Immunological aspects of benign melanocytic tumours and melanoma in situ

Summary Extracts were prepared from 15 malignant melanomas, 10 naevi, 8 pieces of skin containing atypical melanocytes and 4 samples of normal skin. Peripheral blood leucocytes from 52 patients with benign naevi that had altered sufficiently to cause the patient to seek attention, 57 patients with malignant melanomas, and 98 normal individuals were tested against these extracts in a capillary leucocyte migration test. Melanoma extracts preferentially inhibited the leucocytes of melanoma patients and to a lesser extent those of naevus patients. Melanoma patients' leucocytes were more frequently inhibited by naevus extracts (whether from intradermal or from compound naevi) than were the leucocytes of naevus patients and control donors. Extracts of skin containing atypical melanocytes (perimelanomatous skin and lentigenous skin) selectively inhibited melanoma patients' leucocytes.     

Specific Immunodiagnosis of Hepatocellular Carcinoma by Leucocyte Adherence Inhibition

The leucocyte adherence inhibition test provides a rapid, reliable, and specific technique for the immunodiagnosis of primary hepatocellular carcinoma (malignant hepatoma). The patient''s blood leucocytes are tested in vitro for cell-mediated immunity against tumour-associated antigens and the serum is tested for blocking factor which interferes with the immunological reaction. Specific reactivity of both leucocytes and serum was consistently detected in patients with malignant hepatoma, and negative reactions were obtained in other liver diseases including secondary tumours of the liver. The test has provided positive evidence for the presence of hepatoma when more conventional methods gave doubtful or negative results. A positive result preceded the clinical appearance of tumour by up to three years in two patients.     

Indicators of spontaneous and stimulated nitroblue tetrazole test of polymorphous-nuclear leucocytes in acute dysentery patients

140 healthy individuals and 93 sick with acute dysentery were subjected to an examination by spontaneous and by bacterial preparations stimulated reaction with nitroblue tetrazole (NBT test). Indicators in healthy persons were normal in the spontaneous, and increased in the NBT test, stimulated by bacterial preparations. Indicators of the spontaneous NBT test in patients with acute dysentery were raised with a maximum in the period of early convalescence. Stimulation by a live shigella culture--the dysentery vaccine--revealed by means of Sonne diagnostic high, and when endotoxin from Serratia marcescens and dysenterin was used as an inductor, mild indicators of NBT test activity. When a polyvalent agglutinating dysentery serum was used as a stimulator, the activity increased considerably, and a simultaneous use of serum and vaccine had an inhibiting effect on the indicators of the stimulated NBT test. The obtained results testify the sufficient high reserve possibilities of leucocytes towards complete phagocytosis and the efficiency of the NBT test, stimulated by bacterial preparations for the study of functional and metabolic activity of leucocytes in the process of acute bacterial dysentery.     

Cytochemical and electron microscopic research on the peripheral blood leukocytes and skin fibroblasts in mucopolysaccharidoses

Cytochemical studies of the peripheral blood leucocytes in patients suffering from mucopolysaccharidoses (MPS) have revealed metachromatic granules in the cell cytoplasm. Electron microscopy of these cells has shown multiple cytoplasmic vacuoles. It is supposed that metachromatic granules in blood leucocytes of patients with MPS observed in the photo-optical studies correspond to the vacuoles found under electron microscope. The obtained data have shown that peripheral blood leucocytes and skin fibroblasts have the common ultrastructure in MPS patients. The data of electron histochemical studies testify to that the vacuoles of skin fibroblasts are filled with glycosaminoglycans.     

