Isolation of purified populations of T- and B-lymphocytes under clinical conditions]

The authors present comparative characteristics of the methods of isolation of T- and B-lymphocytes in patients with chronic renal insufficiency. T-lymphocytes were obtained on columns with neilon cotton wool. B-cells were fractionated by repeated centrifugation in the gradient of phycol-urotrast density after staging E-rosette formation with the total lymphocyte pool. Methodical peculiarities of single-stage and two-stage B-lymphocyte fractionation were revealed.     

Lymphocyte subsets and specific T-cell immune response in thalassemia

Infection is very common in thalassemia and is one of the major causes of death. To date, it is not quite clear why these patients are susceptible to infection. In this study, lymphocyte immunophenotyping for CD3(+) (T-cells), CD3(+)CD4(+) (T-helper/inducer cells), CD3(+)CD8(+) (T-suppressor/cytotoxic cells), CD3(-)CD19(+) (B-cells), and CD3(-)CD16/56(+) (natural killer cells) subsets and expression of the activation antigen CD69 on CD3(+)CD4(+) and CD3(+)CD8(+) T-cells were determined in the whole blood of thalassemia patients, using a three-color flow cytometric technique. Results showed that only splenectomized beta-thalassemia/hemoglobin (Hb) E patients displayed a marked increase in absolute number of all lymphocytes. In addition, splenectomized beta-thalassemia/Hb E showed a significantly lower percentage of CD3(+) cells, with a corresponding increase in CD19(+) cells. These differences, when compared with normal subjects and other thalassemia patients, may be attributed to splenectomy. alpha-thalassemia patients, on the other hand, showed no significant difference from the normal group. While lymphocyte subsets in splenectomized beta-thalassemia/Hb E patients showed an abnormal distribution, T-cell activation in these patients was not different from the activation seen in normal subjects. This implies that thalassemia patients, during the steady state of disease, appear to have normal T-lymphocyte function with only moderate abnormalities of T- and B-lymphocyte subsets.     

Stem cells and immunological parameters in mice during the latency period and after the development of chemically induced leukemia

T-cell leukemias have been induced in adult BDF1 mice by 12 or 15 weeks of exposure to butylnitrosourea (BNU) in the drinking water. This led to a depression of CFU-S numbers and reduced T- and B-cell responses to mitogens. These parameters were then studied during the BNU-free preleukemic latency period in individual mice. At the same time, leukemic cells were traced in the thymus, the spleen, and the bone marrow by transplantation. In mice without leukemia and mice with leukemic cells in only one organ, there was a general tendency to normal CFU-S numbers and T- and B-cell responses with time after BNU, although control levels were reached in only a few of the mice. The reaction of mixed lymphocyte cultures (MLC) remained low during the latency period. In the thymus an imbalance of the Con A, PHA, and MLC responses was observed. Out of 25 mice with induced leukemia, 8 had leukemic cells in the thymus only and 2 in the marrow only. In mice with leukemic cells in all 3 hemopoietic organs and an enlargement of the spleen, a shift of CFU-S from the marrow to the spleen was observed.     

Immunologic deficiency during experimental Chagas' disease (Trypanosoma cruzi infection): role of adherent, nonspecific esterase-positive splenic cells

The involvement of adherent splenic cells in the production of deficient lymphocyte responses during the acute phase of experimental Chagas' disease was investigated. When cultured together, purified adherent splenocytes from mice acutely infected with Trypanosoma cruzi caused a significant reduction in the responses of normal mouse spleen cells to T and B cell-specific mitogens. Similar observations were made when infected mouse adherent splenocytes were co-cultured with normal mouse nonadherent cells. Exchange of adherent cells in infected mouse spleen cells suspensions for adherent cells from uninfected mice resulted in increased responses to stimulation with the T and B cell mitogens tested. Treatment of infected mouse cell suspensions with indomethacin improved the responsiveness of these cells to the mitogens. These results support the concept that the immunosuppression that is characteristic of experimental acute Chagas' disease is at least in part mediated by an adherent cell population and is dependent on a prostaglandin-mediated mechanism.     

Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.     

Quantitation of T- and B-lymphocytes in peripheral blood of patients with solid tumours. I. Relation to other parameters of in vivo and in vitro immune competence.

