Cyclins D1, E, A expression and synergistic cytotoxicity to a cholangiocarcinoma cell line from recombinant TNF-alpha and PHA supernate.

We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.

Inhibition of lymphocyte proliferative responses to Helicobacter pylori by plastic adherent cells.

A study was carried out on 49 H. pylori-positive and 11 H. pylori-negative patients to determine the reactivity of peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) and acid glycine extract (AGE) of H. pylori, and to identify cells responsible for imunosuppression. Based on response to PHA stimulation, cell-mediated immunity of all patients were competent. In some patients, however, response to AGE of H. pylori was suppressed by plastic adherent cells. This study provided evidence of the presence of plastic adherent suppressor cells which suppressed PBL response to AGE of H. pylori but not to PHA suggesting that immunosuppression is antigen specific. There is also an indication that immunosuppression may be species-specific as PBL devoid of plastic adherent cells only responded to stimulation by AGE of H. pylori but not that to AGE of C. jejuni.

Evaluation of cell mediated immunity in healthy contacts of leprosy.

The cell mediated immunity (CMI) was studied in 50 healthy contacts of leprosy patients and 50 age & sex matched non-contact controls by lepromin test and leucocyte migration inhibition (LMI) test using phytohaemagglutinin (PHA) and lepromin and its association with other risk factors in contacts was assessed. The lepromin positivity correlated well with LMI results. There was no difference in CMI in I, II and III degree of contacts. Amongst direct contacts lepromin test was positive in 67 per cent as compared to 92 per cent in indirect contacts while in LMIT migration index (MI) was significantly increased (0.66 +/- 0.20) in direct contacts. MI was also significantly increased (0.73 +/- 0.20) contacts of less than two years duration which decreased to 0.51 +/- 0.18 in contacts of more than five years duration. Lepromin positivity also increased from 60 per cent to 100 per cent in these contacts. The specific CMI was significantly suppressed in contacts of LL patients (MI:0.74 +/- 0.21) and BL patients (MI: 0.61 +/- 0.01) as compared to healthy controls. B.C.G. vaccinated individuals showed better CMI response. The findings in the study showed specific unresponsiveness to lepromin in LMI in leprosy contacts of less than two years duration, direct contacts, contacts of lepromatous spectrum of index patients and contacts not vaccinated with B.C.G. emphasizing that CMI status is an important parameter in identifying the contact population at the greater risk of acquiring leprosy.

Trifluoperazine reduces the expression of CD69 in phytohemagglutinin-activated lymphocytes

Trifluoperazine (TFP) is a phenothiazine capable of inhibiting lymphocyte proliferation as well as natural killer cells (NK) and lymphokine-activated killer cells (LAK) cytotoxic activity. CD69 is a surface molecule induced by various mechanisms of cellular activation. In the present work the modulation of CD69 expression by TFP was investigated on PHA-stimulated peripheral blood mononuclear cells and compared to that of CD25 (IL-2 receptor) expression. Determination of surface molecules was performed in an indirect immunofluorescence assay using anti-CD69 or anti-CD25 monoclonal antibodies, and analyzed by flow cytometry. The time course of the expression of these two molecules differed: CD69 expression was already declining at 48 h, whereas CD25 was still increasing at 72 h after stimulation. TFP (10 muM) reduced CD69 expression by 71.8 per cent at 24 h, 68.4 per cent at 48 h and 24 per cent at 72 h following activation. In contrast, the same dose of TFP did not significantly affect CD25 expression at 24 h but showed an inhibitory effect at later times. These results suggest that different activation pathways are involved in the expression of CD25 and CD69.

Efeito de Rs1 e Rs2 de soros de pacientes com hanseníase na cultura de linfócitos / The effect of seric Rs1 and Rs2 from Hansen's disease (HD) patients in lymphocytes cultures

