HLA class II haplotypes differentiate between the adult autoimmune polyglandular syndrome types II and III.

BACKGROUND: Genetics of the adult autoimmune polyglandular syndrome (APS) is poorly understood. AIM: The aim of this study was to gain further insight into the genetics of the adult APS types. SITE: The study was conducted at a university referral center. METHODS: The human leukocyte antigen (HLA) class II alleles, haplotypes, and genotypes were determined in a large cohort of patients with APS, autoimmune thyroid disease (AITD), and type 1 diabetes and in healthy controls by the consistent application of high-resolution typing at a four-digit level. RESULTS: Comparison of the allele and haplotype frequencies significantly discriminated patients with APS vs AITD and controls. The HLA class II alleles DRB1*03:01 *04:01, DQA1*03:01, *05:01, DQB1*02:01, and *03:02 were observed more frequently (P<.001) in APS than in AITD and controls, whereas the alleles DRB1*15:01, DQB1*03:01, and *06:02 were underrepresented in APS vs AITD (Pc<.001) and controls (Pc<.01), respectively. The DRB1*03:01-DQA1*05:01-DQB1*02:01 (DR3-DQ2) and DRB1*04:01-DQA1*03:01:DQB1*03:02 (DRB1*04:01-DQ8) haplotypes were overrepresented in APS (Pc<.001). Combination of both haplotypes to a genotype was highly prevalent in APS vs AITD and controls (Pc<.001). Dividing the APS collective into those with Addison's disease (APS type II) and those without Addison's disease but including type 1 diabetes and AITD (APS type III) demonstrated DR3-DQ2/DRB1*04:01-DQ8 as a susceptibility genotype in APS III (Pc<.001), whereas the DR3-DQ2/DRB1*04:04-DQ8 genotype correlated with APS II (Pc<.001). The haplotypes DRB1*11:01-DQA1*05:05-DQB1*03:01 and DRB1*15:01-DQA1*01:02-DQB1*06:02 are protective in APS III but not in type II (Pc<.01). CONCLUSIONS: HLA class II haplotypes differentiate between the adult APS types II and III. Susceptible haplotypes favor the development of polyglandular autoimmunity in patients with AITD.

The complement-mediated prozone effect in the Luminex single-antigen bead assay and its impact on HLA antibody determination in patient sera.

The Luminex xMAP system has become an important tool for HLA antibody screening and identification in sera of transplant patients. Recently, the Luminex single antigen bead assay was shown to be prone to an artefact, the so called prozone phenomenon: Sera with high titer HLA antibodies gave negative results when tested neat, but reacted strongly positive after 1:10 dilution. We also observed such a phenomenon and found that it was most likely caused by the complement component 1 (C1) by competitively displacing the detection antibodies. In this article we review the complement-mediated prozone effect and other mechanisms of interference with solid phase assays, and we discuss possible consequences for HLA antibody testing with the Luminex SAB assay.

Outcomes of surveillance for contralateral breast cancer in patients less than age 60 at the time of initial diagnosis.

BACKGROUND: After an initial diagnosis of breast cancer, the risk of contralateral breast cancer is approximately 0.5% per year. Annual mammography is recommended to identify local recurrences and contralateral new primaries. Because the sensitivity of mammography tends to be lower in younger women, we conducted a retrospective review of the method of detection and pathologic stage of metachronous contralateral primary breast cancers according to age at diagnosis in a cohort of breast cancer patients. METHODS: The Henrietta Banting Database contains information on cases of breast cancer diagnosed at Women's College Hospital from 1987 to 2004. From among 1992 women in the database, 71 patients were identified who were initially diagnosed before age 60 and who subsequently developed a contralateral breast cancer. Medical records were obtained for 53 of the 71 patients. RESULTS: Of the 53 contralateral cancers, 33 (62%) were detected by mammography, including 4 in 16 patients (25%) diagnosed before age 50 and 29 in 37 patients (78%) diagnosed at age 50 or older (p ≤ 0.001). CONCLUSIONS: Mammography has poor sensitivity for the surveillance of contralateral breast cancer in early-onset breast cancer patients. Other imaging modalities should be evaluated in this setting.

Autoimmune polyglandular syndrome type 2 shows the same HLA class II pattern as type 1 diabetes.

