than that of the placebo group (χ2=-2.371, P<0.05). After treatment, the levels of CD3+, CD4+ and CD4+/CD8+ in TCM group were significantly increased and the levels of CD8+ were decreased significantly as compared with those before treatment (P<0.01). After treatment, the levels of CD4+ and CD4+/CD8+ in TCM group were higher (P<0.05，P<0.01)，while the levels of CD8+ were significantly lower than those in ibuprofen group and placebo group (P<0.01). Conclusion:Wenjing Huayu Zhitong therapy can reduce the VAS score and accumulative time of abdominal pain, and improve the dysmenorrhea symptoms in patients with PD. Reversal of the T cell subsets disorder may be one of its mechanisms in treating PD with cold coagulation and blood stasis.

Fishmeal substitution for Arthrospira platensis in juvenile mullet ( Mugil liza) and its effects on growth and non-specific immune parameters / Substitución de harina de pescado por Arthrospira platensis en juveniles de lisa (Mugil liza) y sus efectos en el crecimiento y parámetros del sistema inmune no-específico / Substituição da farinha de peixe por Arthrospira platensis em juvenis de tainha (Mugil liza) e seus efeitos no crescimento e nos parâmetros do sistema imune não-especifico

Abstract Background: Cyanobacterium Athrospira platensis (Spirulina) is a potential fishmeal (FM) substitute in fish diets because of its high protein content, antioxidant and immunostimulant properties. Objective: To evaluate the effects of total and partial substitution of FM with A. platensis (0, 30, 50, 70 and 100% substitution) in juvenile mullet (Mugil liza). Methods: Juvenile mullets (n=210) were maintained in a recirculation system under optimal water parameters for the species. Mullets were fed five experimental diets for 80 days. Each diet was tested in triplicate tanks. At the end of the experimental period growth parameters were measured and samples of blood, liver and spleen were taken to evaluate the immune system. Results: Full replacement (100%) of FM resulted in growth deficits and low survival. The FM replacement induced changes in the proportion of macrophages and lymphocytes. Up to 50% FM replacement increased the expression of CD3 receptors in spleen T lymphocytes (T-Cells), whereas >50% FM replacement decreased the expression of CD3 receptors. We also found that partial FM substitution diminished the apoptotic process. Conclusions: Up to 50% FM substitution with A. platensis can improve performance of non-specific immune system of mullets.

Resumen Antecedentes: La cianobacteria Arthrospira platensis (Spirulina) puede usarse como substituto potencial de la harina de pescado (HP) por su alto contenido de proteína, sus antioxidantes y sus propiedades inmunoestimulantes. Objetivo: Analizar el efecto de la substitución parcial y total de HP por A. platensis (0, 30, 50, 70 y 100% de substitución) en juveniles de lisa (Mugil liza). Métodos: Juveniles de lisa (n=210) se mantuvieron en un sistema de recirculación con parámetros de calidad de agua en niveles óptimos para la especie. Las lisas se alimentaron con las dietas experimentales durante 80 días. Cada dieta fue evaluada en triplicado. Al final del periodo experimental se midieron los parámetros de crecimiento y se colectaron muestras de sangre, hígado y bazo para evaluación del sistema inmune. Resultados: La substitución total (100%) resultó en deficiente crecimiento y baja sobrevivencia. El remplazo de HP produjo cambios en las proporciones de macrófagos y linfocitos. La substitución de hasta un 50% HP aumentó la expresión de receptores CD3 en linfocitos T del bazo. Por otro lado, la substitución mayor a 50% HP disminuyó la expresión de receptores CD3. La substitución parcial de HP disminuyó el proceso de apoptosis. Conclusiones: Proponemos una substitución de HP del 50% por A. platensis, lo cual mejora el desempeño del sistema inmune no especifico de las lisas.

