Bisphenols, but not phthalate esters, modulate gene expression in activated human MAIT cells in vitro.

One route of human exposure to environmental chemicals is oral uptake. This is primarily true for chemicals that may leach from food packaging materials, such as bisphenols and phthalate esters. Upon ingestion, these compounds are transported along the intestinal tract, from where they can be taken up into the blood stream or distributed to mucosal sites. At mucosal sites, mucosal immune cells and in the blood stream peripheral immune cells may be exposed to these chemicals potentially modulating immune cell functions. In the present study, we investigated the impact of three common bisphenols and two phthalate esters on mucosal-associated invariant T (MAIT) cells in vitro, a frequent immune cell type in the intestinal mucosae and peripheral blood of humans. All compounds were non-cytotoxic at the chosen concentrations. MAIT cell activation was only slightly affected as seen by flow cytometric analysis. Phthalate esters did not affect MAIT cell gene expression, while bisphenol-exposure induced significant changes. Transcriptional changes occurred in ∼ 25 % of genes for BPA, ∼ 22 % for BPF and ∼ 8 % for BPS. All bisphenols down-modulated expression of CCND2, CCL20, GZMB and IRF4, indicating an effect on MAIT cell effector function. Further, BPA and BPF showed a high overlap in modulated genes involved in cellular stress response, activation signaling and effector function suggesting that BPF may not be safe substitute for BPA.

MAIT cell activation is reduced by direct and microbiota-mediated exposure to bisphenols.

Oral uptake is the primary route of human bisphenol exposure, resulting in an exposure of the intestinal microbiota and intestine-associated immune cells. Therefore, we compared the impact of bisphenol A (BPA), bisphenol F (BPF) and bisphenol S (BPS) on (i) intestinal microbiota, (ii) microbiota-mediated immunomodulatory effects and (iii) direct effects on mucosal-associated invariant T (MAIT) cells in vitro. We acutely exposed human fecal microbiota, Bacteroides thetaiotaomicron and Escherichia coli to BPA and its analogues BPF and BPS referring to the European tolerable daily intake (TDI), i.e. 2.3 µg/mL, 28.3 µg/mL and 354.0 µg/mL. Growth and viability of E. coli was most susceptible to BPF, whereas B.thetaiotaomicron and fecal microbiota were affected by BPA > BPF > BPS. At 354.0 µg/mL bisphenols altered microbial diversity in compound-specific manner and modulated microbial metabolism, with BPA already acting on metabolism at 28.3 µg/mL. Microbiota-mediated effects on MAIT cells were observed for the individual bacteria at 354.0 µg/mL only. However, BPA and BPF directly modulated MAIT cell responses at low concentrations, whereby bisphenols at concentrations equivalent for the current TDI had no modulatory effects for microbiota or for MAIT cells. Our findings indicate that acute bisphenol exposure may alter microbial metabolism and impact directly on immune cells.

Lifetime prevalence and determinants of hand eczema in an adolescent population in Germany: 15-year follow-up of the LISA cohort study.

BACKGROUND: Hand eczema is a common inflammatory skin disorder in both adolescence and adulthood. OBJECTIVES: We sought to assess the lifetime prevalence of hand eczema and associated exogenous and endogenous risk factors among adolescents in Germany. METHODS: This was a cross-sectional study embedded into a prospective population-based birth cohort in four regions of Germany, which recruited healthy neonates born between November 1997 and January 1999. We included 1736 participants who had completed the 15-year follow-up from birth cohort and 84.6% (1468/1736) had clearly reported whether they have ever had hand eczema. All the data were based on questionnaires and blood tests (immunoglobulin E). Multivariable logistic regression analysis was used to examine endogenous and exogenous factors in relation to the lifetime prevalence of hand eczema among adolescents. RESULTS: One thousand four hundred and sixty-eight adolescents (715 girls, 48.7%) were included in the final analysis. The lifetime prevalence of hand eczema among adolescents at the age of 15 was 10.4% (95% confidence interval [CI]: 8.9%-12.1%), with a significantly higher lifetime prevalence among girls than boys (12.7% vs. 8.2%, P = 0.005). Multivariable logistic regression analysis indicated statistically significant associations between the lifetime prevalence of hand eczema and having ever been diagnosed with atopic dermatitis (aOR = 1.8, 95% CI: 1.1-2.8) or having ever had dry skin (aOR = 1.9, 95% CI: 1.1-3.1), respectively. No statistically significant independent associations were found between asthma, hay fever, allergy-related clinical symptoms, immunoglobulin E positivity and other exogenous factors in relation to hand eczema. CONCLUSION: Our study fills a research gap on the epidemiological burden of hand eczema among adolescents. One out of ten ever suffered from hand eczema until age 15 years indicating that hand eczema constitutes a significant burden in paediatric populations. The role of atopic dermatitis in hand eczema reinforces previous findings. Exogenous risk factors warrant further investigation.

