Autoreactivity, backstimulation and reproducibility in a helper T lymphocyte precursor assay.

Helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis (LDA) have a predictive value for alloreactivity in allogeneic bone marrow transplantation. Methodological problems in LDA include autoreactivity in the responder or stimulator cell populations and interleukin 2 (IL-2) production by the stimulator cells as a response to the responder cells (backstimulation). The extent and impact of these aspects for IL-2 production and HTLp frequency determination were studied by autologous and allogeneic mixed lymphocyte reactions with healthy volunteers and HTLp determinations from bone marrow transplantation donor/recipient pairs. We found that autoreactivity occurred in the unirradiated cells with a reproducible inter-individual variation. The immunogenicity of the stimulator cells was preserved after gamma irradiation with 50 Gy and the risks of autoreactivity and backstimulation were limited. Higher doses of irradiation decreased the immunogenicity. Immune reactions to antigens present in the serum supplement of the culture medium were seen with foetal calf serum and to a lesser extent with pooled human sera. This could be avoided by the use of autologous serum. We were unable to ensure satisfactory culture conditions in serum-free medium. The reproducibility of the HTLp frequency determinations was tested for intra- and inter-assay variation. The coefficients of variation were estimated as 24% and 35%, respectively. This was acceptable considering the range of the HTLp frequencies (1:10(2) to 1:10(7)). The influence of the extent of autoreactivity of the bone marrow donors was investigated in 28 HLA-identical sibling transplantations. We found no correlation between the autoreactivity of the donors and the HTLp frequencies. The extent of autoreactivity of the donor did not correlate with the clinical outcome in terms of acute graft-versus-host disease, treatment-related mortality, risk of relapse and overall survival. In spite of methodological difficulties and interference from autoreactivity and backstimulation, reproducible quantification of clinically significant alloreactivity can be attained.

Fourier-transform infrared spectroscopy as a tool for detecting early lymphocyte activation: a new approach to histocompatibility matching.

Fourier transform infrared (FT-IR) spectroscopy due to its speed and sensitivity is becoming an increasingly powerful tool in the study of cell composition. We outline the potential of FT-IR microspectroscopy in monitoring mitogenic and cell mediated lymphocyte activation. We demonstrate the potential of FT-IR spectroscopy in histocompatibility testing by showing that significant spectral differences (p < 0.001, for the mean integrated intensity of phosphodiester band located in the 1142-996 cm(-1) region) exist between lymphocyte cocultures from pairs of HLA matched and mismatched volunteers after only 90 min of incubation. The preliminary results indicate that early spectral changes measured are due to HLA differences between individuals, although the relative contribution of class I and class II differences has yet to be determined. FT-IR spectroscopy represents a novel approach to histocompatibility matching and the rapidity and sensitivity of the technique indicates a potential role in matching protocols for clinical use, particularly in allogeneic bone marrow transplantation.

Estimation of antigen-responsive T cell frequencies in PBMC from human subjects.

A new method for estimating the frequency of antigen-responsive T cells, using a cell proliferation assay, is described. In this assay, the uptake of tritiated thymidine by peripheral blood mononuclear cells which have been exposed to antigen, is measured for each well on a microtiter plate. Whereas this assay is generally used as part of a limiting dilution assay, here we estimate the frequency of responding cells using a single, carefully chosen cell density. The traditional analysis of such data uses a cut-off to separate wells which contain no responding cells and wells which contain at least one responding cell. The new method uses the scintillation count to estimate the number of responding cells for each well on the plate. We do this by fitting a two-stage model, the first stage being a Poisson model with antigen-specific frequency parameters, and the second stage a linear model with plate-specific parameters.

[Immune status studies after one-time alcohol consumption in healthy subjects]. / Untersuchungen zum Immunstatus nach einmaligem Alkoholkonsum bei gesunden Probanden.

AIM OF STUDY: To ascertain whether once-only intake of a large amount of alcohol causes measurable effects on the immune system of healthy persons. SUBJECTS AND METHODS: Cytokine levels, lymphocyte subpopulations and mitogen stimulation were measured in eleven healthy nonalcohol drinking volunteers (eight men, three women; mean age 30.5 [26-36] years; mean weight 71.4 kg) after a single intake of 80 g ethanol (corresponding to 1.12 g/kg). RESULTS: There were no changes in the concentrations of tumour necrosis factor alpha, interleukins 1 alpha and 1 beta, 3, 6, 10, soluble interleukin-6 receptor, interleukin-1 receptor antagonist, gamma-interferon and granulocyte-macrophage colony stimulating factor. A tendency towards an increase was measured regarding the CD-4 lymphocyte counts as well as the concentration of soluble interleukin-2 receptors and intercellular adhesion molecules (ICAM-1). Interleukins 4 and 8 were low normal. Lymphocyte activity was slightly reduced in the mixed lymphocyte cytotoxicity test. No clear-cut changes were observed in the results of the other mitogen stimulation tests. CONCLUSION: These data show that once-only intake of a large amount of alcohol does not produce measurable abnormalities in the immune system of healthy persons who ordinarily abstain from drinking alcohol.

[The results of determining compatibility in the MLC reaction in hemoblastosis patients undergoing bone marrow transplantation from HLA-identical siblings]. / Rezul'taty opredeleniia sovmestimosti v reaktsii MLC bol'nykh gemoblastozami pri transplantatsii kostnogo mozga ot HLA-identichnykh sibsov.