Resumo Antecedentes: A cianobactéria Athrospira platensis (Spirulina) é um potencial substituto da farinha de peixe (FP) pelo seu alto conteúdo de proteína, antioxidantes e características imune estimulantes. Objetivo: Foram avaliados os efeitos da substituição parcial e total da FP por A. platensis (0, 30, 50, 70 e 100% substituição) em juvenis de tainha (Mugil liza). Métodos: Juvenis de tainha (n=210) foram mantidos em um sistema de recirculação com os parâmetros da água sendo mantidos em níveis ótimos para a espécie. As tainhas foram alimentadas com as dietas experimentais por 80 dias, cada dieta foi testada em triplicata, ao final do período experimental foram avaliados os parâmetros de crescimento e amostras de sangue, fígado e baço foram coletadas para a avaliação do sistema imune. Resultados: A substituição total de FP resultou em redução do crescimento e baixa sobrevivência. A avaliação do sistema imune demostrou que a substituição da FP produz alterações nas proporções de macrófagos e linfócitos. Provou-se que até 50% de substituição da FP incrementa a expressão de receptores CD3. Além disso, a substituição parcial da FP diminui o processo de apoptose. Conclusão: Baseado em nossos descobrimentos, se propõe a substituição de até 50% da FP por A. platensis que melhorará o desempenho do sistema imunológico não específico das tainhas.

Anti-CD3 mAbs induce CD4+FoxP3+Regulatory T( Treg) cells and alleviate acute rejection of the pulmonary al-lografts in mice / 中华胸心血管外科杂志

Objective By using mouse orthotopic lung transplant model, we investigated the immune mechanisms of an-ti-CD3 induced lung allograft protection .Our study intends to further dissect the features of lung transplant immunology and to provide a novel therapeutic insight for the clinical application of anti-CD3 mAbs after lung transplantation.Methods Murine orthotopic allogeneic lung transplants were performed in C57BL/6 wild type(WT) mice using major histocompatibility complex (MHC) fully mismatched BALB/c donors.Syngeneic transplants were also performed in WT C57BL/6 mice using C57BL/6 donors.For immunosuppressive therapy, allograft recipients received 50g dose of anti-CD3 by intraperitoneal injection on days 2, 3, 4, 5, 6 and 9 post-operation(n=4).At day 10, histopathologic characteristics and rejection status of the pulmonary grafts were assessed.The severity of acute rejection was graded by the pathological score , and T cell and neutrophil infiltration in the pulmonary grafts was evaluated by immunohistochemical(IHC) staining for CD3 and myeloperoxidase(MPO) respective-ly.Real-time RT-PCR was performed for FoxP3, IL-17A and IFN-γexpression in the pulmonary grafts.The percentage of FoxP3+Treg in total CD4+T lymphocytes from the recipient spleens was analyzed by FACS.Results 10 days after transplan-tation, histopathologic examination demonstrated that there is no apparent acute rejection observed in the pulmonary isografts , whereas allografts from untreated recipients have marked inflammatory cell infiltration and pulmonary parenchyma lesion .IHC staining for CD3 and MPO showed that the allograft-infiltrating cells of perivascular layers are mainly T lymphocytes , and the cells around the small airways are mostly neutrophils .Anti-CD3 treatment significantly alleviated the acute rejection of pulmo-nary allografts, when compared with the untreated group.Real-time RT-PCR showed that the expression levels of IL-17A and IFN-γin allografts were markedly elevated compared to those in isografts, and anti-CD3 increased the expression of FoxP3, and reduced the expression of IL-17A and IFN-γin the pulmonary allografts.FACS analysis of splenocytes showed that the percent-age of Treg in total CD4+T lymphocytes increased significantly in the anti-CD3 treated allograft recipients, as compared with the isograft and untreated allograft recipients.Conclusion Anti-CD3 mAbs may alleviate acute rejection of the pulmonary al-lografts by promoting FoxP3 expression and Treg development.