Phagocytosis and leucocyte enzymes in protein–calorie malnutrition

1. Enzymes pertinent to bactericidal activities of leucocytes were assayed in children suffering from protein-calorie malnutrition. 2. Leucocytes obtained from malnourished and control children contained similar activities for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Granule-bound NADPH oxidase activity was low in leucocytes isolated from malnourished patients and failed to show the phagocytic stimulation which is normally seen in control leucocytes. Further, leucocytes obtained from malnourished patients did not release the acid phosphatase from lysosomes during phagocytosis, unlike those from controls. 3. Treatment of the malnourishment with a diet high in calories and protein resulted in significant increase in the activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADPH oxidase and in releasing the acid phosphatase from the lysosomes into the supernatant fraction during phagocytosis. 4. The significance of these enzyme changes are discussed in relation to the increased susceptibility of these patients to infection.     

Interferon production by human leukaemic leucocytes.

The Sendai virus-induced interferon (IF) production by partially purified human leucocyte suspensions of normal donors and of leukaemic patients have been investigated in vitro. (i) An increased production was observed with leucocytes taken from patients suffering from chronic myelogenous leukaemia (CML) during exacerbation, but the production was approximately normal with cells taken during remission. (ii) IF production was not influenced by large doses of cytostatics (DBM, 5-FU, FUDR, 5-HU, 6-MP, cyclophosphamide) irrespective of whether normal or CML leucocytes were used. (iii) In contrast, production was inhibited in both systems by inhibitors of RNA or protein synthesis (actinomycin D, puromycin, cycloheximide). (iv) CML leucocytes produced IF for a prolonged period of time as compared to normal leucocytes. (v) Leucocytes from children with acute blastic leukaemia and those from adults with chronic lymphoid or acute paramyeloblastic leukaemia produce, in contrast to normal leukocytes, approximately as much IF in the absence as in the presence of serum. It is concluded that the Sendai virus-induced IF synthesis in CML leucocytes requires de novo protein synthesis.     

Effect of the infestation by Lernaea cyprinacea Linnaeus, 1758 (Copepoda, Lernaeidae) on the leucocytes of Schizodon intermedius Garavello & Britski, 1990 (Osteichthyes, Anostomidae)

Differential white blood cell counts from Schizodon intermedius infested by Lernaea cyprinacea were carried out and compared using the Wilcoxon matched pairs test. The observations were performed in fish infested by 16-77 adult Lernaea, fish with parasitic lesions but without attached crustaceans, and non parasitized fish (control group). The specimens of Schizodon intermedius were obtained from the University of Londrina fish farming facilities. The following leucocytes were observed: lymphocytes, neutrophils, monocytes, basophils, eosinophils and immature leucocytes. Intense lymphocytopenia and neutrophilia were observed in the infested hosts. Consistent increasing of monocyte percentage values occurred in the infested fish. The highest values for immature leucocytes counts were recorded from infested fish specimens.     

Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients

Monitoring the bioenergetics of leucocytes is now emerging as an important approach in translational research to detect mitochondrial dysfunction in blood or other patient samples. Using the mitochondrial stress test, which involves the sequential addition of mitochondrial inhibitors to adherent leucocytes, we have calculated a single value, the Bioenergetic Health Index (BHI), which represents the mitochondrial function in cells isolated from patients. In the present report, we assess the BHI of monocytes isolated from the post-operative blood and post-operative pericardial fluid (PO-PCF) from patients undergoing cardiac surgery. Analysis of the bioenergetics of monocytes isolated from patients’ PO-PCF revealed a profound decrease in mitochondrial function compared with monocytes isolated from their blood or from healthy controls. Further, patient blood monocytes showed no significant difference in the individual energetic parameters from the mitochondrial stress test but, when integrated into the BHI evaluation, there was a significant decrease in BHI compared with healthy control monocytes. These data support the utility of BHI measurements in integrating the individual parameters from the mitochondrial stress test into a single value. Supporting our previous finding that the PO-PCF is pro-oxidant, we found that exposure of rat cardiomyocytes to PO-PCF caused a significant loss of mitochondrial membrane potential and increased reactive oxygen species (ROS). These findings support the hypothesis that integrated measures of bioenergetic health could have prognostic and diagnostic value in translational bioenergetics.     