The absolute number of T- and B-lymphocytes in the peripheral blood was measured in 22 patients with disseminated nonlymphoid solid malignant tumours. Patients with normal absolute lymphocyte counts had normal absolute numbers of T- and B-lymphocytes; patients with low counts had low numbers. The percentage of T- and B-lymphocytes was similar, in both groups of patients, to that of healthy control subjects. Patients with a normal absolute lymphocyte count had better in vitro lymphocyte responses to mitogenic agents than did patients with low absolute lymphocyte counts. Improvement of depressed in vitro lymphocyte responses to normal on culture of cells in AB plasma was most likely in patients with normal absolute lymphocyte counts. The absolute lymphocyte count by itself, then, provides useful information about the immune status of patients with solid malignant tumours.     

Increased B Cell and Cytotoxic NK Cell Proportions and Increased T Cell Responsiveness in Blood of Natalizumab-Treated Multiple Sclerosis Patients

Background

Changes in the blood lymphocyte composition probably both mediate and reflect the effects of natalizumab treatment in multiple sclerosis, with implications for treatment benefits and risks.

Methods

A broad panel of markers for lymphocyte populations, including states of activation and co-stimulation, as well as functional T cell responses to recall antigens and mitogens, were assessed by flow cytometry in 40 patients with relapsing multiple sclerosis before and after one-year natalizumab treatment.

Results

Absolute numbers of all major lymphocyte populations increased after treatment, most markedly for NK and B cells. The fraction of both memory and presumed regulatory B cell subsets increased, as did CD3^-CD56^dim cytotoxic NK cells, whereas CD3^-CD56^bright regulatory NK cells decreased. The increase in cell numbers was further associated with a restored T cell responsiveness to recall antigens and mitogens in functional assays.

Conclusions

Our data confirms that natalizumab treatment increases the number of lymphocytes in blood, likely mirroring the expression of VLA-4 being highest on NK and B cells. This finding supports reduction of lymphocyte extravasation as a main mode of action, although the differential effects on subpopulation composition suggests that cell-signalling may also be affected. The systemic increase in T cell responsiveness reflects the increase in numbers, and while augmenting anti-infectious responses systemically, localized responses may become correspondingly decreased.     

Determination of the surface area and viscosity of membranes in T- and B-lymphocytes by means of fluorescent probes

The surface area of lymphocyte membranes was measured by registering F?rster's energy transfer on fluorescent probes. Pyrene served as donor, 4-(n-hydroxystyryl)-N-tetradecylpiridinium (HSP) was the acceptor. The surface area B-lymphocyte membranes was shown to be 1,2 times larger than that of T-lymphocytes. The mean value of lymphocyte membranes viscosity was measured using the excimerization effect of pyrene. This value was the same in all the cells investigated Fluorescence of the probe 3-methoxybenzanthrone (MBA) was 2-2.5 times higher in B-lymphocytes and was not proportional to the surface area of T- and B-cells membranes. MBA fluorescence may imply some differences in physical structures of these cells which are not connected with the viscosity of their membrane lipid phase.     

Serial immunologic assessment during a randomized trial of chemoimmunotherapy in acute myelogenous leukemia

Summary Thirty-one adults who had acute myelogenous leukemia and in whom remission had been induced and consolidated with chemotherapy were randomized to receive one of three maintenance schedules: (A) BCG + chemotherapy [1, 3-bis-(2-chlorethyl)-1-nitrosourea (BCNU) and cytosine arabinoside]; (B) splenectomy, followed 1 week later with BCG and chemotherapy; or (C) allogeneic leukemic cells, BCG, and chemotherapy. Serial immunologic assessments were performed at the onset of maintenance and every 3 months.No differences were found in duration of remission (median 209 days) or survival (median 454 days) among the three schedules. Six patients remain in remission after from 2–4 + years. Skin test responses, mitogen responses, mixed lymphocyte culture responses, antibody responses, and T and B lymphocyte numbers were depressed at the onset of maintenance therapy. Therapy clearly improved the state of anergy as defined by recall antigen responsiveness, and induced in vivo and in vitro PPD reactivity. However, immunotherapy resulted in a reduction of the number of T or B cells and of the in vitro lymphocyte response to mitogens and allogeneic cells. Serum obtained at diagnosis and during remission inhibited in vitro blastogenic responses in more than half the patients. These data indicate that chemoimmunotherapy given as described tended to be more immunosuppressive than stimulatory.     