Cluster Containing More Than 20 CD3-Positive Cells in Bone Marrow Biopsy Is a Candidate Prognostic Indicator in Peripheral T-Cell Lymphoma, Not Otherwise Specified

Assessment of bone marrow (BM) involvement in peripheral T-cell lymphoma, not otherwise specified (PTCL) is straightforward in cases of extensive involvement but difficult in cases of minimal to partial involvement. We evaluated the usefulness of CD3 as an immunohistochemical marker for assessing BM involvement in PTCL patients. BM biopsies of 92 PTCL patients were immunohistochemically stained for CD3, CD4, CD8, CD20, and CD56, and evaluated by two hematopathologists. CD3 positivity was graded according to the proportion of CD3-positive cells and the number of CD3-positive cells in a cluster. These criteria were used to determine the cut-offs at which significant differences in progression-free survival (PFS) and overall survival (OS) were observed. Multivariate analysis controlling the International Prognostic Index (IPI) score and its individual factors revealed that >20 CD3-positive cells in a cluster adversely affected PFS (relative risk [RR], 2.1; 95% confidence interval [CI], 1.0–4.3; P=0.047) and OS (RR, 2.4; 95% CI, 1.1–5.1; P=0.028) independent of IPI score. A cluster with >20 CD3-positive cells is a candidate indicator for BM involvement in PTCL.

Expect the unexpected – Loss of surface CD3 on flow cytometry in hepatosplenic T-cell lymphoma: An eye opener

Hepatosplenic T-cell lymphoma (HSTCL) is a rare extranodal T-cell lymphoma that shows preferential sinusoidal infiltration of spleen and liver. It usually shows bright expression of surface CD3 (sCD3) with restriction for γδ-T cell receptors (TCR). We present a case of a 34-year-old male who presented with hepatosplenomegaly and B symptoms. His peripheral blood and bone marrow (BM) was involved by atypical lymphoid cells that were CD2+, CD7+, CD56+, cytoplasmic CD3+, and sCD3− on immunophenotyping by flow cytometry. As sCD3 is a lineage marker for T-cell lymphomas, the loss of sCD3 posed a diagnostic dilemma. However, typical pattern of sinusoidal BM and liver involvement by CD3+ cells and TCR gene rearrangement positivity led to final diagnosis of HSTCL. The differential diagnosis, workup, and clinical course of the case are discussed. To the best of our knowledge, only one case of de novo HSTCL with negative sCD3 has been reported before in the literature.

Polyclonal anti T-lymphocyte antibody therapy monitoring in kidney transplant recipients: comparison of CD3+ T cell and total lymphocyte counts / Monitoramento da terapia com anticorpo policlonal antilinfócitos T em transplantados renais: comparação da contagem de células T CD3+ e de linfócitos totais

ABSTRACT Objective: To investigate the correlation between total lymphocyte and CD3+ T cell counts in peripheral blood in renal transplant patients treated with anti-thymocyte globulin, and discuss related outcomes. Methods: A single-center, retrospective study involving 226 patients submitted to kidney transplant between 2008 and 2013, and treated with anti-thymocyte globulin for induction or treatment of cellular rejection. Doses were adjusted according to CD3+ T cell or total lymphocyte counts in peripheral blood. Results: A total of 664 paired samples were analyzed. The Spearman's correlation coefficient was 0.416 (p<0.001) for all samples combined; the overall Kappa coefficient was 0.267 (p<0.001). Diagnostic parameters estimated based on total lymphocyte counts were also calculated using the number of CD3+ T cells (gold standard), with a cut off of >20 cells/mm3. Conclusion: Total lymphocyte and CD3+ T cell counts in peripheral blood are not equivalent monitoring strategies in anti-thymocyte globulin therapy.