Conocimiento y prácticas relacionadas con control y prevención de COVID-19 en trabajadores de la salud / Knowledge and practice regarding COVID-19 infection, control and prevention among healthcare workers

La enfermedad por coronavirus 2019 (COVID-19) ha afectado a los trabajadores de la salud (TS) quienes suman riesgo de exposición en la comunidad y el trabajo. El conocimiento y preparación son fundamentales, sin embargo, durante la pandemia se han suspendido las actividades presenciales de formación-capacitación. El objetivo de este estudio fue determinar si la educación en línea genera una diferencia en el conocimiento y práctica de prevención y control de infecciones (PCI) para la COVID-19 en TS. Previo consentimiento, se administró un cuestionario antes-después a los participantes de un curso de PCI en línea. Para la evaluación de conocimientos y prácticas se diseñaron preguntas tipo Likert con valores de 1 a 5, realizando el análisis con la prueba no paramétrica de rangos con signo de Wilcoxon para muestras relacionadas. En 345 participantes, la mediana fue 30 (RIC 15) años, femenino (224, 65%), residentes en el departamento de Guatemala (221, 57%), labora en sector público (155, 44.9%), sector privado (154, 44.6%), y seguro social (29, 8.4%). La evaluación antes-después mostró diferencia significativa de la brecha existente en el conocimiento y las prácticas de PCI (p < .05). La mayor brecha se observó en el autocuidado de la salud física, mental y nutricional. En las percepciones, uno de cada tres encuestados manifestó temor a sufrir estigma en caso de resultar infectados. La educación y entrenamiento en PCI es esencial ante una enfermedad altamente contagiosa que amenaza la salud y seguridad de los TS, principalmente en entornos sanitarios con recursos limitados.

The coronavirus disease 2019 (COVID-19) has affected health workers (HCWs) who adds risk of exposure in the community and at work. Knowledge and preparation are essential. However, during the pandemic, face-to-face training activities have been suspended. The objective of this study was to determine if online education generate a difference in the knowledge and practice of infection prevention and control (IPC) of COVID-19 in HCWs. With prior consent, a before-after questionnaire was administered to participants of an online PCI course. For the evaluation of knowledge and practices, Likert-type questions were designed with values from 1 to 5, performing the analysis with the non-parametric test of Wilcoxon signed ranges for related samples. In 345 participants, the median was 30 (IQR 15) years, female (224, 65%), living in the department of Guatemala (221, 57%), working in the public sector (155, 44.9%), private sector (154, 44.6%), and social security (29, 8.4%). The before-after assessment showed a significant difference in the gap between knowledge and practices. The largest gap was observed in self-care of physical, mental, and nutritional health. About perceptions, one out of every three respondents expressed fear of suffering stigma if they get infected. Education and training in IPC are essential in the face of a highly contagious disease that threatens the health and safety of HCWs, mainly in healthcare settings with limited resources.

MAIT cell activation in adolescents is impacted by bile acid concentrations and body weight.