Fluorescence assay to monitor phagocytosis by bloodclot derived polymorphonuclear leucocytes. 2. Study of patients with psoriasis and the effect of exposing leucocytes to psoriatic skin scales

Comparisons of phagocytic parameters were carried out by a recently developed fluorescence test which is reproducible, simple and fast. Phagocytosis by polymorphonuclear leucocytes (PMNs) obtained from patients with psoriasis was compared with that of healthy individuals. Psoriatic skin scales, non-sterile and sterile, were tested for stimulatory effect on PMNs and compared with the effect of normal skin scrapings. Results confirm enhanced phagocytosis of bacteria by PMNs from patients with psoriasis over that of PMNs from healthy volunteers. Furthermore, the supernatant fluid from suspensions of psoriatic skin scales, non-sterile and sterile, stimulated PMNs activity.     

Defective PGE reactivity in leucocytes of multiple sclerosis patients

Leucocyte migration inhibition in vitro, in response to antigen or mitogen, is suppressed by PGE₂ (0.01–1.0 μg/ml).The susceptibility of leucocytes to such inhibition by PGE₂ has been compared using cell preparations obtained from normal individuals, multiple sclerosis patients and from patients with other neurological diseases. The results indicate defective reactivity of leucocytes from multiple sclerosis patients.     

In-vitro Inhibition of Leucocyte Migration in Crohn's Disease by a Sarcoid Spleen Suspension

A Kveim suspension has been shown to inhibit the migration of leucocytes in vitro from 12 out of 18 patients with Crohn''s disease but to have no comparable effect on leucocytes from patients with ulcerative colitis or from a group of patients with other diseases. These findings provide further evidence of cross-reactivity or of a possible aetiological link between Crohn''s disease and sarcoidosis and suggest a further immunological distinction between Crohn''s disease and ulcerative colitis.     

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. II. Presence of the NADPH2 oxidizing activity in a myeloperoxidase-deficient subject.

1. The biochemical properties of leucocytes from a myeloperoxidase-deficient subject were compared with those of leucocytes from healthy subjects. 2. Myleoperoxidase-deficient leucocytes responded to phagocytosis of heat-killed bacteria with increased respiration, increased oxidation of glucose through the hexose monophosphate shunt and increased production of H2O2 as normal leucocytes do. 3. The ability of granules isolated from myeloperoxidase-deficient leucocytes to oxidize nicotinamide coenzymes was comparable to that of granules isolated from normal leucocytes. 4. The results argue against the hypothesis that oxidation of NADPH2 in leucocytes is performed by myeloperoxidase.     

Expression of oncornavirus antigens in peripheral leucocytes of patients with chronic myeloid leukaemia.

Peripheral leucocytes from 16 patients with chronic myeloid leukaemia (CML) were examined for the presence of oncornavirus p30 antigens by indirect cytoplasmic immunofluorescence. The leucocytes of 12 patients who could be kept in balance by chemotherapy proved to be negative or contained the p30 antigen of mammalian endogenous oncornaviruses as the only viral antigen. In the leucocytes of four patients being in blastoid crisis, an antigen related to the p30 antigen of mammalian leukaemia-sarcoma viruses was detected. In five of six patients decrease in sensitivity to chemotherapy, or blastoid crisis, was preceded by expression of leukaemia-sarcoma virus p30 antigen(s). Leucocytes from 15 CML patients kept in balance by chemotherapy and those from seven being in blastoid crisis, were examined by indirect membrane immunofluorescence for the presence of antigen(s) related to the gp70 antigen of the simian and murine leukaemia-sarcoma virus. All tests proved to be negative.     

Dose-response relationship in the migration inhibition test using peritoneal exudate cells and blood leucocytes of tuberculin sensitive guinea pigs

A comparison of the direct migration inhibition using peritoneal exudate cells and peripheral blood leucocytes from the same tuberculin sensitive guinea pigs was performed. Three antigen concentrations: 3, 15, and 75 microgram of PPD per ml were used. Both type of cells provided similar results except at early incubation intervals when leucocytes, in the presence of lower doses of the antigen, displayed stronger inhibition than peritoneal cells. Thus, in our study, peripheral blood leucocytes are at least equivalent to peritoneal exudate cells in the migration inhibition test.     