RESUMO Objetivo: Investigar a correlação entre a contagem de linfócitos totais e células T CD3+ no sangue periférico em receptores de transplante renal submetidos a tratamento com globulina antitimocitária, e discutir resultados relacionados. Métodos: Estudo retrospectivo de centro único envolvendo 226 pacientes submetidos a transplante renal entre 2008 e 2013 e tratados com globulina antitimocitária, para fins de indução ou tratamento de rejeição celular. As doses foram ajustadas de acordo com a contagem de células T CD3+ ou linfócitos totais no sangue periférico. Resultados: No total, 664 amostras pareadas foram analisadas. O coeficiente de correlação de Spearman para as amostras em geral foi de 0,416 (p<0,001) e o coeficiente Kappa, de 0,267 (p<0,001). Os parâmetros diagnósticos estimados com base na contagem de linfócitos totais foram recalculados, empregando-se o número de células T CD3+ (padrão-ouro) e adotando-se o ponto de corte >20 células/mm3. Conclusão: A contagem de linfócitos totais no sangue periférico não substitui a contagem de células T CD3+ enquanto estratégia de monitorização da terapia à base de globulina antitimocitária.

Construção de um vetor de expressão para fragmentos de anticorpos anti-cd3 humanos com potencial imunorregulatório / Construction of an expression vector for fragments of human anti-cd3 antibodies with immunoregulatory potential

Construiu-se um novo vetor para expressão de fragmentos de anticorpos anti-CD3 humano (pMIRES FvFc R ­ Fc mutado) com potencial imunoregulatório. Foram utilizadas técnicas de clonagem no vetor pUC 57 e no vetor de expressão pMIRES FvFc R por meio de transformação bacteriana por choque térmico; seguida de preparação do DNA plasmidial em pequena e média escala; digestão dos mesmos com enzimas de restrição, e posterior análise e extração do DNA plasmidial por eletroforese. A clonagem foi confirmada pela técnica de sequenciamento de Sanger. Este trabalho resultou na construção de um novo cassete para expressão de fragmentos de anticorpos anti-CD3 humano contendo duas mutações na região variável e uma mutação na porção Fc, que se acredita que melhore a produção heteróloga do anticorpo. A construção deste novo vetor de expressão, com a adição da sequência de exportação de RNA e mutações na porção Fc, buscou contribuir para a caracterização e aprimoramento das características ligantes e efetoras dos anticorpos monoclonais anti-CD3.

Preparation of biotin-grafted pullulan acetate nanoparticles conjugated with CD3 antibodies and their uptake of T cells / 医学研究生学报

Objective Cytotoxic T lymphocytes are the main effector cells of anti-tumor immunity. Active targeting of nanoparticles to T cells and activation of T cells can be achieved by conjugation with specific antibodies. We prepared the biotin-grafted pullulan acetate nanoparticles conjugated with CD3 (Bio-PA-CD3 NPs), and explored their effects on the proliferation, cytokine secretion and uptake of CD8+T cells.Methods We prepared Bio-PA NPs by the dialysis method, conjugated CD3 antibodies to the surface of NPs to make Bio-PA-CD3 NPs, and measured the diameter and Zeta potential of the NPs. We evaluated the effects of the NPs on the proliferation of CD8+T cells and the secretion of cytokines by CCK-8 assay and ELISA, respectively, and quantitatively analyzed the cellular uptakes of the Bio-PA-CD3 NPs by the flow cytometry.Results The Bio-PA-CD3 NPs exhibited regular spherical shapes of even size and with no adhesion. The content of CD3 antibodies on the surface of the NPs decreased with the increased degree of biotin substitution. The CD3 contents of the Bio-PA-CD3 NPs with biotin substitution degrees of 1.6%, 5.4% and 6.3% were (36.1±4.4), (21.4±4.3) and (10.3±4.7) μg/mg, respectively. Compared with Bio-PA NPs, Bio-PA-CD3 NPs at a certain concentration significantly enhanced the proliferation of CD8+T cells in vitro and promoted the secretion of IFN-γ, TNF-β and IL-2 cytokines. The Bio-PA-CD3 NPs manifested a higher cellular uptake with the increased content of CD3 antibodies.Conclusion The Bio-PA-CD3 NPs we prepared could be a promising agent to enhance the immune effect of T cells.