Autoimmune polyglandular syndrome (APS) type 2 is defined by the manifestation of at least two autoimmune endocrine diseases. Only few data exist on genetic associations of APS type 2. In this controlled study, 98 patients with APS type 2, 96 patients with type 1 diabetes (T1D), and 92 patients with autoimmune thyroid disease, both as a single autoimmune endocrinopathy, were tested for association with alleles of the human leukocyte antigen (HLA) class II loci DRB1, DQA1, and DQB1. Patients with APS type 2 had significantly more often the alleles DRB1*03 (P(c) < 0.0001), DRB1*04 (P(c) < 0.000005), DQA1*03 (P(c) < 0.0001), and DQB1*02 (P(c) < 0.05), when compared with controls. Less frequent in APS were DRB1*15 (P(c) < 0.05), DQA1*01 (P(c) < 0.0005), and DQB1*05 (P(c) < 0.005). With regard to frequency and linkage of these alleles, the susceptible haplotypes DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*0401/04-DQA1*0301-DQB1*0302 were deduced. Protective haplotypes in this study were DRB1*1501-DQA1*0102-DQB1*0602 and DRB1*0101-DQA1*0101-DQB1*0501. Comparing APS patients with vs without AD, no significant differences regarding HLA class II alleles were noted in our collective. Patients with T1D as a singular disease had the same susceptible and protective HLA alleles and haplotypes. The prevalence of DRB1*03 and DRB1*04 in APS patients was not because of the presence of diabetes, as the APS type 2 patients without diabetes had the same allele distribution. In conclusion, these data suggest a common immunogenetic pathomechanism for T1D and APS type 2, which might be different from the immunogenetic pathomechanism of other autoimmune endocrine disease.

Upper respiratory tract infection in athletes: influence of lifestyle, type of sport, training effort, and immunostimulant intake.

Epidemiological evidence suggests that heavy acute or chronic exercise is related to an increased incidence of upper respiratory tract infections in athletes, while moderate exercise is believed to be protective. During the past years, many groups have investigated the association between changes within the immune system and exercise at different intensity levels. Although following strenuous exercise, some immunologic alterations were quite consistent and reproducible, e.g. neutrophilia, lymphopenia, and depression of natural killer cell activity, some findings were divergent or strongly dependent on the study design and athletes investigated. Lately, interesting results in the field of psychoneuroimmunolgy as well as new insights in the relationship between macro- and micronutrient and the immune system have brought up new fields of research interest. There is growing evidence that e.g. lifestyle factors, the coping with daily stress, and dietary behavior are important cofactors in the immune response to exercise. The present work gives a short review on the literature dealing with URTI in athletes with special reference to the above mentioned cofactors. In addition, the results of a recent investigation concerning training and associated lifestyle patterns in German athletes are presented.

HSP expression in human leukocytes is modulated by endurance exercise.

PURPOSE: Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression. METHODS: The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control. RESULTS: After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01). CONCLUSIONS: Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training.

Similarities and differences of the immune response to exercise and trauma: the IFN-gamma concept.

Similar to physical fitness, fitness of the immune system requires training. Animals that have been raised under sterile conditions have a poor immune system and fail to thrive. "Immune training" is normally provided by contact with live microorganisms or immunizations. Increasing evidence has suggested that moderate sports can decrease the frequency of infections while excessive, exhausting exercise can lead to the opposite, a situation that has been described by a J-curve. Following prolonged exhausting exercise, a transient partial suppression of several immune functions can be shown, and it has been suggested that this period provides a window for invasion of microbes. On the basis of data showing that endotoxin-inducible interferon-gamma (IFN-gamma) production is virtually abrogated for a short period following excessive exercise, we present the hypothesis that the rigorous regulatory blockade of one of the ways of IFN-gamma induction may be critically involved in causing the transient immunosuppression following exhaustive exercise stress.

Zinc, iron, and magnesium status in athletes--influence on the regulation of exercise-induced stress and immune function.

Intense physical exercise has been shown to be associated with immunosuppression and increased rate of infection. The immunosuppressive effect of exhaustive exercise has been attributed to a reduced helper/suppressor T-cell ratio, low salivary levels of immunoglobulin-A, decreased lymphocyte proliferative response and natural killer cell activity, and elevation of stress hormones. Yet some athletes can withstand intense training periods without health problems while others are prone to infections. Thus it has been postulated that other factors may interfere with immunoregulation. The notion that macro- and micronutrients are involved in the regulation of immunological processes and the ability to cope with muscular and systemic exercise stress has been gaining attention. Particularly trace elements have been shown to be related to cell mediated and humoral immunity such as NK-cell activity, T- and B-cell functions, and cytokine release. Many investigations have reported decreased concentrations of trace elements in blood and tissues after training and competition. However, the magnitude of losses is highly dependent on the type and intensity of exercise, the individual regulatory state, and most important, nutrition. This paper reviews the data on zinc, iron, and magnesium status in athletes and summarizes the consequences of deficiencies in these trace elements regarding exercise tolerance and immune function. These elements were chosen since there is evidence they are related to exercise-induced stress and immune function.

Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153.

In the present study, we show that Fas receptor ligation or cellular treatment with synthetic C6-ceramide results in activation or phosphorylation, respectively, of the small G-protein Rac1, Jun N-terminal kinase (JNK)/p38 kinases (p38-K), and the transcription factor GADD153. A signaling cascade from the Fas receptor via ceramide, Ras, Rac1, and JNK/p38-K to GADD153 is demonstrated employing transfection of transdominant inhibitory N17Ras, N17Rac1, c-Jun, or treatment with a specific p38-K inhibitor. The critical function of this signaling cascade is indicated by prevention of Fas- or C6-ceramide-induced apoptosis after inhibition of Ras, Rac1, or JNK/p38-K.

Effect of exhaustive exercise stress on the cytokine response.

Fifteen athletes were investigated 24 h before, 1 h after, and 20 h after an exhaustive exercise stress test (mean duration 68 min). Testing for cytokines was done in serum, urine, and the supernatants of whole blood cell cultures, which were stimulated with lipopolysaccharide (LPS), concanavalin A (Con A), or phythaemagglutinin (PHA). Elevated levels of interleukin 6 (IL-6) and soluble IL-2 receptor (sIL-2R) were found 1 h after the run in both serum and urine samples. TNF-alpha in serum was also increased, whereas IL-2 in urine was decreased after the exercise. All other testings in serum and urine (including IFN-gamma) gave borderline or negative results. In cell cultures, the LPS-induced release of the inflammatory cytokines TNF-alpha, IL-1, and IL-6 was suppressed 1 h after exercise. Also, the Con-A-induced and LPS-induced release of IFN-gamma, and the PHA-induced release of IL-2 were suppressed 1 h after exercise. In contrast, Con-A-induced release of IL-2 was mildly increased after the run. We conclude that exercise of the intensity and duration described here causes an activation of the immune system, which is immediately counter-regulated. Twenty hours after the exercise, most of the observed changes were back to pre-exercise levels, indicating only a short duration for this suppressive counter-regulation.

Essential fatty acids, immune function, and exercise.

The immunologic response to exercise comprises numerous alterations within the immune system, but how these processes are regulated is still largely unknown. Exercise-related immunological changes include signs of inflammation, such as release of inflammatory mediators, activation of various white blood cell lines and complement, and induction of acute phase proteins. Nevertheless, signs of immunosuppression, such as decreased T and B cell function or impaired cytotoxic or phagocytic activity, can also be observed. Some data suggest that essential fatty acids help regulate inflammatory processes, modulating both cytokine release and the acute phase response. Positive effects of changing dietary essential fatty acids have been demonstrated in chronic inflammatory diseases. In contrast, little is known about the contribution of fatty acids to the exercise-induced immunologic reaction. Essential fatty acids may determine alterations within the immune system following exercise. Therefore, future studies are necessary to evaluate the influence of the fatty acid composition on the inflammatory or immunosuppressive component following heavy exertion.

Impaired production of cytokines in a case of human leishmaniasis.

A patient presented with the unique clinical picture of diffuse cutaneous and mucosal leishmaniasis caused by Leishmania tropica. Elevated serum levels of several cytokines including interleukin (IL) 2, interferon gamma (IFN-gamma), and tumor necrosis factor alpha were found. All cytokine levels returned to normal during therapy. No IL-10 or IL-4 levels were detectable. In whole blood cultures, induction of IFN-gamma by lipopolysaccharide (LPS) was completely negative, even after therapy. Concanavalin A (Con A)-induced release of IFN-gamma, like Con A-induced release of the other cytokines, was only initially impaired but returned to normal during therapy. Induction of the other cytokines by LPS was never impaired. The low expression of human leukocyte antigen DR on monocytes increased during IFN-gamma therapy but dropped when IFN-gamma treatment was ceased. We conclude that in this patient one or more of the routes of IFN-gamma production was impaired, thus resulting in insufficient IFN-gamma production in the infected lesions (although IFN-gamma was systemically present).

The cytokine response to strenuous exercise.

Several groups have now investigated the cytokine response to strenuous exercise. In this article we try to summarize known data on this topic. Significant, albeit mild increases in plasma levels of the monokines IL-1, TNF-alpha, IL-6, and of soluble IL-2 receptor have been reported following strenuous exercise. Increased excretion of cytokines after exercise can also be shown in the urine of athletes. Modulation of cytokine release by strenuous exercise can also be demonstrated using in vitro cell cultures. Several authors have shown an increase in endotoxin-stimulated monokine release following exercise. In contrast, using whole blood cultures we found strongly depressed production of interferon gamma (in response to mitogen or endotoxin) following strenuous exercise. The potential significance of cytokine modulation for exercise-related immunological problems is discussed.