X-chromosome methylation ratios as indicators of chromosomal activity: Evidence of intraindividual divergencies among tissues of different embryonal origin

To test whether the differentiation events that lead to the embryonal layers and their derived organs produce divergent X-chromosome activation ratios among the different tissues, the X-chromosome activation ratios in leucocytes and muscle (mesodermal origin), thyroid gland (endodermal origin) and medulla of the suprarenal glands (ectodermal origin) from ten deceased females were surveyed. Analysis of the degree of methylation of the polymorphic alleles recognized by the probes M27 and pSPT-PGK showed that the ratios for the medulla of the suprarenals correlated well with those of all other tissues except for leucocytes; the thyroid gland showed limited correlation with muscle, whereas leucocytes showed correlation only with muscle. The results of this preliminary study suggest that differentiation events result in considerable variation in the activation ratios in different tissues. As a consequence caution should be taken in extrapolating from the activation ratios observed in leucocytes or fibroblasts to tissues of endodermal or ectodermal origin.     

Mixed Leucocyte Reaction in Multiple Sclerosis

Astorga and Williams¹ have demonstrated that leucocytes from some patients with rheumatoid arthritis (RA) have an impaired one-way mixed leucocyte reaction² (MLR) when stimulated with leucocytes from other such patients. No definite explanation of this phenomenon is given. We have investigated other diseases in which auto-immune aetiology is suspected to see whether similar phenomena are associated with them. Here we report our investigations of multiple sclerosis (MS).     

Detection of human leucocytes by cyclic voltammetry and its application to monitoring of allergic reaction

Cyclic voltammetry was applied to the detection of human leucocytes and the monitoring of allergic reactions. A basal plane pyrolytic graphite electrode with attached leucocytes on a porous nitrocellulose membrane filter was employed as a working electrode. An anodic peak current appeared at 0.33 V versus the saturated calomel electrode (SCE) when the potential of the working electrode was scanned in the range of 0-1.0 V versus SCE. This peak current was attributed to the electrochemical oxidation of serotonin. When egg white was added to leucocytes obtained from patients who were allergic to egg, the peak current decreased owing to degranulation of leucocytes leading to serotonin release. The peak current decreased with increasing allergen concentration in the range of 5-50 micrograms ml-1. Leucocytes did not respond to other allergens such as soybean, milk and dinitrophenylated bovine serum albumin (DNP-BSA).     

急性白血病患者外周血白细胞诱生干扰素的能力和淋巴细胞转化功能的研究

用微量细胞病变抑制法测定了53例急性白血病患者外周血白细胞诱生干扰素的水平,以及18例急性淋巴细胞性白血病患者的淋巴细胞转化功能。发现前者体外诱生干扰素的水平显著下降,并与病程转归有密切关系;后者淋巴细胞转化指数低于正常,但与干扰素水平之间无平行关系。本文还对急性白血病患者白细胞体外诱生干扰素水平下降的可能原因进行了讨论。     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Cortisol binding in human breast cancer: Correlation with antitumor immunity

Summary The intensity of cortisol binding was measured in the cytosol fraction of the primary tumor obtained from 50 patients with stage I and II breast cancer. The state of cellular antitumor immunity of the same patients was investigated by the tube leucocyte adherence inhibition (LAI) test, performed with peripheral blood leucocytes 1–2 days preoperatively. It was found that the intensity of tumor cortisol binding correlates negatively with LAI values. Patients with high cortisol binding in their tumors have low LAI values, while low tumor cortisol binding is associated with higher antitumor immunity. The results suggest that high cortisol binding in the tumor might inhibit the tumor recognition process and/or the cellular immune defense mechanism and thus facilitate cancer development.