Phenotypes of CIK cells prepared by ATG-F culture system and its killing effect against K562 cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the function of CIK (cytokine induced killer) cells cultured using ATG-F (anti-human T lymphocyte rabbit immunoglobulin-Fresenius) and IFN- γ, IL-2 system and its feasibility in clinical practice. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and were used to culture CIK cells by different activating antibodies; the total cell count was calculated on Day 7 and 14. The CIK cell composition, cell surface activation and proportion of inhibitory receptor molecular in ATG-F group, CD3 group and TG (Thymoglobulin) group were analyzed by Flow cytometry, and the cytotoxicity of CIK cells against K562 cells were also determined by flow cytometry at day 14 in ATG-F high-dose group, CD3 group and TG group. Results: CIK cells were successfully cultured by ATG-F, IFN-γ, IL-2 system. The proliferation rate of ATGF high-dose group was significantly higher than that in TG group (27.25±1.25 vs 16.60±1.72, P <0.01), but the proportion of CD3+ CD56+ cells showed no statistical difference compare with the CD3 group ( P >0.05). The percentage of CD3-CD56+ NK cells in ATG-F high-dose group was significantly higher than that in TG group and CD3 group [(11.19±2.60）% vs（5.66±1.00）%,（1.42± 0.51）% ， P <0.01], while the proportion of CD4+T cells was significantly lower than that in CD3, TG group [(4.35±1.47）% vs （26.88±5.01）%,（14.52±6.22）%, P <0.01]; the proportion of CD56+CD94+, CD56+CD158a+, CD56+CD158b cells was significantly higher than those in CD3 group (all P <0.01). The ATG-F high does group showed significantly higher cytotoxicity against K562 cells than that of CD3 group at the target/effect ratio of 1∶10. Conclusion: CIK cells cultured by ATG-F culture system has higher NK cell proportion than other ordinary culture system, and its activated receptor has more stronger cytotoxicity against K562 cells.

Inhibition of C2CD3 regulates the proliferation, invasion and migration of epithelial ovarian cancer through inhibiting Hedgehog pathway / 上海交通大学学报（医学版）

Objective • To explore the relationship between the expression of C2 calcium dependent domain containing protein 3 (C2CD3) in ovarian cancer and clinicopathological parameters, and its effect on the proliferation and invasion of ovarian cancer SKOV3 cells and possible mechanisms. Methods • The expression of C2CD3 protein in ovarian tissue was detected by immunohistochemistry. The proliferation, migration and invasion abilities of SKOV3 cells were detected by EdU assay, wound healing assay and Transwell assay respectively. Western blotting was performed to investigate the expression of C2CD3, Shh, Ptch1, Smo and Gli1 proteins. Results • C2CD3 protein was located in cytoplasm in ovarian cancer. C2CD3 was highly expressed in ovarian cancer compared to normal ovarian tissue. C2CD3 immunostaining was significantly higher in tumor samples in advanced stages (stage III / ) than in early stages (stage / Ⅱ). The staining intensity was significantly correlated with the grade (grade3 vs. grade1+2). The association between C2CD3 expression and age (or tumor type) was not significant. Inhibition of C2CD3 gene significantly weakened the proliferation, invasion and migration abilities of SKOV3 cells, and the expression of Shh, Ptch1, Smo and Gli1 proteins was significantly decreased. Conclusion • The expression of C2CD3 protein is increased in ovarian cancer tissues. Inhibition of C2CD3 gene weakens the proliferation, invasion and migration capacities of ovarian cancer SKOV3 cells, which may be related to the inhibition of Shh, Ptch1, Smo and Gli1 proteins in Hedgehog pathway.

