Lymphocyte Function Tests Research Articles

CHRONIC MUCOCUTANEOUS CANDIDIASIS: IS NOT WHAT WE HAVE THOUGHT

Introduction Chronic mucocutaneous candidiasis is only treated symptomatically, most cases do not have a diagnosis of certainty, however, at least half of the cases are caused by transducer and activator of transcription 1 (STAT1) mutations. Case Description Female of 12 years; mother with systemic lupus erythematosus, oral and vaginal candidiasis. Medical history: refers to 8 cases of urinary tract infection from 3 years of age with a microbiological report of Candida spp., from birth vulvovaginal infections of recurrence, aphthae and oral candiadiasis rebellious to treatment with frequent recurrences. Starts study protocol in 2015 with profile of immunoglobulins and immunoglobulin G subclasses normal, subpopulations of lymphocytes normal, initiates prophylactic treatment with itraconazole, presenting recurrences. Lymphocyte function tests were performed with normality report, initiate immunomodulation with transfer factor, partial response. Start treatment with intravenous immunoglobulin at a dose of 400 mg/kg/dose with a decrease in infectious processes; 2016 molecular study were performed with diagnosis of STAT1 mutation with gain of function. Discussion In patients with chronic mucocutaneous candidiasis the presence of STAT1 mutations with gain of function must be considered, responsible for the regulation of the Th17 response, which is characterized by the production of interleukin 17 and 22, crucial for the epithelial defense; the use of the transfer factor confers an immunoregulation factor, which is why it was granted to the patient. In our country, the report of these entities will help to increase the index of suspicion, favor the detection and grant the early treatment of this entity.

Granulomatous disease in common variable immunodeficiency.

Granulomatous lesions are occasionally found in the lymphoid or solid organs of patients with common variable immunodeficiency. To examine the clinical and immunologic conditions in patients with common variable immunodeficiency who have granulomas. Case series. Large tertiary care medical center. 17 hypogammaglobulinemic patients with common variable immunodeficiency whose organ or tissue biopsy samples contained noncaseating granulomas. Results of lymphocyte function tests. Eight of 17 patients had granulomas at some point before hypogammaglobulinemia was diagnosed. Sixteen of the 17 had deficient T-cell proliferation to mitogens. Although 14 patients received standard treatment with intravenous immunoglobulin, they have had substantial illness, including frequent autoimmune disease. Dysregulated T-cell function or macrophage activation may have been involved in formation of granulomas and increased illness in hypogammaglobulinemic patients with common variable immunodeficiency. Delay in recognition of antibody deficiency may have contributed to the severity of illness in these patients.

Effects of recombinant murine tumor necrosis factor-alpha on immune function.

TNF-alpha is a macrophage-derived cytokine with diverse biologic activities, including potent immunomodulatory effects. In vitro studies have implied that TNF-alpha has predominantly proinflammatory and immunostimulatory effects, but paradoxically in vivo studies have demonstrated that administration of TNF-alpha suppresses murine lupus. To assess the effects of TNF-alpha on immune function in normal mice, we treated C57BL/6 mice with recombinant murine TNF-alpha (10 micrograms i.p.) or PBS on alternate days for up to 8 wk. Administration of TNF-alpha decreased the percentage of splenic T and B cells and increased the percentage of splenic macrophages without significantly altering the total number of mononuclear cells. Administration of TNF-alpha also caused progressive inhibition of splenic lymphocyte function, out of proportion to the quantitative reduction in B and T cells. After 8 wk of therapy, the proliferative responses of splenic lymphocytes to Con A, PHA, and LPS were reduced by 100, 90, and 60%, respectively, in treated mice compared with control mice. The reduction in T cell proliferation was due primarily to alteration of accessory cell function rather than direct inhibition of T cell function. Treatment with TNF-alpha markedly inhibited T cell cytotoxicity induced by immunization with allogenic target cells, and it virtually ablated NK cell activity. Inhibition of these in vitro tests of lymphocyte function correlated with inhibition of delayed type hypersensitivity in vivo. In contrast, treatment with TNF-alpha did not impair humoral immunity. These findings imply that TNF-alpha may affect cell-mediated immunity more profoundly than humoral immunity. This observation may be relevant to the mechanism whereby TNF-alpha suppresses murine lupus.