No presente trabalho foi analisado efeito de receptores solúveis para E de pesos moleculares distintos (Rs1 e Rs2), presentes em níveis elevados em soros de pacientes com hanseníase, na cultura de linfócitos estimulados com fitohemaglutinina (PHA). Foram realizadas culturas de linfócitos de pacientes com hanseníase na forma lepromatosa (LV), na forma tuberculóide (LT) e linfócitos de indivíduos normais (LN), tratados com fraçöes de Rs1 e Rs2 adsorvidas com E (para adsorver Rs) e com hemácias de carneiro tripsinizadas (ET). Foi utilizado meio RPMI, contendo 10 porcento de soro bovino fetal, penicilina, estreptomicina, herpes a 37§C, com 5 porcento de tensäo de CO2, durante 72 horas. Os resultados obtidos demonstraram maior resposta proliferativa nas fraçöes Rs1 e Rs2 absorvidas com E, em relaçäo às fraçöes absorvidas com ET. Adicionalmente foram realizadas cultura de linfócitos com Rs1 e Rs2 previamente purificados, observando-se também inibiçäo na resposta linfoproliferativa à PHA. Esses resultados sugerem que tanto Rs1 como Rs2 apresentam atividade inibitória na resposta linfoproliferativa à PHA, tanto com linfócitos de pacientes com hanseníase na forma lepromatosa ou tuberculóide, bem como linfócitos de doadores normais

Actividad citolítica NK en cancer renal y prostático / NK cytolitic activity in renal and prostatic cancer

Natural killer cytolitic activity, the basis of cancer immunotherapy that uses cytolytic cells, may be impaired in cancer. The aim of this work was to study in vitro the natural killer cytolitic activity and its response to the immunomodulators interleukin-2 interferon and phytohemagglutinin stimulated lymphocyte proliferation in a group of 9 patients with renal cell cancer and 6 with prostatic cancer. The results were compared with those of 20 normal volunteers. Twelve patients were operated and were studied twice 48 h and 14 days after surgery. Natural killer cytolitic activity was significantly lower in renal cell and prostatic cancer patients than controls (3.3 ñ 1.6, 4.9 ñ 2.2 and 20.6 ñ 3.7 per cent of specific lysis respectively). This activity was not modified in cancer patients by interleukin-2 50 Ul/ml or interferon 3000 Ul/ml and did not differ in the two postoperative pèriods. Phytohemagglutinin stimulated lymphocyte proliferation was also lower in cancer patients, compared to controls (stimulation index of 18 ñ 3 and 26.5 ñ 5 respectively). It is concluded that these patients have a low immunological level and this study is the first step towards an immunological characterization of cancer patients that are candidate to adoptive immunotherapy

Efecto de dosis profiláctica de cloroquina sobre la respuesta linfoproliferativa a mitógenos / Effect of prophylactic doses of chloroquine upon mitogen-induced lymphoproliferative response

Se describe el efecto de la cloroquina administrada en dosis profiláctica de 300 y 600 mg base semanales, sobre la respuesta linfoproliferativa a los mitógenos fitohemaglutinina y concanavalina A. A todos los individuos se les realizó el estudio antes del tratamiento y a las 6 semanas de la administración de la droga. Se encontró una disminución estadísticamente significativa (p < 0,01) entre los valores obtenidos con ambos mitógenos, antes del tratamiento y a las 6 semanas, en el grupo que recibió la dosis a 600 mg base semanales. Con la concanavalina A también se observó este efecto, aunque en menor medida (p < 0,05), en el grupo que ingirió 300 mg base semanales

Influencia del coralán sobre la respuesta in vitro de células inmunocompetentes / Coralan influence on the in vitro response of inmunocompetent cells

El coralán, aislado de la Pseudopterogorgia americana, un coral blando, es una glicoproteìna sulfatada que contiene 1% de sulfato, se ha informado que tienen influencia en el rechazo del trasplante del tumor ascìtico de Ehrlich. Diferentes concentraciones de esta sustancia se probaron en células mononucleares de sangre periférica (CMN) frente a la PHA, se observó un incremento significativo de la respuesta, lo que indica que una de las vías por las que este producto ajerce su actividad es por la vía de los linfocitos T. Tambièn se produce un ligero incremento de la actividad de las células NK frente a las células K 562 cuando las CMN son preincubadas con el producto 2 horas antes de realizarse el experimento, datos que en nuestras condiciones fueron comparables al procucido por el IFN, que se ha descrito que aumenta esta actividad.

Immunological studies in leprosy--II: Study of lymphoblast transformation to PHA in different types of leprosy patients.

Ninety cases of different types of leprosy were studied for lymphoblast transformation response using PHA-M. The difference of percentage was statistically analysed and found to be highly significant when total leprosy cases were compared with control cases and with different types of leprosy cases (P = less than .005), except control and TT where it was significant only (P = less than .05). The difference between TT and other types was also calculated and has been observed to be highly significant (P = less than .005). There was gradual decrease in lymphoblast transformation percentage from tuberculoid pole to lepromatous pole.