Establishment of a method for culturing cynomolgus T lymphocytes induced by human CD3Ab / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To establish a method for in vitro isolation and culture of T lymphocytes from peripheral blood of cynomolgus monkeys that induced by human CD3 antibody based on the foundation of protein homology of CD3 from human, cynomolgus monkey and porcine. Methods: The amino acid sequences of human, cynomolgus monkeys and porcine CD3 proteins were obtained from NCBI, and the sequence, homology and phylogenetic tree were analyzed by DNAMAN software. Western blotting was used to detect the expression of CD3 protein on T cell membranes from the three species. PBMCs of healthy cynomolgus were isolated and divided into three groups: group A was stimulated with anti-human CD3Ab alone, group B was stimulated with IL-2 alone, and group C was costimulated with human CD3Ab and IL-2. Cell morphology and growth status were observed under inverted microscope and the cell growth curve was plotted. Cell viability was detected by trypan blue staining and the expressions of CD3, CD4 and CD8 on T cell surface were detected by flow cytometry. Results: The homology of the amino acid sequence of human CD3 protein to cynomolgus monkey and porcine were 86.9% and 65.6% respectively. The expression levels of CD3 protein on cynomolgus and porcine T cell membrane were 79% and 17% contrast to human, respectively. Cells of group A did not proliferate. Proliferation, viability and CD3 expression [(93.8±3.6)% vs (70.3±4.7)%, P<0.01] in T cells of group C were significantly higher than those in group B. Growth curve of T cells in group C showed an S-shape, which is consistent with Logistic growth curve. T cells in group C exhibited high purity and expressed high level CD3; moreover, the CD8+T cell took a high proportion. Conclusion: The membrane of T lymphocytes from peripheral blood of cynomolgus can express CD3 protein that highly homological to human. Co-stimulation of human CD3Ab, IL-2 and 1% PHAcan induce the proliferation and differentiation of T lymphocytes of cynomolgus, and obtain T lymphocytes with good growth status, high proliferation ability and high purity.

Effect of CD3+, CD4+, CD8+ T lymphocytes and CD19+ B lymphocytes on the pathogenesis of acute respiratory distress syndrome / 中华急诊医学杂志

Objective To investigate the roles of CD3+,CD4+,CD8+T lymphocytes and CD19+B lymphocytes on the pathogenesis of acute respiratory distress syndrome(ARDS).Methods According to Berlin definition Of ARDS in 2012,34 patients with ARDS admitted in the Department of ICU of Central Hospital of Baoji from January,2016 to January,2017 were enrolled in this study as study group(ARDS group).At the same time,22 healthy subjects were recruited as control group.Clinical data of ARDS patients were collected,and the survivors were followed up.The ARDS patients were divided into moderate group(n=20) and severe group (n=14) according to clinical settings on the first day after diagnosis of ARDS and Berlin Definition of ARDS in 2012,and at the same time they were also dividedinto two groups according to the outcome followed up for 28 days:non-survival group(n=14) and survival group(n =20).Sample of 3 mL peripheral venous blood of ARDS patients was collected on an empty stomach in the early morning on the first day after diagnosis of ARDS and the blood samples of healthy subjects were also collected on the first day to measure the level of CD3+,CD4+,CD8+T cells and CD19+ B cell in peripheral venous blood by flow cytometry.Comparison of CD3+,CD4+,CD8+ T cells and CD 19+ B cell numbers were carried out between ARDS group and control group on the first day after diagnosis of ARDS,and between moderate group and severe group as well as between survival group and nonsurvival group.The risk factors associated with ARDS were analyzed using logistic regression analysis.Results On the first day after diagnosis of ARDS,there were significant differences in serum Lac and pre-albumin between survival group and non-survival group(P＜0.05).The numbers of CD3+,CD4+T cells and CD19+B cell of peripheral venous serum in ARDS group were significantly lower than those in control group(P＜0.05),while there was no significant difference in CD8+ T cell number between ARDS group and control group (P＞0.05).There were statistically significant differences in numbers of CD3+,CD4+,CD8+T cells and CD19+B cell between moderate group and severe group and as well as between survival group and non-survival group(P＜0.05).Logistic regression analysis showed that CD19+B cell (OR=0.614,95％CI:0.416-0.907,P=0.014) level on the first day after diagnosis of ARDS was related with the risk of prognosis of ARDS.The ROC of CD19+B cell had area under curve(AUC) of 0.907,and the cut-off value of CD19+B cell in the survival followed up for 28 day's was 12.59％.Conclusions CD3+,CD4+,CD8+T cells and CD19+B cell level of peripheral venous serum in ARDS patients can be helpful for the assess of ARDS severity of patients in the early stage,and for prognosis judgment,especially CD 19+B cell is more remarkable.