E1Lymphocytopenia in pseudo-lupus-erythmetaosus syndrome]

Seven female patients with pseudo-lupus erythematosus (LE)-syndrome had markedly reduced lymphocyte counts in their peripheral blood during the active phase of the disease. One patient, we were able to study during the active phase of her disease, had a diminuation of spontaneous rosettes-forming (T-)lymphocytes, using neuraminidase-treated sheep red blood cells for the test. In this case the percentage of surface-immunoglobulin-bearing (B-)lymphocytes determined by an indirect immunofluorescence technique was augmented. In comparison with 20 normal controls 6 other patients did not show any alterations in the relation of B- and T-lymphocytes in the peripheral blood. 5 patients had circulating lymphocytotoxic antibodies in their serum. A specificity of these antibodies for T-lymphocytes could not be realized.     

Pituitary-testicular interrelationships in mumps orchitis and other viral infections

Leydig-cell function was assessed in 27 men with acute mumps orchitis by measuring plasma testosterone concentrations before and after the administration of human chorionic gonadotrophin (HCG). The test was also performed on groups of patients with other febrile viral infections and mumps without orchitis and on healthy euspermic men. The concentrations both before and after HCG were significantly lower in patients in the acute phase of mumps—but not in those with other viral infections and mumps without orchitis—than in the healthy men. Basal concentrations of follicle-stimulating hormone (FSH) and luteinising hormone (LH) were significantly increased in patients with acute mumps orchitis, while an exaggerated response to LH-releasing hormone was noted in four patients after the acute phase of the disease. Raised plasma LH concentrations were also found in several patients with viral infections, including mumps without orchitis. There appeared to be no particular merit of any of the treatments used (aspirin, prednisolone, and cold baths). In patients reevaluated three to five and 10 to 12 months after the acute phase of their disease the basal testosterone concentrations were similar to those of the healthy men, but several of the patients showed a severely impaired response to HCG. Mean basal FSH and LH concentrations were significantly increased 10 to 12 months after the acute phase, while the mean LH concentration was also raised at three to five months.It is concluded that mumps orchitis impairs Leydigcell function during the acute phase of the disease but may also have a more permanent damaging effect, similar to that found in the germinal epithelium.     

Impact of stress, gender and menstrual cycle on immune system: possible role of nitric oxide

Stress is a factor found to be involved in the etiology of many diseases. Gender and menstrual cycle phases are other factors affecting the predisposition of individuals for certain diseases. Results from animal and human studies suggest that the distribution of immune system cells may change at different phases of the menstrual cycle. Acute mental stress in humans alters immune variables, too. The increase in the number of natural killer (NK) cells is the most consistent finding among the immune variables, though there are controversies for the other lymphocyte groups. Nitric oxide (NO) as an immune mediator has an unsettled role whether it causes the redistribution of the immune cells, or is an end product of lymphocyte activation. This study was planned to investigate the effect of mental stress on lymphocyte subtypes and the role of NO, for men and women at different phases of the cycle. For this purpose, healthy men (n = 10) and women (n = 10), during the follicular and luteal phases underwent Stroop colour-word interference and cold pressor tests. The immune system responses before and after the tests were determined by cell counts with the flowcytometer. Menstrual cycle phase was ascertained by plasma estrogen and progesterone measurements. Stress response was evaluated by blood pressure (BP) and heart rate (HR) measurements throughout the tests and plasma cortisol and urinary metanephrine and vanillylmandelic acid (VMA) measurements before and after the tests. Plasma and urinary NO determinations were performed before and after the test was completed. All the results were analysed with the appropriate statistical methods. The luteal phase differed from the other groups due to the presence of suppressed immune response to acute stress, including decreased CD4/CD8 ratio and NK cell percentage. On the other hand, acute stress caused a shift from cellular to humoral immunity in men. As indicated by these results, individual reaction towards stress is affected by gender and menstrual cycle phase. NO appears to be a possible effector molecule for these differences.     