Pulmonary Consequences of Congenital and Acquired Primary Immunodeficiency States

A sophisticated system of pulmonary host defense strives to maintain the functional integrity of the lung against the threats of infections, toxins, and malignancy. Congenital and acquired defects in the immune mechanisms of this host defense are associated with a variety of pulmonary disorders that include infections with usual or opportunistic organisms; inflammatory disorders; and malignancies. The age of the patient, associated abnormalities, family history, and the type of pulmonary and systemic diseases that are present provide clues to the specific underlying disorder. Laboratory tests including immunoglobulin levels, lymphocyte subset enumeration, and tests of lymphocyte function can help to confirm the clinical impression. Determination of the specific disorder allows the physician to anticipate possible complications, initiate appropriate prophylactic measures, and, in an increasing number of diseases, offer specific therapy.

The effects of 9-ene-tetrahydrocannabinol on hormone release and immune function

We investigated effects of 9-ene-tetrahydrocannabinol (THC) on endocrine and immunological function. Seventeen male volunteers entered into a double blind, randomized study to receive oral THC (10 mg t.i.d. for 3 days and on the morning of the fourth day) or placebo, after at least 2 weeks of abstinence. Plasma prolactin, ACTH, cortisol, luteinizing hormone and testosterone were not altered during or after THC, compared with baseline concentrations. Tests of lymphocyte function showed no differences compared to baseline between THC and placebo groups. As the relatively low dosing regimen of THC (10 mg t.i.d.) resulted in no alterations, another group of 6 men were administered higher doses of THC by inhalation (18 mg/marijuana cigarette) following the same dosing regimen. No endocrine or immunological alterations were observed. When the subjects were grouped according to their history of THC use prior to admission, heavy THC users had lower prolactin concentrations than light users. No differences were observed in concentrations of other hormones or in tests of immune function.

In vitro cell-mediated immunity after thermal injury is not impaired. Density gradient purification of mononuclear cells is associated with spurious (artifactual) immunosuppression.

Mononuclear cells isolated by density gradient centrifugation from the peripheral blood of burn patients, but not healthy volunteers, are contaminated with large numbers of nonmononuclear cells. These contaminating leukocytes could cause artifactual alterations in standard in vitro tests of lymphocyte function. Thus, we compared the in vitro blastogenic response of density gradient purified leukocytes and T-cell purified lymphocytes from 13 burn patients to mitogenic (PHA) and antigenic stimuli. The mitogenic and antigenic response of the patients' density gradient purified leukocytes were impaired compared to healthy volunteers (p less than 0.01). However, when the contaminating nonlymphocytes were removed, the patients' cells responded normally to both stimuli. Thus, density gradient purified mononuclear cells from burn patients are contaminated by leukocytes that are not phenotypically or functionally lymphocytes. Since the lymphocytes from burn patients respond normally to PHA and alloantigens after the contaminating nonlymphocyte cell population has been removed, it appears that in vitro assays of lymphocyte function using density gradient purified leukocytes may give spurious results.

Indomethacin sensitive suppressor-cell activity in head and neck cancer patients. The role of the adherent mononuclear cell.