GLTSCR1, ATM, PPP1R13L and CD3EAP Genetic Variants and Lung Cancer Risk in a Chinese Population / 华中科技大学学报（医学）（英德文版）

Genetic variants in glioma tumor suppressor candidate region gene 1 (GLTSCR1)and ATM serine/threonine kinase (ATM) have been associated with various cancer risks.Epidemiological studies also revealed the association of variants of GLTSCR1 and ATM genes with different brain tumors.However,little is known about the relationship between both gene polymorphisms and lung cancer risk.We conducted a Chinese hospital-based casecontrol study involving 384 lung cancer cases and 387 cancer-free controls.No significant differences in the single polymorphism (GLTSCR1 rs1035938 and ATM rs11212592)association were found in five genetic models (co-dominant,dominant,recessive,overdominant and log-additive models) (adjusted by smoking duration).Join effect of three SNPs (PPP1R13L rs1970764,CD3EAP rs967591,GLTSCR1 rs1035938) on chromosome 19q 13.3 showed that the designated haplotype2 (rs 1970764<rs967591A-rs 1035938c) [OR (95％ CI)=1.60 (1.11-2.32),P=0.012] and haplotype8 (rs 1970764G-rs967591G-rs 1035938T)[OR (95％ CI)=2.45 (1.17-5.12),P=0.018] were associated with increased risk of lung cancer (adjusted by smoking duration).The analysis ofmultifactor dimensionality reduction revealed that two 3-way models were the best fit models in analyses of 2 loci (P＜0.001)or 4 loci (P=0.015-0.016).The entropy-based analysis indicated the strongest synergistic effect between PPP1R13L rs1970764 and ATM rs11212592 in analysis of four genes.In conclusion,our study suggests that haplotypes consisting of PPP1R13L rs1970764-CD3EAP rs967591-GLTSCR1 rs1035938 on Chr19q13.3,interaction of smoking and GLTSCR1 rs1035938-ATM rs11212592,and synergistic action of PPP1R13L rs1970764 and ATMrs11212592 may associate with lung cancer risk in the Chinese population.

GLTSCR1, ATM, PPP1R13L and CD3EAP Genetic Variants and Lung Cancer Risk in a Chinese Population / 华中科技大学学报（医学）（英德文版）