Bile acids (BAs) are produced by liver hepatocytes and were recently shown to exert functions additional to their well-known role in lipid digestion. As yet it is not known whether the mucosal-associated invariant T (MAIT) cells, which represent 10-15% of the hepatic T cell population, are affected by BAs. The focus of the present investigation was on the association of BA serum concentration with MAIT cell function and inflammatory parameters as well as on the relationship of these parameters to body weight. Blood samples from 41 normal weight and 41 overweight children of the Lifestyle Immune System Allergy (LISA) study were analyzed with respect to MAIT cell surface and activation markers [CD107a, CD137, CD69, interferon (IFN)-γ, tumor necrosis factor (TNF)-α] after Escherichia coli stimulation, mRNA expression of promyelocytic leukemia zinc finger protein (PLZF) and major histocompatibility complex class I-related gene protein (MR1), the inflammatory markers C-reactive protein (CRP), interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1α as well as the concentrations of 13 conjugated and unconjugated BAs. Higher body weight was associated with reduced MAIT cell activation and expression of natural killer cell marker (NKp80) and chemokine receptor (CXCR3). BA concentrations were positively associated with the inflammatory parameters CRP, IL-8 and MIP-1α, but were negatively associated with the number of activated MAIT cells and the MAIT cell transcription factor PLZF. These relationships were exclusively found with conjugated BAs. BA-mediated inhibition of MAIT cell activation was confirmed in vitro. Thus, conjugated BAs have the capacity to modulate the balance between pro- and anti-inflammatory immune responses.

Stressful life events in childhood and allergic sensitization.

Stressful life events evidently have an impact on development of allergic diseases, but the mechanism linking stress to pathological changes of immune system function is still not fully understood. The aim of our study was to investigate the relationship between stressful life events, neuropeptide and cytokine concentrations in children as well as the association between early stressful life events and atopic eczema (AE). Within the LISA plus (Life style - Immune system - Allergy) study, blood samples from children of 6 years of age were analyzed for concentration of the neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM), substance P (SP) and the Th1/Th2 cytokines IFN-γ and IL-4. Life events such as severe disease or death of a family member, unemployment or divorce of the parents were assessed with a questionnaire filled in by the parents. Furthermore, lifetime prevalence of AE and incidence after the assessment period of life events were compared. Our data suggest that separation/ divorce of parents increase childrens risk of developing AE later in life. Children with separated/divorced parents showed high VIP levels and high concentrations of the Th2 cytokine IL-4 in their blood. Severe diseases and death of a family member were neither associated with neuropeptide levels nor with cytokine concentrations. Unemployment of the parents was associated with decreased IFN-γ concentrations in childrens blood but not with neuropeptide levels. Thus, the neuropeptide VIP might be a mediator between stressful life events and immune regulation contributing to the Th2-shifted immune response in children with separated/divorced parents.

Maternal cytokine status may prime the metabolic profile and increase risk of obesity in children.

BACKGROUND: The maternal inflammation status during pregnancy has been associated with metabolic imprinting and obesity development in the child. However, the influence of the maternal Th2 cytokines, interleukin-4 (IL4), IL5 and IL13, has not been studied so far. METHODS: We investigated the relationship between maternal innate (IL6, IL8, IL10 and tumor necrosis factor-α (TNFa)) and adaptive (interferon-γ, IL4, IL5 and IL13) blood cytokine levels at 34 weeks of gestation and children's overweight development until the age of 3 years in 407 children of the German longitudinal LINA (Lifestyle and Environmental Factors and their Influence on Newborns Allergy risk) cohort. Children's body weight and height were measured during the annual clinical visits or acquired from questionnaires. Body mass index (BMI) Z-scores were calculated according to the WHO reference data to adjust for child's age and gender. Cytokine secretion was stimulated with phytohemagglutinin or lipopolysaccharide and measured by cytometric bead assay. Furthermore, we assessed metabolic parameter in blood of 318 children at age 1 using the AbsoluteIDQ p180 Kit (Biocrates LIFE Science AG). RESULTS: Applying logistic regression models, we found that an increase of maternal IL4 and IL13 was associated with a decreased risk for overweight development in 1- and 2-year-old children. This effect was consistent up to the age of 3 years for IL13 and mainly concerns children without maternal history of atopy. Children's acylcarnitine concentrations at 1 year were positively correlated with maternal IL13 levels and inversely associated with the BMI Z-score at age 1. CONCLUSIONS: We were able to show for the first time that the maternal Th2 status may be linked inversely to early childhood overweight development accompanied by an altered metabolic profile of the fetus. However, our data do not support a direct mediating role of acylcarnitines on maternal IL13-induced weight development.

Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study.

BACKGROUND: Vitamin D levels are known to be associated with atopic disease development; however, existing data are controversial. The aim of this study was to investigate whether corresponding maternal and cord blood vitamin D levels are associated with atopic outcomes in early infancy. METHODS: Within the LINA cohort study (Lifestyle and environmental factors and their Influence on Newborns Allergy risk), 25(OH)D was measured in blood samples of 378 mother-child pairs during pregnancy and at birth. Information about children's atopic manifestations during the first 2 years of life was obtained from questionnaires filled out by the parents during pregnancy and annually thereafter. Cord blood regulatory T cells (Treg) were detected by methylation-specific PCR using a Treg-specific demethylated region in the FOXP3 gene. RESULTS: The median maternal 25(OH)D(3) level was 22.19 ng/ml (IQR 14.40-31.19 ng/ml); the median cord blood 25(OH)D(3) 10.95 ng/ml (6.99-17.39 ng/ml). A high correlation was seen between maternal and cord blood 25(OH)D(3) levels, both showing a seasonal distribution. Maternal and cord blood 25(OH)D(3) was positively associated with children's risk for food allergy within the first 2 years. Further, higher maternal 25(OH)D(3) resulted in a higher risk for sensitization against food allergens at the age of two. Cord blood 25(OH)D(3) levels were negatively correlated with regulatory T cell numbers. CONCLUSION: Our study demonstrates that high vitamin D levels in pregnancy and at birth may contribute to a higher risk for food allergy and therefore argues against vitamin D supplement to protect against allergy.

The LINA cohort: indoor chemical exposure, circulating eosinophil/basophil (Eo/B) progenitors and early life skin manifestations.

BACKGROUND: Hematopoietic progenitor cells, especially those committed to the Eo/B lineage, are known to contribute to allergic inflammation. OBJECTIVE: The aim of the present study was to investigate whether environmental factors are associated with changes in numbers of circulating Eo/B progenitors at 1 year of age. METHODS: Peripheral blood from 60 1-year-old children enrolled in the LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) birth cohort was assessed for Eo/B progenitor cells (Eo/B CFU) using standardized and validated methylcellulose assays. Frozen peripheral blood mononuclear cells (PBMC) were cultured in the presence of IL-3, IL-5 or GM-CSF, and Eo/B CFUs enumerated. Clinical outcomes and exposure to environmental tobacco smoke (ETS) were documented by standardized questionnaires, and indoor volatile organic compound (VOC) concentrations were assessed by passive sampling. RESULTS: Children with skin manifestations (atopic dermatitis or cradle cap) within the first year of life had higher numbers of circulating IL-3-, IL-5- or GM-CSF-stimulated Eo/B CFUs (P < 0.05) at 1 year. In children with cradle cap, a positive correlation was found between Eo/B CFUs and exposure to ETS-related VOCs during pregnancy or at 1 year of age (P < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: This is the first demonstration that environmental exposures are positively associated with levels of circulating Eo/B progenitors. The recruitment and differentiation of Eo/B progenitors in response to environmental triggers may play a role in the development of skin manifestations during the first year of life.

Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.