Head and neck cancer (H&N CA) patients have known depression of cell-mediated immunity. There is suggestive evidence that prostaglandin (PGE2)-secreting cells may be a major factor. The authors have sought to determine the role of PGE2-releasing monocytes-macrophages in this immune depression by determining the effects of adherent cell depletion and by measuring the effects of indomethacin, a PGE2 synthetase inhibitor, on selected tests of lymphocyte function. Lymphocyte stimulation with phytohemagglutinin (PHA) (T-cell stimulant) and Staph phage lysate (SPL) (B-cell stimulant) was done in the presence of varying concentrations of indomethacin; the effect of adherent cell depletion also was determined. The study population included 45 patients with localized or locoregional squamous CA of the H&N and 40 controls. Results included the following: (1) lymphocyte stimulation responses to PHA and SPL were generally depressed in the CA patients versus controls; (2) incubation with indomethacin produced bivalent effects in both controls and CA patients, depending on the concentration of indomethacin and lymphocyte stimulant; incubation with optimum concentrations of indomethacin generally produced augmented responses in both study groups whereas high concentrations of indomethacin were suppressive; (3) the immune potentiating effects were not observed in older patients with advanced disease; and (4) removal of adherent leukocytes (mainly monocytes) also restored depressed lymphocyte responses. Although other factors also are operative, our data suggest that PGE2-secreting monocytes-macrophages may have a major role in the immune depression of H&N CA patients. Age and host effects of the cancer and the malnutrition common to these patients probably are involved also, although their singular contribution has not been measured. This depression is largely reversible by a PGE2 synthesis inhibitor, indomethacin, which suggests the potential value of in vivo administration of indomethacin to H&N CA patients as an adjunct.

Recurrent eruption following a solitary envenomation by the cnidarian Stomolophous meleagris

J.W. Burnett and G.J. Calton. Recurrent eruption following a solitary envenomation by the cnidarian Stomolophous meleagris. Toxicon 23, 1010–1014, 1985. — The case history of a patient who developed a linear erythematous eruption on the ankle after envenomation by Stomolophous meleagris is described. This eruption subsided within 20 hr and recurred without subsequent envenomation 7 days later. The recurrent eruption was similar in morphology to the original lesion, but was accompanied by stiffness and edema. Examination of biopsied skin from the recurrent eruption showed a perivascular mononuclear cell dermal infiltrate. No abnormal deposits of immune globulins or complement were found in this specimen. The patient's serum contained antibodies to Stomolophous antigen and two other jellyfish. Two screening tests of lymphocyte function were negative upon exposure to Stomolophous antigen. This was the first reported case of recurrent local eruption to a single human envenomation by this coelenterate, which usually is regarded as relatively harmless to man.

Partial characterization of bone marrow hemopoiesis in mice after cisplatin administration

After cessation of cisplatin ( cis-dichlorodiammineplatinum) chemotherapy, selective recovery of certain cell lineages occurs during bone marrow hemopoiesis. To further investigate the process of selective hemopoiesis, we combined the use of buoyant density gradient separation, morphology, and lymphocyte function assays to characterize changes in the hemopoiesis of immature and mature marrow cells after exposure to cisplatin. A single, cytotoxic dose of cisplatin was administered to B6D2F 1 male mice, and marrow cell suspensions were taken from these mice 3 and 7 days later to characterize hemopoietic recovery. Morphology and buoyant density separation of marrow cells revealed that the most significant changes in cellular composition occurred at Day 3. At this time, there was an increase in immature white blood cells (WBCs) and immature polymorphoneutrophils (PMNs) with a concomitant reduction in immature red blood cells (RBCs). By Day 7, the normal proportion of immature RBCs, immature WBCs, and PMNs was restored; however, the buoyant distribution patterns for PMNs indicated that a greater proportion of immature PMNs was still present relative to marrow suspensions from normal mice. Fewer lymphocytes were also observed in marrows from the Day 7 group when compared with controls. Lymphocyte function tests indicated reduced mitogen responsiveness of lymphocytes from both Day 3 and Day 7; however, more immature lymphocytes were present after 7 days than were seen with either normal or Day 3 marrow suspensions. Overall, the results indicated that hemopoiesis proceeded through a specific hierarchy which began with the restoration of the erythrocyte line followed by the leukocyte cell lines. Lymphocyte recovery lagged behind the restoration of all the cell lineages examined.