A rapid technique for measuring calcium uptake in mitogen-induced T and B lymphocytes.

The procedures for lymphocyte activation and for removing the cells from the radioactive loading solution in incubation medium were modified to routinely obtain significant and reproducible 45Ca2+ uptakes in mitogen-induced mouse T and B lymphocytes. Factors such as mouse strain, lymphocyte origin, and media pH were not critical to the 45Ca2+ uptake measurements. In contrast, factors such as lymphocyte cell concentration during mitogenic activation, filtering the 45Ca2+:3H2O mixtures, and the nature and purity of the B-cell mitogens were critical for obtaining maximal and reproducible 45Ca2+ uptakes. Centrifugation through silicone oil into sucrose was an efficient and rapid procedure for separating the cells from the radioactive loading solution in the incubation medium. Using optimal conditions, an approximate twofold increase in 45Ca2+ uptake (representing an influx of approximately 97 amol per lymphocyte and an increase in average cellular Ca2+ of approximately 0.72 mM) was routinely obtained with purified mouse lymphocytes activated with a variety of T- and B-cell mitogens (using concentrations resulting in maximal [3H]thymidine incorporation). A larger 45Ca2+ uptake was routinely obtained with mitogenic concentrations of A23187, a divalent cation ionophore stimulating T cells. Experiments employing [14C]sucrose and [14C]inulin with control and mitogen-induced lymphocytes showed that the trapped extracellular fluid measurements in the cell pellets should be used to correct the magnitude of the 45Ca2+ uptake measurements.     

Are diagnostic tests repeated unnecessarily on hospital admission?

After admission to hospital, patients are often subjected to laboratory tests that may duplicate testing already done. For nearly 2800 patients at three hospitals the numbers and types of tests done in the week before and the week after admission were determined. A team of general practitioners judged the necessity of tests that had been repeated, with only those tests that had yielded normal results at both times being labelled as unjustifiably duplicated. Only 246 patients had had tests done in the week before entering the hospital, but 192 (71%) of them had had tests repeated after admission, 35 (14%) unjustifiably. Of the 743 laboratory tests performed before admission 447 (60%) were subsequently duplicated, but only 85 (11%) of them unjustifiably. Duplication, whether justifiable or unnecessary, was more likely to involve the hemoglobin determinations and urinalyses done routinely in hospitals.     

The entry of T and B lymphocytes into rat popliteal lymph nodes undergoing a graft-versus-host reaction

The entry of radiolabeled blood-borne T and B lymphocytes into resting popliteal lymph nodes and popliteal lymph nodes stimulated with semiallogeneic lymphocytes was investigated in rats. Thoracic duct lymphocytes separated into T- and B-lymphocyte populations on nylon-wool columns were radiolabeled with 51chromium and equal numbers of T or B lymphocytes were injected intravenously. While the ratio of T and B lymphocytes in the blood is approximately 3:1 it was found that the ratio of T to B lymphocytes migrating into lymph nodes was approximately 9 T to 1 B lymphocyte in both resting and antigenically stimulated lymph nodes. Since the ratio of T to B lymphocytes in thoracic duct lymph is similar to that of blood, there is a disparity between the number of T cells entering and leaving lymph nodes. These results suggest that some T lymphocytes may return to the blood directly and/or there is increased T lymphocyte death in lymph nodes.     

Blood eicosanoids and immune indices during fasciolosis in water buffaloes

The effects of trickle infections of water buffaloes with Fasciola hepatica (60 metacercariae daily during a period of 20 days) on the blood plasma levels of prostaglandin E₂ (PGE₂), 6-keto-prostaglandin F₁ (6-keto-PG F₁) and thromboxane B₂ (TXB₂) were assessed. F. hepatica specific IgG and T- and B-lymphocyte ratios were evaluated as indicators of the immune response. Although the applied mode of infection did not result in clinical disease, changes in the plasma eicosanoid pattern were observed. Plasma PGE₂ values were significantly elevated in the infected water buffaloes 11 weeks post-infection (w.p.i.). In contrast, transiently but significantly lower TXB₂ values than in the uninfected controls were recorded in the phase of chronic fasciolosis. Plasma 6-keto-PGF₁ values were not considerably altered by the infection throughout the study period. F. hepatica-specific IgG were detected from 4 to 21 w.p.i. The proportion of peripheral T- and B-lymphocytes shifted towards B-cells from 2 to 12 w.p.i., gradually returning to control values afterwards. Although the water buffaloes appeared to be rather resistant to trickle infection with F. hepatica, moderate changes in plasma eicosanoid patterns were observed, indicating tissue damage and/or inflammation. Induction of the immune response could be monitored by an increase of F. hepatica-specific IgG, which was paralleled by a relative increase of the B-lymphocyte population.     