Blastogênese de linfócitos em doentes de lepra e familiares / Blastogenesis of the lymphocites in leprosy patients and their relatives

Estudou-se o teste de transformaçäo de linfócitos (LTT) sob estímulo de fito-hemaglutinina (PHA), lepromina e M. leprae em doentes de lepra e em seus irmäos, com o objetivo de verificar se existiria ou näo traço familiar herdado ligado ao LTT. Investigou-se, também, a distribuiçäo familiar da reaçäo de Mitsuda e a correlaçäo entre esta reaçäo e LTT em doentes e em seus irmäos. Paralelamente ao LTT foi feita a determinaçäo de proteínas séricas, pela eletroforese, para observar se há correlaçäo entre resultados da blastogênese e nível de globulinas. O teste foi feito em 34 doentes (17 de forma lepromatosa (L) e 17 da tuberculóide (T) e 34 irmäos de doentes, previamente pareados, isto é, cada doente L e T foi pareado com irmäo näo afetado, tendo em conta sexo e idade. Linfócitos foram cultivados em meio Eagle com 20% de plasma autólogo em concentraçäo de 0,4 x 10**6 células por ml, durante 5 dias a 37-C e os resultados da blastogênese avaliados pela incorporaçäo de 3H timidina. Linfócitos de doentes L apresentaram menor atividade blastogênica sob estímulo de pHA, lepromina e M. leprae do que linfócitos e seus irmäos e essa depressäo de resposta talves esteja associada a presença da moléstia. Näo se evidenciou caráter familiar herdado que determinasse semelhante no LTT. Os níveis das globulinas alfa1, ß e gama foram semelhantes em doentes L e irmäos e näo se relacionaram a presença da doença ou herança familiar. Entretanto, alfa2 globulina tende a concentraçöes diferentes nos pares de irmäos, o que poderia estar associado a traço familiar que selecionaria doentes e näo doentes. Näo houve correlaçäo entre níveis de globulinas e resultados do LTT. A reaçäo da Mitsuda foi negativa em todos os doentes L enquanto a maioria de seus irmäos (14/17) reagiu positivamente...

Análisis citogenético por estimulación mitogénica diferencial PHA: PWM / Citogenetic analysis by diferential mitogenetic stimulation PHA: PWM

The mitogenic selectivity of phytomitogens: phytohemaglutinin (PHA) and pokeweed mitogen (PWM) has been investigated, using, two mosaic lyphocyte cell lines from human donors: UGMC-0036 (45.X0/46.XX) and UGMC-0281 (46,XY/47,XXY) respectively, as a model to test karyotypic variation in terms of previously described heterogeneity of PHA and PWM responses of such cells onto the established T and B lymphocyte categories. A fixed peripheral blood leukocyte population was cultured by duplicated for each cell line (8 x10 a la 3 - 85 x 10 a la 2 cell/ml). Set of cultures were stimulated in paralell using, separately, PHA 0.25 ml/10 ml and PWM 0.1 ml/10 ml and incubated for 2.3.4.5. and 6 days. Mitotic Index calculations and Thymidine uptake (3H-TdR. 0.8 *Ci/ml) were used as a-measure of proliferative activity. Karyotypic analysis (n-20) were performed for each given experimental situation and pertinent metaphases recorded during maximum response periods to each polyclonal lymphocyte stimulant. Studies of the time course of thymidine incorporation in PHA as well as in PWM cultures, demostrate several differences in responce profiles elicited by them on both cell lines. To test the null hypothesis (H: p1-p2=0), which assumes no differences in the pairwise comparisons for karyotypic frequencies in the two cell lines stimulated separately, the test on the difference between the parameters of two binomial distribution was employed. Data analysis do not allowed, with the number of null hypothesis at the significance level set (D=0.05) in case of cell line UGMC-0036. However, values for cell line UGMC-0281 showed a significant heterogeneity in the 144 hrs. incubation period; so that, PHA-cultures gave a significant increase of 46.XY metaphases, whereas in PWM-cultures a significant increase for the 46.XXY clonus is demostrated. These observations suggest selective responses of T and B Lymphocytes, which may carry different sexual chromosome complement as result of nondisjuntion mitotic events during their clonal differentiation