Compatibility in mixed lymphocyte culture (MLC) test was determined for 60 hemoblastosis patients with their 71 HLA-identical sibs to transplant allogenic syngeneic bone marrow. A protocol for the MLC radioisotope micromethod with culturing in plates is described for the pair donor-recipient in the direct and reverse variants covering about 20 variants of mixed cultures. This secures the results' reliability. Plotting of the MLC compatibility results for each pair has been proposed. Incompatibility by MLC was revealed in 5 out of 71 pairs (7%). Bone marrow transplantation was performed in 30 patients from their HLA-identical/MLC-compatible sibs. The secondary disease due to incompatibility missed in the MLC reaction occurred in 5 cases (16.7%), transplant rejection in 2 cases (6.7%), which is less in number than literature data. It was established that determination of compatibility by MLC may serve a reliable criterion of assessing a genotypic pair HLA identity and should be conducted before each allogenic and syngeneic transplantation of bone marrow. The protocols of the method performance and compatibility parameters evaluation warrant high significance of the compatibility definition confirmed by clinical responses with minimal probability of secondary disease.

Interferón gamma y factor necrosante de tumores/linfotoxina en el cultivo de linfocitos de candidatos y donadores de trasplante renal

Basados en el papel central que ejercen el interferón gamma (INF-gamma) y el factor necrazante de tumores (FNT) en los procesos de acitivación e inflamación y en los efectos antivirales de éstos, nos propusimos determinar sus niveles en cultivos mixtos de linfocitos (CML) de parejas de candidatos receptores/donadores de trasplante renal. Mediante radioinminoanálisis y ensayo biológico correlacionamos estos niveles con la presencia de episodios de rechazo e infección por Citomegalovirus (CMV) en el período postrasplante

[Study of possible influence of mixed lymphocyte reaction on experimental models used in biological dosimetry]. / Etude de l'influence possible d'une réaction mixte lymphocytaire sur les modèles expérimentaux utilisés en dosimétrie biologique.

The study aimed to investigate whether "mixed lymphocyte reactions" could interfere with the results of experiments in which irradiated blood from one person is mixed with non-irradiated blood from another. This experimental model had been designed to simulate, in vitro, non-homogeneous in vivo exposure such as could occur after accidents with ionizing radiation. The results show that the experimental models gives valid results for donors with related blood characteristics.

Histocompatibilidad, citomegalovirus y trasplante renal

Se utilizó el cultivo mixto de linfocitos (CML) para tratar de establecer la correlación entre alorreactividad, rechazo e infección por citomegalovirus (CMV) y la posible reactivación del virus in vitro. Se practicaron CML clásicos para estudiar la respuesta proliferativa y en paralelo, CML para los estudios de reactivación viral. El período de seguimineto clínico de los pacientes osciló entre tres meses y un año y durante el cual se pudo corroborar la fuerte asociación entre rechazo e infección por CMV (p=0.001). Se probaron, por aislamiento viral, 144 sobrenadantes de combinaciones de CML con resultados negativos. En general, la correlación entre CML y HLA fue pobre; si bien fue significativa para HLA-(A+B), no fue la DR. Tampoco hubo asociación entre el rechazo y los parámetros clínicos rutinarios para el seguimineto de los pacientes (BUN, creatinina, depuración de creatinina), al igual que con la clase y el número de transfusiones. El porcentaje promedio de respuesta relativa (por cientoRR) fue mayor cuando la relación con el donante era paternal que cuando era fraternal (p=0.05) y la mayoría de los episodios de infección por CMV y rechazo se presentaron en los pacientes que recibieron trasplante de origen paterno

Efecto de la gamaglobulina humana intravenosa sobre el cultivo mixto de linfocitos

Se estudio el efecto de la gamaglobulina humana intravenosa (GGHIV)(Sandoblogulin) sobre la reacción alogénica in vitro mediante el cultivo mixto de linfocitos (CML) en individuos normales. LA GGHIV en concentraciones superiores a 0.5 mg/ml inhibió en 61 por ciento y 99 por ciento la respuesta proliferativa. Se descartó que la inhibición fuese debida a factores citotóxicos presentes en la GGHIV comercial y/o deficit nutricional en los medios de cultivo. La incubación de los CML con 4 mg/ml de GGHIV durante 4 h/37 grados Centigrados produjo un 74 por ciento de inhibición. Se observó un efecto más acentuado al preincubar las células estimuladoras. La adición de GGHIV al CML, ocasionó una inhibición dependiente del tiempo de adición, mayor entre las 0 horas (h)(90 por ciento) y las 48 h (40 por ciento). El fenómeno inhibitorio no fue específico sobre la reacción alogénica puesto que también se encontró sobre la respuesta proliferativa a concavalina A (Con A) (63 por ciento), fitohematoglutinina A (PHA) (62 por ciento) y derivado protéico purificado (PPD) (84 por ciento). La inhibición sobre el CML, se revirtió parcialmente con la adición de 100 U de IL-2rH en las primeras 24h. La GGHIV redujo en un 79 por ciento la expresión del receptor de IL-2 al quinto día del CML. Los resultados de cuantificación de IL-1B en los sobrenadantes de CML con y sin GGHIV no arrojaron datos concluyentes. Se discuten los posibles mecanismos de acción y futuras utilidades de la GGHIV como inmunomoduladora en los transplantes de tejidos.