BACKGROUND: Regulatory T cells (Tregs) with stable FOXP3 expression are characterized by a specific demethylated region in the FOXP3 gene (Treg-specific demethylated region, TSDR). The aim of this study was to analyse the influence of prenatal factors on cord blood Treg numbers, as detected by changes in the TSDR demethylation, and the subsequent risk for allergic diseases. METHODS: Analyses were performed within the LINA study in blood samples from pregnant women (34th gestational week) and in cord blood (n = 346 mother-child pairs). Treg numbers were detected via DNA demethylation in the FOXP3 TSDR. At age 1, total and specific IgE was measured in children's blood. In addition, maternal cytokine production (Th1/Th2/Th17) was analysed. Exposure and disease outcomes were assessed by questionnaires. RESULTS: Boys had lower Treg numbers compared with girls (P < 0.001). Parental atopy history, particularly maternal hay fever and paternal asthma were related to lower Treg numbers in cord blood (adj. MR = 0.81, 95% CI = 0.68-0.97; adj. MR = 0.60, 95% CI = 0.45-0.81). Maternal cytokines (IL-13, IL-17E and IFN-γ) and maternal smoking/exposure to tobacco smoke during pregnancy were also associated with decreased cord blood Treg numbers (adj. MR = 0.89, 95% CI = 0.97-1.00). Children with lower Treg numbers at birth had a higher risk to develop atopic dermatitis (adj. OR = 1.55, 95% CI = 1.00-2.41) and sensitization to food allergens (adj. OR = 1.55, 95% CI = 1.06-2.25) during the first year of life. CONCLUSIONS: These results indicate that both genetic and environmental factors presumably influence the development of foetal Tregs. Low cord blood Treg numbers may predict early atopic dermatitis.

Maternal immune status in pregnancy is related to offspring's immune responses and atopy risk.

BACKGROUND: The influence of maternal immune responses in pregnancy on children's immune competence and the development of atopic diseases later in life are poorly understood. To determine potential maternal effects on the maturation of children's immune system and resulting disease risks, we analysed immune responses in mother-child pairs in a prospective birth cohort study. METHODS: Within the Lifestyle and Environmental factors and their Influence on Newborns Allergy risk (LINA) study, concentrations of Th1/Th2/Th17 and inflammatory cytokines/chemokines as well as IgE were measured in phytohemagglutinin and lipopolysaccharide stimulated maternal blood in the 34th week of gestation and in corresponding children's blood at birth and 1 year after (n = 353 mother-child pairs). Information on atopic outcomes during the first year of life was obtained from questionnaires. RESULTS: Concentrations of inflammatory markers, excepting TNF-α, were manifold higher in cord blood samples compared with maternal blood. Th1/Th2 cytokines were lower in children's blood with a Th2 bias at birth. Maternal inflammatory parameters (MCP-1, IL-10, TNF-α) in pregnancy showed an association with corresponding cytokines blood levels in children at the age of one. High maternal IgE concentrations in pregnancy were associated with increased children's IgE at birth and at the age of one, whereas children's atopic dermatitis (AD) was determined by maternal AD. CONCLUSIONS: Maternal inflammatory cytokines during pregnancy correlate with children's corresponding cytokines at the age of one but are not related to IgE or AD. While maternal IgE predicts children's IgE, AD in children is only associated with maternal disease.

Reduced maternal regulatory T cell numbers and increased T helper type 2 cytokine production are associated with elevated levels of immunoglobulin E in cord blood.

BACKGROUND: There is evidence that the basis of an atopic-skewed immune response is acquired early in life, perhaps at the fetal stage. Thus, we hypothesized that the development of the fetal immune system might be influenced by maternal regulatory T cells (Treg) and maternal T cell cytokine production during pregnancy. The aim of the present study was to assess the influence of maternal Treg and cytokine production during pregnancy on Treg and atopy at birth. METHODS: Within the mother-child study LINA (Lifestyle and Environmental factors and their Influence on Newborns Allergy risk), we determined the frequency and function of Treg and the total IgE concentration in pregnant women in the 34th week of gestation and in corresponding cord bloods at birth (n=24). Furthermore, we assessed how maternal mitogen-induced T-helper type 1/T-helper type 2 and inflammatory cytokines influence the level of cord blood Treg and IgE. RESULTS: Frequencies of CD4(+)CD25(high) T cells were higher (P=0.001), whereas percentages of FOXP3+ T cells were lower (P<0.001) in cord blood cells compared with maternal blood. Reduced maternal CD4(+)CD25(high) Treg frequencies correlated with increased total IgE concentrations at the 34th week of gestation (r=-0.32, P=0.028) and with increased IgE concentrations in cord blood (r=-0.50, P<0.001). Elevated maternal mitogen-induced Th2 cytokine production was related to increased total IgE levels in the serum of corresponding cord bloods (IL-4, r=0.53; IL-5, r=0.43; IL-13, r=0.52). CONCLUSIONS: Because cord blood IgE has been shown to be predictive for allergic diseases in early childhood, our results indicate that reduced maternal Treg numbers and increased Th2 cytokine production during pregnancy might influence the allergy risk of the child.