Development of anergy to delayed-type hypersensitivity antigens following renal allotransplantation

A study is reported of patients receiving 180 renal allotransplants and at risk for 1 or more years who had pretransplant and posttransplant skin tests using delayed type hypersensitivity (DTH) recall antigens. Pretransplant skin testing revealed an incidence of 14% anergic patients prior to a first transplant, and 35% were anergic prior to a second allograft. There was a statistically significant correlation between pretransplant anergy and age, as well as a significant correlation between pretransplant anergy and time of dialysis-dependent uremia. The conversion to the anegic state posttransplant in those patients with surviving allografts increased annually for the first 5 years as a linear function, with 69% of the population at risk with their first transplant exhibiting anergy by 5 years. In those patients receiving second renal transplants, all were anergic by 4 years posttransplant. In vitro tests of lymphocyte function using mitogen stimulation did not reveal significant differences between anergic and responsive patients. In this series of 180 allografts there were 14 late deaths. Eleven of these were in anergic patients and in 10 instances were related to sepsis or malignancy. Three patients who were skin-test responsive at the time of death died from cardiovascular causes. It is concluded that there is an increasing risk of the development of anergy to DTH antigens posttransplant and that the type of late mortality may be marked by this hyporeactive state.

Cell-Mediated Immunity Is Depressed Following Cardiopulmonary Bypass

Sequential in vitro lymphocyte function tests in 13 patients undergoing cardiac operation were performed to determine factors that contribute to depressed cell-mediated immunity following operation. Lymphocytes were stimulated with phytohemagglutinin (PHA), pokeweed mitogen, concanavalin A (Con A), and mitomycin-treated, pooled, allogeneic lymphocytes (MLC). Mitogen responses were measured by 3 H-labeled thymidine incorporation. Circulating levels of T, B, and Fc-receptor lymphocytes were determined by counting E, EAC, and EA rosettes. Serum cortisol was measured by radioimmunoassay. The T-cell-dependent lymphocyte responses (PHA, Con A, and MLC) were significantly decreased 24 hours after operation, and this was accompanied by a 60% decrease in circulating T-cell levels. The PHA, Con A, and MLC responses, and circulating T-cell levels returned to preoperative values one week following operation. Lymphocyte responses to mitogens remained significantly decreased when the number of T cells in the postoperative cultures were adjusted to preoperative levels. This indicates that the T cells remaining after operation were functionally impaired. We conclude that lymphocyte proliferative responses and antigen recognition are significantly depressed following cardiac operation, and that these responses are related to decreased numbers of circulating T lymphocytes and depressed function of the remaining T lymphocytes.

A simple method for determining the extent of cellular contamination in peripheral blood lymphocyte preparations

A method is described for calculating, from the size distribution of the cells, the extent of cellular contamination of lymphocyte concentrates prepared from venous blood. The precision of this method was checked by direct comparison with differential leucocyte counts obtained by direct microscopy on 29 samples chosen because they showed a wide range of intensity of contamination. By virtue of its technical simplicity, the method has proved useful in a diagnostic service laboratory for checking the purity of lymphocyte preparations either before performing lymphocyte function tests or after a period of 24 h in tissue culture.

Impaired thymus-derived lymphocyte function in patients with malignant brain tumour

Cell-mediated immunity was evaluated in 28 patients with malignant glioma, using in vivo and in vitro tests of lymphocyte function. The results were compared to those found in patients with carcinomatosis (11 subjects), benign brain tumours (9), other neurological disorders (20) and normal, healthy controls (21). Significant impairment of delayed hypersensitivity responses to common antigens was found in patients with malignant glioma and in those with generalised malignancy. A less significant depression of lymphocyte responses was also detected in patients with meningioma. The impairment in cell-mediated immunity was shown not to be due to a serum blocking factor. Our data indicate that there is defective T cell function in patients with glioma, similar to that reported in cases with malignancies outside the central nervous system. This impaired immunity may have clinical significance.

PRE-ECLAMPSIA—A STATE OF MOTHER-FETUS IMMUNE IMBALANCE

Serial lymphocyte counts and function tests were done during and after pre-eclamptic pregnancies, and in the offspring. Compared with normal pregnancies, pre-eclamptic pregnancies were associated with lower B-lymphocyte counts in the fathers; lower T-lymphocyte count, lower B-lymphocyte count, and impaired T-lymphocyte function in mothers; a low response in mixed lymphocyte culture (MLC) tests between the mother and father; and an increased B-cell count in the children. Follow-up of one pre-eclamptic woman throughout pregnancy showed important immune changes at the beginning of the second trimester. These findings suggest that pre-eclampsia is caused by a combination of maternal and paternal hyporesponsiveness together with fetal hyperresponsiveness.

