ADCC (K-Cell) Lysis of Human Erythrocytes Sensitized with Rhesus Alloantibodies

An ADCC system has been developed using anti-D and papainized group O rhesus (D) positive red cells as the targets. Monocyte depleted mononuclear cell suspensions were effective in lysing appropriately sensitized red cells and papainization considerably enhanced the degree of specific lysis. Variation in culture volume and incubation in tubes or microplates were not critical to the degree of specific lysis obtained provided that the number of effector cells and target cells per culture was constant and the anti-D not diluted below the optimal concentration. Cytolytic activity was seen down to levels of 3 ng anti-D per culture. Specificity for lysis resided with the anti-D and not the effector cells. Several sources of anti-D were effective in inducing lysis of D positive red cells although individual variation was noted. Anti-c and anti-E were also shown to be effective in inducing specific lysis of red cells with the appropriate antigens.     

Lymphoid Cell Dependent (K-Cell) Lysis of Human Erythrocytes Sensitized with Rhesus Alloantibodies

S ummary . An in vitro homologous system using human Rhesus alloantibodies and target erythrocytes labelled with ⁵¹Cr has been used to demonstrate the ability of normal peripheral blood lymphoid cells to lyse antibody-coated human red cells. The results obtained suggest that this type of mechanism may be relevant to certain haemolytic diseases in man.     

Characteristics of Different Rhesus Alloantibodies in Lymphocyte Dependent (K Cell) Cytotoxicity to Human Erythrocytes

The property of rhesus alloantibodies to elicit antibody-dependent, cell-mediated cytotoxicity (ADCC) against target erythrocytes carrying various Rh genotypes was studied. The killer activity of normal peripheral lymphocytes on human erythrocyte target cells carrying the appropriate antigens elicited by alloantisera was measured by 51Cr release at 18 h. There was no correlation between ADCC and antibodies directed to the antigens present on the surface of different genotypes of Rh-positive red blood cells. The agglutinin titre of different Rh antibodies showed no correlation with the level of ADCC although the degree of cellular cytotoxicity was different with different anti-D sera. Anti-C + D+ E antibody caused higher ADCC than anti-C + D and the lowest cytotoxicity was observed with anti-D and anti-D+ E. This raised the possibility that ADCC was elicited by antibodies directed to other specificities. K cell lysis of human red cells by human peripheral blood lymphocytes in vitro suggests that a similar mechanism may operate in vitro in the destruction of erythrocytes coated by allo or autoantibodies.     

IgG(1) antiendomysium and IgG antitissue transglutaminase (anti-tTG) antibodies in coeliac patients with selective IgA deficiency. Working Groups on Celiac Disease of SIGEP and Club del Tenue

BACKGROUND: In selective IgA deficiency (IgAD), there is no reliable screening test for coeliac disease (CD). AIM: To evaluate the usefulness of IgG(1) antiendomysium and IgG antitissue transglutaminase tests for CD diagnosis in IgAD. METHODS: IgA and IgG antigliadin antibodies (IgA- and IgG-AGA), IgA and IgG(1) antiendomysium antibodies (IgA- and IgG(1)-EMA), and IgA and IgG antitissue transglutaminase (IgA- and IgG-anti-tTG) were assayed in: (a) 20 untreated IgAD/CD patients; (b) 34 IgAD/CD patients on a strict gluten free diet (GFD); (c) 10 IgAD/CD patients not on a strict GFD; (d) 11 untreated CD patients without IgAD; (e) 10 healthy IgAD patients; and (f) 25 healthy controls. RESULTS: In all untreated IgAD/CD patients, IgG(1)-EMA, IgG-anti-tTG, and IgG-AGA were positive whereas IgA antibodies against these antigens were negative. IgAD/CD patients on a strict GFD did not produce IgG-AGA or IgG(1)-EMA but four of 34 produced IgG anti-tTG. IgAD/CD subjects not on a strict GFD produced IgG-AGA whereas 5/10 and 4/10 were IgG(1)- EMA and IgG-anti-tTG negative, respectively. Untreated CD patients without IgAD were AGA (IgA and IgG), EMA (IgA and IgG(1)), and anti-tTG (IgA and IgG) positive. Healthy controls were AGA and EMA negative whereas two of 10 apparently healthy IgAD subjects and one of 25 healthy negative control were IgG-anti-tTG positive. CONCLUSIONS: Both IgG(1)-EMA and IgG-anti-tTG tests appear to be useful for identification of IgAD/CD patients whereas they are less satisfactory for monitoring dietary compliance in these subjects. In addition, our findings seem to suggest that IgG-EMA autoantibodies produced by coeliac patients are mainly of the IgG(1) subtype.     