Association between suppressors of cytokine signalling, T-helper type 1/T-helper type 2 balance and allergic sensitization in children.

BACKGROUND: Suppressors of cytokine signalling (SOCS) family members have been shown to play an important role in the balance of cytokines that determine the onset of T-helper type 1 (Th1)- and Th2-mediated immune responses. In particular, for cytokine-induced Src-homology 2 protein (CIS), SOCS1, SOCS3 and SOCS5, a role in the regulation of T cell differentiation has been discussed. However, only few data exist so far in the human system. OBJECTIVES: The aim of the present study was to analyse the relationship between these suppressors and Th1/Th2 regulation as well as allergic sensitizations within a population-based study. METHODS: Within the Lifestyle-Immune system-Allergy plus cohort study, mRNA was prepared from blood samples of 6-year-old children for the analysis of cytokines, transcription factors for T cell regulation and SOCS molecule expression by quantitative real-time polymerase chain reaction. In addition, total and specific IgE concentrations have been measured by the Pharmacia CAP System. A complete data set from 248 children was available. Results Among the SOCS molecules investigated, only SOCS1 showed a strong positive correlation to allergic sensitizations. In addition, an up-regulated SOCS1 expression correlated with down-regulated T-box expressed in T cells (Tbet) and higher expression levels of GATA-binding protein 3 (GATA-3) and IL-4. No association between SOCS1 and forkhead box P3 (FOXP3) was observed. For SOCS3, SOCS5 and CIS, a contradictory picture was found. The expression of these SOCS molecules was positively correlated with Tbet and FOXP3 and (with the exception of CIS) negatively with IL-4. CONCLUSIONS: Our data suggest that SOCS3, SOCS5 and CIS, which correlate with an up-regulated Th1 and regulatory T cell activity, are without relevance for the allergic status. In contrast, SOCS1 might be involved in the development of a Th2-skewed immune response and subsequent allergic sensitizations.

Association of neuropeptides with Th1/Th2 balance and allergic sensitization in children.

BACKGROUND: Among neurogenic factors, the neuropeptides have an important regulatory influence on immune system activity and may lead to allergic sensitization. OBJECTIVE: The aim of our study was to investigate the relationship of the neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM) and substance P (SP) on modulation of Th1/Th2 balance and allergic sensitization in children. METHODS: Within the LISAplus (Life style-Immune system-Allergy) study, blood samples of 321 six-year-old children were analysed for concentration of neuropeptides, Th1 and Th2 cytokines, transcription factors for T cell regulation and suppressors of cytokine signalling. In addition, samples were screened for specific IgE against inhalant and food allergens. RESULTS: Children with high SOM values showed a Th2 polarization and a reduced expression of FOXP3, the marker for regulatory T cells. High (VIP) levels correlated inversely with the expression of T cell transcription factors (Tbet and SOCS3). In contrast, elevated levels of SP were associated with reduced GATA3 and SOCS3 expression and with increased IFN-gamma concentrations. Allergic sensitization was more prevalent in children with higher SOM and VIP concentrations but not associated with SP levels. CONCLUSION: Our data reveal an association between neuropeptides and modulatory effects on immune cells in vivo, especially on Th1/Th2 balance with a correlation to allergic sensitization in children. We suggest that elevated SOM and VIP concentrations and the inducing factors should be considered as allergy risk factors.

Hybrid cell vaccination for cancer immune therapy: first clinical trial with metastatic melanoma.