<i>In vivo</i> and <i>in vitro</i> Assays of Immunocompetence in Bronchogenic Carcinoma

To determine the degree of correlation among the various in vivo and in vitro assays that could be used to assess immunocompetence in bronchogenic carcinoma, the response to common recall skin antigens, primary sensitization to DNCB and lymphocyte function tests based on blastogenic response to mitogens and in mixed lymphocyte culture (MLC) were tested in 48 patients with bronchogenic carcinoma. The values were compared to responses in 94 age-matched healthy control subjects. There were impaired skin tests reactions among squamous cell carcinoma patients, with little impairment of their lymphocyte blastogenesis reactions. Anaplastic carcinoma patients had notable defects in lymphocyte function tests but less impairment of the skin test reactions. These data suggest that the mitogen concanavalin A, phytohemagglutinin, and the MLC are more useful screening assays of in vitro immunocompetence than are the other commonly used mitogens.

Thymic Hormones

The thymus produces several polypeptides, which induce lymphocyte differentiation in vitro and in vivo. Several of these polypeptides have been chemically characterized, and three of them have been sequenced and synthesised (alpha 1 thymosin, thymopoietin and the serum thymic factor). Thymic hormones do not act identically on all T-cell subsets: they alter preferentially post-thymic precursor cells, and among mature T cells cytotoxic cells and suppressor cells. Their mode of action at the cellular level involves binding to specific cellular receptors and interaction with adenyl cyclase. Preliminary clinical trials with crude extracts have provided promising results in immunodeficient and cancer patients. The differentiation of T cells from stem cells has been the matter of considerable investigation over the last two decades, since it has been realized that the thymus and its products, the thymus-derived cells (T cells) play a central role in the generation of effector cells in cell-mediated immunity and in the regulation of the various categories of immune responses. That the thymus could act by the intermediate of humoral substances was precociously suggested by MILLER and OSOBA before the observation that thymuses grafted within a cell-impermeable Millipore diffusion chamber restored the immunocompetence of neonatally thymectomized (Tx) mice (1). However, although this experiment was ultimately confirmed by using chambers with well-controlled impermeability (2), MILLER did not pursue the idea of the humoral function of the thymus. Probably, the striking results obtained by DAVIES (3) and other workers, indicating direct migration of functional T cells from the thymus and the poor results initially obtained in trying to reconstitute the immune system of neonatally Tx mice by cell-free thymic extracts contributed to this disappointment. A new impetus was given to the subject in the early 70's when in vitro tests of lymphocyte function became available and when purified extracts of the thymus proved capable of restoring antigen-specific and non-specific immunocompetence of Tx mice. More recently, completely defined synthetic thymic hormones have been obtained. The question is no longer to decide whether thymic hormones exist, but rather to elucidate their biological significance and potential clinical applications. The multiplicity of available factors has created some confusion. It will be the aim of these few pages to review critically the various factors reported in the literature, giving particular emphasis to their pharmacology and their potential use in the modulation of immune responses.

A longitudinal study of in vitro tests for lymphocyte function in rheumatoid arthritis

In vitro tests of lymphocyte function have been performed in 61 patients with ;classical' or ;definite' rheumatoid arthritis. In vitro lymphocyte function was assessed by lymphocyte transformation responses to phytohaemagglutinin (PHA), Pokeweed mitogen (PWM), Candida antigen, and herpes simplex type I (HSV1). Follow up data were available after 6 months of treatment in 32 of these patients. Spontaneous lymphocyte transformation was assessed in all patients. Results obtained in patients with rheumatoid arthiritis were compared to those seen in a normal control population. Disease activity of patients with rheumatoid arthritis was assessed using standard clinical methods. Lymphocytes from patients with rheumatoid arthritis showed a similar degree of spontaneous transformation to that seen in normal subjects. In contrast, lymphocytes from patients with rheumatoid arthritis responded less well to PHA and Candida and HSV1 antigens when compared to normal patients. In patients with rheumatoid arthritis the response to PWM was markedly enhanced compared to normals. Clinical improvement was noted in 19 of the 32 patients seen at follow up, all of whom had received gold or penicillamine therapy. The abnormal responses of PHA and PWM seen before treatment became normal in those patients who improved clinically. The responses to Candida and HSV1 antigens not only returned to normal following treatment but were increased above those seen in normal controls. A statistically significant association was seen between clinical improvement and improvement of in vitro tests of lymphocyte function.