Prevention of endotoxin-induced monokine release by human low- and high-density lipoproteins and by apolipoprotein A-I.

Interaction of endotoxin (lipopolysaccharide [LPS]) with human lipoproteins is known to prevent the LPS-induced activation of human monocytes and release of cytokines (monokines). LPS was exposed to lipoprotein classes separated by ultracentrifugation and to apolipoprotein A-I. Then monocytes were added, and the LPS activation of monocytes was determined by measuring the induced monokines. Failure of LPS to induce monokine release was called LPS inactivation caused by lipoproteins or apolipoproteins. The LPS inactivation is shown to be a function of low-density lipoproteins. High-density lipoproteins inactivate LPS to a much lesser extent. The very-low-density lipoproteins cannot inactivate LPS. Lipid components seemed not absolutely required for LPS inactivation, because purified human apolipoprotein A-I without its physiological lipid complement also inhibits LPS-induced monokine release.

Production of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 in the perfused rat liver.

The kinetics of the production and release of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6) were investigated in the perfused rat liver and in primary cultures of Kupffer cells after stimulation with lipopolysaccharide (LPS). A small and transient accumulation of TNF-alpha could be detected immunohistochemically and by cytotoxicity assay in the intracellular space about 1 h after addition of LPS to the cultured cells. TNF-alpha release in the perfused liver followed similar kinetics as those found in the serum of LPS-treated rats and in primary cultures of rat Kupffer cells. The cytotoxic TNF-alpha activity of the perfusate attained its maximum (11.5 +/- 2.6 U/ml) 90 min after LPS stimulation and remained nearly constant for further 150 min. 2 microM dexamethasone reduced the production of TNF-alpha by 10 g of liver during 240 min from 46 to 16 x 10(3) units. The production of IL-1 and IL-6 by 10 g of liver during the initial 240 min was 3 and 530 x 10(3) IU, respectively. The maximal concentrations of IL-1 (1.4 +/- 0.7 IU/ml) and IL-6 (157 +/- 60 IU/ml) were found 240 min after LPS addition. The production of IL-1 was totally suppressed by 2 microM dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of lipoproteins in the inactivation of endotoxin by serum.

The lipopolysaccharide (LPS)-induced release of cytokines from human monocytes can be prevented by previous interaction of serum with LPS. This inactivation is a function of lipoproteins. Here we show that LPS can be inactivated by low-density lipoproteins (LDL) as well as by high-density lipoproteins (HDL). The effects of heparin and EDTA on LPS inactivation by serum are also described.

Low density lipoproteins inhibit endotoxin activation of monocytes.

Human serum and low density lipoproteins (LDLs) were shown to inactivate endotoxin (lipopolysaccharide [LPS]) by testing the effect of LPS interactions with serum or LDL on the activation of human monocytes. Sera and LDL preparations from four patients with familial hypercholesterolemia were used to demonstrate the inhibition of LPS from inducing interleukin-1 release. Before LDL removal by immunoapheresis, the patients' sera were able to inactive approximately fivefold more LPS than after LDL removal. The LPS-inactivating capacity lost during apheresis could essentially be retrieved in the LDL-rich eluate from the immunoadsorption columns. Because patients were treated frequently with immunoapheresis, their LDL levels before LDL removal were not markedly elevated. These patients' sera before LDL removal were shown to inactivate amounts of LPS comparable to those inactivated by the sera from three healthy volunteers. LDL prepared by ultracentrifugation showed similar LPS inactivation as LDL prepared by immunoapheresis. We conclude that the inhibition of LPS-induced monocyte activation by human serum is dependent to a large extent on the LDL fraction. LDLs were demonstrated to inhibit LPS from inducing interleukin-1 release by human monocytes.

The role of lipoproteins in inactivation of endotoxin by serum.

Lipopolysaccharide (LPS)-induced release of cytokines from human monocytes can be prevented by previous interference of serum with LPS. This inactivation is a function of lipoproteins. Here we show that low-density lipoproteins as well as high-density lipoproteins can inactivate LPS. The effects of heparin and EDTA on LPS inactivation by serum are also described.

Development of cell surface linkage complexes in cultured fibroblasts.

The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identified by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-to-extracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular alpha-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, alpha-actinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface alpha-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membrane-associated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.