Genetic variants in glioma tumor suppressor candidate region gene 1 (GLTSCR1)and ATM serine/threonine kinase (ATM) have been associated with various cancer risks.Epidemiological studies also revealed the association of variants of GLTSCR1 and ATM genes with different brain tumors.However,little is known about the relationship between both gene polymorphisms and lung cancer risk.We conducted a Chinese hospital-based casecontrol study involving 384 lung cancer cases and 387 cancer-free controls.No significant differences in the single polymorphism (GLTSCR1 rs1035938 and ATM rs11212592)association were found in five genetic models (co-dominant,dominant,recessive,overdominant and log-additive models) (adjusted by smoking duration).Join effect of three SNPs (PPP1R13L rs1970764,CD3EAP rs967591,GLTSCR1 rs1035938) on chromosome 19q 13.3 showed that the designated haplotype2 (rs 1970764<rs967591A-rs 1035938c) [OR (95％ CI)=1.60 (1.11-2.32),P=0.012] and haplotype8 (rs 1970764G-rs967591G-rs 1035938T)[OR (95％ CI)=2.45 (1.17-5.12),P=0.018] were associated with increased risk of lung cancer (adjusted by smoking duration).The analysis ofmultifactor dimensionality reduction revealed that two 3-way models were the best fit models in analyses of 2 loci (P＜0.001)or 4 loci (P=0.015-0.016).The entropy-based analysis indicated the strongest synergistic effect between PPP1R13L rs1970764 and ATM rs11212592 in analysis of four genes.In conclusion,our study suggests that haplotypes consisting of PPP1R13L rs1970764-CD3EAP rs967591-GLTSCR1 rs1035938 on Chr19q13.3,interaction of smoking and GLTSCR1 rs1035938-ATM rs11212592,and synergistic action of PPP1R13L rs1970764 and ATMrs11212592 may associate with lung cancer risk in the Chinese population.

Presencia del marcador lmp-1 del virus epstein-barr en linfomas de caninos / Presence of lmp-1 protein of epstein-barr virus in lymphomas of canine origin

RESUMEN El virus Epstein-Barr (EBV) es un virus de alta prevalencia en humanos que se asocia con tumores de la línea linfoide B. En caninos se dispone de pocos reportes sobre la presencia del EBV y su rol en esta especie. El objetivo del presente estudio fue determinar la presencia de la proteína latente de membrana del EBV (LMP-1) en tejidos obtenidos de 20 linfomas de caninos cuyo diagnóstico se había realizado durante un periodo de 10 años, entre 2004 y 2014. Los linfomas se reclasificaron mediante las nuevas clasificaciones histopatológicas para linfomas y se sometieron a inmunohistoquímica (IHQ) con los anticuerpos anti-CD79a, anti-CD3, anticuerpos específicos para linfocitos B y T, además de un anti-LMP-1 como marcador de la presencia del EBV. Se encontró que el linfoma más común fue el linfoma nodal de zona T con un 75% de los casos. Al realizar la inmunomarcación se encontraron 18 casos positivos a CD3, 2 casos positivos a CD79a y 6 casos positivos a LMP-1, lo que representa el 30% de positividad del EBV en linfomas. El análisis Ji cuadrado demostró significancia estadística entre la presencia del virus y la presencia del linfoma lo que sugiere, no solamente que el virus está circulando en la población canina, sino que además puede tener relación con la ocurrencia de esta neoplasia. Con relación a las variables demográficas, sólo en la raza Golden Retriever se demostró relación con la presencia del linfoma, pero no con la presencia del virus.

ABSTRACT Epstein Barr virus (EBV) is a human high prevalent virus associated with lymphoid B cells tumors development. In canines, few reports have been published regarding the presence of the virus in dogs but its role in this species remains unclear. The aim of this study was to determine the presence of LMP-1 protein of EBV in 20 canine lymphomas tissues which were previously diagnosed in a period of time between 2004 -2014. Lymphomas were reclassified in accordance with the new histopathological classifications for lymphomas and were stained by IHQ with anti-CD79a, anti-CD3 and anti-LMP-1; in addition, specific antibodies for B lymphocytes, T lymphocytes and EBV biomarker, respectively. It was found that the most common lymphoma was T-zone lymphoma in 75% of the cases of the study. The distribution of the cases regarding the immunostaining was: 18 positive cases with anti-CD3, 2 positive cases with anti-CD79a and 6 positive cases with anti- LMP-1. Positive cases of LMP-1 as a biomarker of the presence of EBV corresponded to the 30% of the cases of the study. Chi-square test showed statistical significance between the presence of the virus and the presence of lymphomas, which suggests not only that the virus is circulating in the canine population but also that could have implications in the development of the disease. Regarding demographic parameters, only the Golden Retriever breed showed a relationship with the presence of lymphoma, but not with the presence of the virus.