Adjuvant-induced thymus-derived suppressor cells of cell-mediated tumour immunity

THE use of immunoadjuvants in enhancing immune responses to various antigens and particularly in tumour therapy is based largely on the concept that an increased level of specific immunity may be achieved by nonspecific stimulation of the immune system1. However, the tumour-protective effect of adjuvants has not been entirely consistent in all systems studied, and also, an immune suppressive effect of adjuvants has been demonstrated mainly by using in vitro assays of cell-mediated immunity2. It remains to be determined whether a defect measured by such general tests of lymphocyte function may also represent an indication of depressed cell-mediated immunity against the progressive growth of tumours. Moreover, the target cell in the immune system for adjuvant effect has not been characterised clearly. Our laboratory has made extensive study of immune response to SV40-induced syngeneic transplantable MKSA cells of BALB/c mice3–5. This tumour has been shown to possess tumour-associated antigens which can immunise specifically against the growth of MKSA cells. The role of T lymphocytes in the immune response against SV40-induced tumour-associated antigens was shown by in vivo tumour cell neutralisation assay (Winn)6 and by in vitro 51Cr release assay7. We now report that injection of complete Freund's adjuvant (CFA), one of the most useful adjuvants in enhancing immune responses, markedly inhibited the ability of immune spleen cells to prevent SV40-induced tumour growth in the Winn assay and to lyse SV40-transformed cells in the cytotoxicity assay. Moreover, untreated mice which reject suboptimal numbers of tumour cells die of progressive tumour growth following treatment with CFA and tumour cells; adult thymectomy reverses the suppression caused by CFA; and the target cell for CFA effect is the T lymphocyte. These data suggest that treatment with CFA causes depression of specific anti-tumour cell-mediated immunity by activation of suppressor thymus-derived lymphocytes.

In Vitro Tests of Lymphocyte Structure and Function

In Vitro Tests of Lymphocyte Structure and Function

Effect of adriamycin and high-dose methotrexate chemotherapy on in vivo and in vitro cell-mediated immunity in cancer patients.

Adjuvant chemotherapy with adriamycin (ADR) and high-dose methotrexate (HDM) with citrovorum rescue appears to hold promise for preventing recurrence of sarcomas following surgery. However, the effect of prolonged treatment with these agents on both in vivo and in vitro cell-mediated immunity (CMI) is unknown. To assess in vitro CMI, cryopreserved lymphocytes from 18 patients, collected before and at monthly intervals following initiation of biweekly ADR and HDM therapy, were tested simultaneously for stimulation to phytohemagglutinin, poke-weed mitogen, Concanavalin A, and pooled allogeneic lymphocytes by a micro-test technique. In vivo CMI was determined by serial skin-testing with 2,4,-dinitrochlorobenzene (DNCB). No significant overall change was noted for any of the in vitro lymphocyte function tests. DNCB skin test ratings, peripheral leukocyte counts and absolute lymphocyte counts did not change significantly during the study. Lymphocytes from a second group of patients were tested before, at 24 and 48 hours after HDM therapy. In vitro lymphocyte function tests were significantly depressed 24 hours following treatment, but returned to pretreatment levels at 48 hours. Long-term adjuvant ADR and HDM therapy does not appear to alter in vivo and in vitro CMI, although transient depressions were noted. The preservation of intact CMI may account in part, for the effectiveness of these agents as surgical adjuvants.