Hybrid cell vaccination is a new cancer immune therapy approach that aims at recruiting T cell help for the induction of tumour specific cytolytic immunity. The vaccines are generated by fusion of the patients' tumour cells with allogeneic MHC class II bearing cells to combine the tumour's antigenicity with the immunogenicity of allogeneic MHC molecules. Safety and anti-tumour activity of this treatment were assessed in a clinical trial that has yielded one complete and one partial remission, and 5 cases of stable disease among 16 patients with advanced stage metastatic melanoma. As evidenced by histology, the vaccination induced T cell relocation into tumour nodules. Stable disease could be maintained by repeated booster injections for more than 24 months in some patients. The side effects were minor. Occasional occurrences of vitiligo spots after vaccination were indicative of a restricted therapy induced auto-immune reactivity. The results suggest that hybrid cell vaccination is a safe cancer immune therapy potentially effective for induction of acute anti-tumour response as well as long-term maintenance.

SM5-1: a new monoclonal antibody which is highly sensitive and specific for melanocytic lesions.

Antibodies such as HMB-45 and anti-S100 protein have been widely used as markers of malignant melanoma despite evidence that HMB-45 has a sensitivity of only 67-93% and S100 is nonspecific for melanoma. Using a subtractive immunization protocol in a mouse model of human melanoma, we have generated several monoclonal antibodies with putative specificity for melanoma. After initial screenings, the antibody SM5-1 was chosen because of its intriguing reactivity with melanocytic tumors in both frozen and paraffin sections. The immunohistochemical staining of SM5-1 was studied in paraffin-embedded specimens of 401 melanomas (n = 401; 250 primary melanomas, 151 metastases), melanocytic nevi of the skin (n = 16), nonmelanocytic neoplasms (n = 84). The results were compared with HMB-45 and anti-S100 staining. All antibodies reacted with nevi and 97-99% with primary melanomas. Whereas both SM5-1 and anti-S100 stained 96% (146/151) of melanoma metastases, HMB-45 correctly identified only 83% (126/151). All HMB-45-negative metastases were positive for SM5-1. Whereas neither SM5-1 nor HMB-45 stained any of 84 specimens from 40 different nonmelanocytic neoplasms, anti-S100 was positive in 21/84 (25%). While the staining pattern of SM5-1 was mostly homogeneous, small tumor areas in some metastases remained unstained. Staining with SM5-1 was also observed in perivascular dendritic cells, in plasma cells, some myofibroblasts and the secretion of eccrine sweat glands. Nonactivated epidermal melanocytes, keratinocytes, endothelial cells, smooth muscle cells and peripheral nerves were all negative for SM5-1. These results suggest that SM5-1 is highly specific, as well as sensitive, for melanocytic lesions and is useful in the immunohistochemical evaluation of melanoma.

Catalase, myeloperoxidase and hydrogen peroxide in cystic fibrosis.

An oxidant-antioxidant imbalance with damaging consequences for the bronchial epithelium has been hypothesized in the airways of patients with cystic fibrosis (CF). It is based on the assumption that neutrophils entering the lumen of the infected airways undergo activation and release toxic oxygen metabolites and myeloperoxidase (MPO), an enzyme which transforms hydrogen peroxide (H2O2) into highly toxic oxygen metabolites. Our aims were to substantiate this hypothesis. H2O2 levels were measured in breath condensates of 63 CF patients and 51 normal subjects. In CF sputum samples, activities and concentrations of MPO and catalase (CAT) were determined. MPO/H2O2-mediated cytotoxicity of CF sputum was measured in cell culture assays. H2O2 levels were similar in CF patients and normal subjects (mean +/-SD) 0.97 +/- 0.69 versus 1.11+/-0.78 microM; p=0.427). Concentrations and activities of CAT (0.31+/-0.18 microM; 105+/-69 units) and MPO (5.93+/-4.8 microM; 87.8+/-75 units) were detectable in 38 CF sputa. Addition of H2O2 to in vitro cells preincubated with CF sputum did not induce cytotoxicity even when CAT was removed from sputum. Sputum MPO together with H2O2 did not inactivate alpha-proteinase inhibitor. Preincubation of MPO with sulphated glycoconjugates or deoxyribonucleic acid (DNA) totally inhibited its cytotoxic effect. In conclusion, catalase, sulphated glycoconjugates and deoxyribonucleic acid may prevent myeolperoxidase-mediated oxygen radical generation in cystic fibrosis sputum.