Effect of Various Treatments of Gamma-Globulin (IgG) for Achieving Intravenous Tolerance on the Capacity to Interact with Human Monocyte Fc Receptors: A Comparative Study

Gamma-globulins for intravenous application (IgG-IV), processed by various methods, were tested for their ability to interact with human monocyte Fc receptors by determining the dose required to inhibit monocyte Fc receptor-mediated rosette formation and phagocytosis by half (ID50). Since dimeric and oligomeric IgG were found to be 2-3 times and 5-15 times more potent, respectively, than monomeric IgG, the varying proportions of polymeric IgG in intact IgG-IV were corrected for by calculation. The results of the rosette formation and phagocytosis tests were closely correlated, and insignificant differences between preparations processed by the same procedure were noted, while considerable differences were found between different procedures. The decreasing order of inhibitory activity was DEAE-Sephadex-treated IgG, acid-treated IgG, plasmin-digested IgG, polyethylene glycol(PEG)-precipitated IgG, IgG subjected to reduction/alkylation, IgG that underwent sulfitolysis, IgG treated with beta-propiolactone, and finally pepsin-treated IgG. Thus, while mild procedures preserve the capacity of IgG to interact with monocyte Fc receptors, chemical modification severely interferes with this important effector function.     

Hormonal effects of gonadotropin-releasing hormone (GnRH) agonist in the human male. III. Effects of long term combined treatment with GnRH agonist and androgen

Chronic treatment with agonist analogs of GnRH results in reversible oligospermia in man, but leads to impotence and decreased libido due to a concomitant fall in serum testosterone (T) concentrations. We, therefore, assessed the effects of combined treatment with a potent GnRH agonist and T on gonadotropins and spermatogenesis in normal men, anticipating that addition of androgen would prevent agonist-induced changes in libido. Seven normal men were treated with 200 micrograms of the GnRH agonist D-(Nal2)6GnRH (GnRH-A), sc, daily for 16 weeks. In addition, 200 mg T enanthate were administered every 2 weeks for the entire 16-week treatment period. Basal LH, FSH, and T concentrations were measured every week during a 5-week control period, daily on treatment days 0, 1-10, 14, 18, 22, 26, and 28, every week thereafter until day 56, and every 2 weeks thereafter for the remainder of the treatment and recovery phases. Detailed analysis of LH and FSH over the 24-h period was performed by multiple blood sampling on days 0, 1, 10, 28, 56, 84, and 112. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. The mean sperm count declined by 83%, to a nadir of 16.6 +/- 6.2 (+/- SEM) million/ml. One subject had no significant decrease in sperm count. Azoospermia was not achieved in any subject. Basal serum LH concentrations, after an early phase of stimulation, declined to near baseline by day 14. However, basal, 24-h integrated serum LH concentrations, and 24-h urinary LH excretion were not significantly lowered by combined treatment. Bioassayable serum LH concentrations, however, declined significantly from 20.4 +/- 6.3 to 4.5 +/- 0.5 mIU/ml, and the bioassayable to immunoassayable LH ratio decreased from 2.1 +/- 1.0 to 0.7 +/- 0.1 after 16 weeks of GnRH-A treatment. Basal and 24-h integrated FSH concentrations, after an initial period of stimulation, declined progressively to baseline by days 5-6 and were significantly below baseline by day 112. Serum T concentrations did not fall into the hypogonadal (less than 250 ng/dl) range in any subject at any time during the treatment period. After discontinuation of treatment, LH, FSH, and sperm counts returned to normal in all subjects. Thus, single daily injection of GnRH-A and T failed to predictably induce azoospermia in normal men over the 16-week treatment period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human immunodeficiency virus (HIV) and HIV infected cells in saliva and salivary glands of a patient with systemic lupus erythematosus.