Characterization of low-density granulocytes in COVID-19

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.     

The effects of short-term resistance training on endocrine function in men and women

This investigation examined hormonal adaptations to acute resistance exercise and determined whether training adaptations are observed within an 8-week period in untrained men and women. The protocol consisted of a 1-week pre-conditioning orientation phase followed by 8 weeks of heavy resistance training. Three lower-limb exercises for the quadriceps femoris muscle group (squat, leg press, knee extension) were performed twice a week (Monday and Friday) with every other Wednesday used for maximal dynamic 1 RM strength testing. Blood samples were obtained pre-exercise (Pre-Ex), immediately post-exercise (IP), and 5 min post-exercise (5-P) during the first week of training (T-1), after 6 weeks (T-2) and 8 weeks (T-3) of training to determine blood concentrations of whole-blood lactate (LAC), serum total testosterone (TT), sex-hormone binding globulin (SHBG), cortisol (CORT) and growth hormone (GH). Serum TT concentrations were significantly (P ≤ 0.05) higher for men at all time points measured. Men did not demonstrate an increase due to exercise until T-2. An increase in pre-exercise concentrations of TT were observed both for men and women at T-2 and T-3. No differences were observed for CORT between men and women; increases in CORT above pre-exercise values were observed for men at all training phases and at T-2 and T-3 for women. A reduction in CORT concentrations at rest was observed both in men and women at T-3. Women demonstrated higher pre-exercise GH values than men at all training phases; no changes with training were observed for GH concentrations. Exercise-induced increases in GH above pre-exercise values were observed at all phases of training. Women demonstrated higher serum concentrations of SHBG at all time points. No exercise-induced increases were observed in men over the training period but women increased SHBG with exercise at T-3. SHBG concentrations in women were also significantly higher at T-2 and T-3 when compared to T-1 values. Increases in LAC concentrations due to exercise were observed both for men and women for all training phases but no significant differences were observed with training. These data illustrate that untrained individuals may exhibit early-phase endocrine adaptations during a resistance training program. These hormonal adaptations may influence and help to mediate other adaptations in the nervous system and muscle fibers, which have been shown to be very responsive in the early phase of strength adaptations with resistance training. Accepted: 11 December 1997     

In vitro functions of lymphocytes during high-dose medroxyprogesterone acetate (MPA) treatment

Summary The number and function of T and B lymphocytes were studied in endometrial cancer patients during 3 months' treatment with high-dose medroxyprogesterone acetate (MPA). No significant changes were observed in the proportions of T and B cells or in the function of B cells during MPA treatment. At 3 months, lymphocyte blastogenic responsiveness to mitogens was slightly reduced both in patients treated with standard therapy alone (intracavitary radium and surgery) and in patients receiving additional MPA therapy. These results indicate that other factors than progesterone are responsible for the suppression of lymphocyte blastogenesis induced by mitogens during high-dose progestin treatment.     

Nonlymphoid cells interacted with mitogens fail to elaborate macrophage migration inhibitory factor (MIF).

The ability of a number of normal and transformed nonlymphoid cells to produce macrophage migration inhibitory factor (MIF) was studied. MIF was not detectable in either native or concentrated supernatants of unstimulated cultures or in supernatants of cells interacted with T- or B-lymphocyte mitogens. These results are considered in the context of literature reports of release of MIF-like materials by nonlymphoid cells. It is concluded that more stringent criteria are required to ascertain whether the inhibition of migration induced by supernatants of nonlymphoid cell cultures is in fact due to lymphokine-like materials.