DNA repair after UV and gamma irradiation. II. Human lymphocytes

A technique of an in vitro microculture system has been set up in order to standardize DNA repair in adult human lymphocytes after ultraviolet and gamma irradiation measuring tritiated thymidine uptake. The results obtained in DNA unscheduled synthesis were different if ultraviolet or gamma rays were employed.     

Free ribosomes in physiologically nondividing cells. Human peripheral lymphocytes

Ribosomes of physiologically nondividing human peripheral blood lymphocytes were studied by ultraviolet absorbance measurements of cytoplasmic extracts subjected to ultracentrifugation in sucrose gradients at high and low ionic strength. At least 70% of the total cytoplasmic ribosomes were free ribosomes which sedimented at 80 S in low salt and dissociated to 40 S and 60 S subunits in high salt. These particles were not involved in protein synthesis. The remaining ribosomes were equally divided among native subunits, active monosomes, and larger polysomes. Free ribosomes were shown to exist as 80 S particles in the intact cell, and labeling studies indicated that they did not freely return to the pools of protein-synthesizing components. New ribosomes appeared first as native subunits and in polysomes. After a lag of 15 to 20 min, the particles began to enter the free ribosome pool. Thus, free ribosomes arise in the resting cell by a unidirectional flow which continuously removes particles from the protein-synthesizing pool and sequesters them as an accumulation of inactive 80 S particles. The transition from native subunits to free ribosomes is accompanied by functional changes in association behavior of subunits and by alteration of sedimentation behavior of the subunits. These changes may be due to absence of a protein or proteins from the free ribosomes which is required to permit effective dissociation of subunits prior to initiation of translation. Deficiency of this dissociation factor may be responsible for the continuous formation of free ribosomes in resting cells. Our data also suggest a limitation of the rate of initiation of protein synthesis which may result from deficiency of an initiation factor.     

DNA synthesis in tonsil lymphocytes. I. Changes in cell population during culture

Allergic asthma-like attacks were induced in guinea pigs by intraperitoneal injections of Ovalbumin and following inhalation of Ovalbumin aerosol. The animals were narcotized, tracheotomized and a small catheter was placed in mid-oesophagus. The quantities: oesophageal pressure, respiratory flow and tidal volume were measured and recorded on magnetic tape during spontaneous breathing. A technique was applied for automated computation of the parameters of respiratory mechanics using a digital computer. The parameters compliance, resistance, work of breathing and power of breathing showed typical pathological variances and characteristic response-curves in the experimental phases during and after the asthma attacks. These parameters became normal after the injection of Alupent.     

Quantitative computerized image analysis of immunostained lymphocytes. A methodological approach

A methodological approach by computerized image analysis to quantify immunostained objects in histological sections is described. We have investigated antibodies against CD4, CD8, CD20, CD23 and CD25 in frozen sections of human nasal mucosa; however, the methodology of standardization is of general validity. The study was designed particularly to investigate the following points: 1) light intensity, 2) the grey level for counter staining intensity, 3) the grey level threshold value for positive objects, 4) the minimal acceptable size of a positive object, 5) the influence of the brightness of the light on both the number and the area of objects. Furthermore, random sampling and determination of 6) the area per section, and 7) the number of histological sections to be measured per biopsy. Finally, a study of reproducibility of immunostaining intensity was performed. The influence of the different parameters mentioned above was studied and the values (eg. threshold value) for our particular setting of microscope, image analysis equipment, computer software etc, were defined. The method was then tested for intra- and interindividual variation which was found to be less than 5%. Correlation analysis of the reproducibility gave coefficients of correlation of 0.99, both concerning number of immunopositive objects and immunopositive area. We emphasize the importance of a highly standardized methodology if the numeric data obtained from computer assisted image analysis are to be more accurate than semiquantitative assessments by experienced observers. With a thorough standardization as described in this method it is possible to obtain numeric values, and data with low deviations, which are two obvious and important advantages.     

The binding of phytohemagglutinin M to rat spleen lymphocytes. Quantitative studies

Phytohemagglutinin M (PHAM) has been purified from the commercial mixture of proteins produced by Phaseolus vulgaris, using a Sepharose-thyroglobulin column. The protein gave one band on gel electrophoresis and two bands on SDS-gel electrophoresis (mol. wt. 33 700 and 32 100, respectively). Molecular weight determination by ultracentrifugation gave a value of 61 200 +/- 700. The protein had a minimum sugar content of 16%. Binding studies of PHAM to purified rat spleen lymphocytes have been performed at 0, 25, and 37 degrees C. It was shown that the cells bound about the same amount of lectin at 0 and 37 degrees C, but less protein was bound at 25 degrees C. The binding phenomenon showed saturability at all temperatures. Data were analyzed by Scatchard plots and two kinds of binding sites were found. High-affinity sites and low-affinity sites have been characterized in terms of association constants and (apparent) number. It was also shown that cells treated with trypsin or sodium azide bound less lectin. Bound concanavalin A did not appear to affect the amount of bound PHAM, but its influence was reflected in the value of the association constant for the binding of PHAM. Unlabelled PHAM was shown to displace radioactive PHAM from the cells, but could not remove bound concanavalin A. The significance of these results is discussed in terms of the fluid plasma membrane model and cellular metabolism.     