A young woman with systemic lupus erythematosus (SLE) was infected with human immunodeficiency virus 1 (HIV-1) and about 6 years later developed persistent bilateral parotid gland enlargement. It was unclear whether this represented salivary gland involvement as a component of her SLE (secondary Sj?gren's syndrome) or the initial clinical manifestation of her HIV-1 infection. HIV proviral DNA was found in individual salivary glandular secretions and in whole saliva. Additionally, cells positive for HIV RNA were isolated from whole saliva. A parotid gland biopsy revealed infiltrating lymphocytes containing large amounts of HIV RNA.     

Isolation of infectious human T-cell leukemia/lymphotropic virus type III (HTLV-III) from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) and from healthy carriers: a study of risk groups and tissue sources.

Acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) are thought to be caused by human T-cell leukemia/lymphotropic virus type III (HTLV-III). Since the fall of 1982, independent isolates of HTLV-III have been obtained in this laboratory, in collaboration with several clinical groups, from 101 AIDS and ARC patients and healthy donors at risk for AIDS. Most isolates were from peripheral blood T lymphocytes established in cell culture, but some were obtained from bone marrow, lymph node, brain tissue, and cell-free plasma and from cells associated with saliva, cerebrospinal fluid, and semen. Virus was isolated from approximately 50% of AIDS patients, 85% of ARC patients, and 30% of healthy individuals at risk for AIDS. The risk groups included homosexuals, promiscuous heterosexuals, i.v. drug users, recipients of blood or blood products, and spouses and offspring of AIDS patients and others at risk for AIDS. A high correlation was seen between persistent levels of serum antibody and the ability to isolate virus from patient or donor leukocytes. Immunologic and nucleic acid analysis demonstrated that the virus isolates were highly related, although substantial diversity was observed in the restriction enzyme cleavage patterns of those studied in detail. Biological analysis of cells from infected patients and donors as well as from normal peripheral blood mononuclear cells exposed to virus in vitro demonstrated that OKT4/Leu3a+ (helper/inducer) lymphocytes were preferentially infected and were subjected to a characteristic cytopathic effect. The availability of multiple isolates of virus from a number of different patients and donors will greatly facilitate the characterization of HTLV-III and the study of possible biological and/or biochemical variants of the virus responsible for the development of AIDS, ARC, and related diseases.     

The Effect of Iron(III) on the Activity of Selenoenzymes and Oxidative Damage in the Liver of Rats. Interaction with Natural Antioxidants and Deferiprone

Iron is an essential element which, under certain conditions, can have prooxidant and cancerogenic effects. The effect of iron and iron chelators on the activity of selenoenzymes has been studied. Acute experiments in male Wistar rats demonstrated the prooxidant effect of iron on the selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). These enzymes represent an important part of the antioxidant defense system. Deferiprone (L1) has been shown to abolish the stimulating effect of iron on lipid peroxidation and reduced glutathione (GSH) levels, and also to inhibit the influence of iron on the activity of TrxR and GPx. Similarly, the flavonoid quercetin abolished the influence of iron on the activity of TrxR. The activity of both selenoenzymes has also been shown to be stimulated using L1, naringin (NAR), quercetin (QUE) and myricetin (MYR) in the absence of iron. Further studies including the combination of synthetic and natural compounds (e.g., flavonoids) are being considered for their possible development and clinical use in antioxidant therapies.     

Soluble Fc gamma receptor III (CD 16) and eicosanoid concentrations in gut lavage fluid from patients with inflammatory bowel disease: reflection of mucosal inflammation.

BACKGROUND--Activated neutrophils cause tissue injury in inflammatory bowel disease (IBD). Upon activation, they shed soluble Fc gamma IIIb receptors (sFc gamma RIIIb). The subsequent inflammatory response is modulated by several mediators, including neutrophil derived leukotriene B4 (LTB4), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2). The aim of this study was to determine the value of gut lavage sFc gamma RIII and eicosanoid measurements for the assessment of mucosal inflammation in IBD. METHODS--A total of 18 patients with active IBD, 10 ulcerative colitis (UC), and eight Crohn's disease (CD), and 12 control patients underwent whole gut lavage. Disease activity, endoscopic appearance, and histopathology were graded. Samples were processed for the determination of sFc gamma RIIIb, LTB4, PGE2, and TXB2. RESULTS--Soluble Fc gamma RIIIb concentrations were increased in both IBD groups. Significant correlations were seen between sFc gamma RIIIb and LTB4 values with histology scores. Mean eicosanoid lavage fluid concentrations in control patients were 14.1 pg/ml for LTB4, 5.6 pg/ml for PGE2, and 397 pg/ml for TXB2. Concentrations of all eicosanoids in IBD patients were significantly increased: LTB4 in UC: mean 73.2 pg/ml, in CD: 96.4 pg/ml (both p < 0.01 v controls). PGE2 in UC: 20.2 pg/ml, in CD: 43.4 pg/ml (p < 0.01). TXB2 in UC: 719.3 pg/ml, in CD: 180.6 pg/ml (both p < 0.05). CONCLUSIONS--Whole gut lavage fluid analysis is an effective method to study mucosal eicosanoid production. Soluble Fc gamma RIIIb concentrations in gut lavage fluid closely correlate with histological signs of mucosal inflammation and with lavage LTB4 concentration. These data suggest that lavage Fc gamma RIIIb assessment may be used as a simple assay to estimate mucosal neutrophil infiltration in IBD.     

Cytoplasmic transfer of the mtDNA nt 8993 T-->G (ATP6) point mutation associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio.

A point mutation in the mtDNA-encoded ATP6 gene (T-->G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP/O ratio) but did not affect 2,4-dinitrophenol (DNP)-uncoupled respiration, suggesting a defective mitochondrial H(+)-translocating ATP synthase. Intact mitochondria isolated from patient and control lymphoblastoid cell lines were tested for state III, ADP-limited (state IV), and DNP-uncoupled respiration with various substrates. Mitochondria isolated from patient lymphoblasts harboring 95-100% of mtDNAs carrying the nt 8993 T-->G mutation showed state III respiration rates 26-50% lower than controls while having normal DNP-uncoupled rates. This resulted in state III/DNP ratios of 0.52-0.70 in patient mitochondria versus 0.88-0.97 in controls. The ADP/O ratio was also decreased 30-40% in patient mitochondria. Patient lymphoblasts heteroplasmic for the nt 8993 mutation were enucleated by using Percoll gradients and the cytoplasts were fused to mtDNA-deficient (rho 0) cells by electric shock. Cybrid clones homoplasmic for the wild-type nucleotide (T) at nt 8993 gave state III/DNP and ADP/O ratios similar to those of control cybrids, whereas cybrid clones homoplasmic for the mutant nucleotide (G) showed a 24-53% reduction in state III respiration, a state III/DNP ratio of 0.53-0.64, and a 30% decrease in the ADP/O ratio. Thus, the reduced state III respiration rates and ADP/O ratios are linked to the T-->G mutation at nt 8993.