Gut intraepithelial T lymphocytes

One of the most prominent features of the early phase of cerebral ischaemia is the immunohistochemical collapse of cytoskeletal proteins. Among these proteins, microtubule-associated protein 2 (MtP2) has been shown to be vulnerable to ischaemic injuries. In order to identify a suitable volatile anaesthetic on the basis of cytoskeletal protein breakdown during cerebral ischaemia, we have compared the effects of isoflurane and halothane on MtP2 degradation in rats. Under equipotent isoflurane or halothane anaesthesia, forebrain ischaemia was induced by occlusion of the bilateral common carotid artery, combined with a decrease in mean arterial pressure to 50 mm Hg. After 20 min of ischaemia, the frontoparietal cortex, brainstem, hippocampus and cerebellum were removed separately and homogenized. MtP2 from each region was measured using an enzyme-linked immunosorbent assay. MtP2 degradation in the frontoparietal cortex and hippocampus was significantly (P < 0.05 and P < 0.01) less with isoflurane anaesthesia (75.6 (SD 10.7)% and 72.3 (12.8)%, respectively) than with halothane (65.0 (13.1)% and 54.7 (13.9)%, respectively).     

Granulated lymphocytes of pregnancy

In pregnant mammals, the antigenically distinctive conceptus implants and grows in a uterine environment governed by the maternal immune system. The uterus per se is not immunologically privileged and large numbers of lymphocytes accumulate at implantation sites. In mice and humans, many of these lymphocytes have been identified as uterine natural killer (uNK) cells and exhibit a characteristic granulated morphology. In this review we focus on uNK cells and discuss their origin, differentiation and possible roles in the maintenance of healthy pregnancies. In species with less invasive placentation (ruminants, pigs), lymphocytes with similar granular morphology also appear during gestation and their identity and possible functions are examined.     

Immunologic injury in measles virus infection. III. Presence and characterization of human cytotoxic lymphocytes

Human peripheral blood leukocytes (PBL) from patients with chronic measles virus infection (SSPE) or from immune, adult humans (convalescent from acute childhood measles virus infection) are cytotoxic for target cells infected with measles virus as measured by a 51Cr assay. Specific release of 51Cr by immune PBL occurred both with and without human antibodies added to measles virus-infected cultures. Maximal killing in the absence of added antibodies to measles virus was usually detected only after 15 to 18 hr of incubation and with a high PBL to target ratio (100:1). When antibody to measles virus was added, PBL-mediated killing of virus-infected cells was not blocked. Instead, killing was enhanced and maximal lysis occurred with fewer PBL and a shorter incubation time. This cytotoxic reaction was inhibited in a dose-response manner upon the addition of Fab fragments of IgG containing antibodies to measles virus. On the average 4 to 5 x 105 antibody molecules bound per infected target cell before initiation of antibody-enhanced PBL killing. Depletion of either glass-adhering or E-rosette-forming cells did not reduce PBL killing of measles virus-infected target cells in either system. In contrast, removal of non-E rosette or of EAC rosette-forming population of PBL almost completely abrogated cytotoxicity. When Fc-bearing cells were removed, killing of virus-infected target cells was concomitantly reduced. Lysis of measles virus-infected target cells did not require histocompatibility between the PBL and the target cell. Further, immunospecific lymphocyte killing was not enhanced by such a histocompatibility fit. These experiments indicate first, that the effector PBL involved in lysis of measles virus-infected targets are not T cells but are probably K cells and, second, that PBL obtained from patients with SSPE are competent in killing measles virus-infected targets. Moreover, sera from SSPE patients did not contain a factor(s) that blocked PBL-mediated cytotoxicity.     

Adenoviral-mediated gene transfer in lymphocytes

Although adenovirus can infect a wide range of cell types, lymphocytes are not generally susceptible to adenovirus infection, in part because of the absence of the expression of the cellular receptor for the adenoviral fiber protein. The cellular receptor for adenovirus and coxsackievirus (CAR) recently was cloned and shown to mediate adenoviral entry by interaction with the viral fiber protein. We show that the ectopic expression of CAR in various lymphocyte cell lines, which are almost completely resistant to adenovirus infection, is sufficient to facilitate the efficient transduction of these cells by recombinant adenoviruses. Furthermore, this property of CAR does not require its cytoplasmic domain, consistent with the idea that CAR primarily serves as a high affinity binding site for the adenoviral fiber protein, and that viral entry is mediated by interaction of the viral penton base proteins with cellular integrins. As a demonstration of their functional utility, we used CAR-expressing lymphocytes transduced with an adenovirus expressing Fas ligand to efficiently kill Fas receptor-expressing tumor cells. The ability to efficiently manipulate gene expression in lymphocyte cells by using adenovirus vectors should facilitate the functional characterization of pathways affecting lymphocyte physiology.     

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.     

Structure and biologic functions of human IgD. X. Enhancement of PHA responsiveness by anti-delta-activated lymphocytes

Human peripheral lymphocytes with the capacity to be stimulated by anti-delta exhibited in PHA responsiveness when cultured with anti-delta 1 or 12 hr before PHA exposure over cells exposed to PHA alone. When these lymphocytes were preincubated with PHA 1 or 12 hr before anti-delta activation, no augmentation of the PHA response was seen. In addition, lymphocytes from donors with a high PHA response (low anti-delta activation) failed to show an enhancing effect on PHA responsiveness when pretreated with anti-delta. Moreover, anti-mu showed no synergistic effect on PHA responsiveness. This study is the first to indicate that anti-delta-activated cells enhance PHA responsiveness.