Activated CD8+ T cells and natural killer T cells in bronchoalveolar lavage fluid in hypersensitivity pneumonitis and sarcoidosis.

BACKGROUND: Sarcoidosis and hypersensitivity pneumonitis are diffuse parenchymal lung diseases characterized by formation of non-caseating granulomas with a bronchocentric distribution. Analysis of the white blood cell differential profile in bronchoalveolar lavage fluid can be a useful supplement in the diagnostic work-up. OBJECTIVE: Diagnostic markers that can improve the discrimination of sarcoidosis and hypersensitivity pneumonitis are wanted. METHODS: Bronchoalveolar lavage fluid fractions of CD4+ and CD8+ T cells expressing the activation marker HLA-DR and fractions of natural killer T cells determined by flow cytometry were investigated in sarcoidosis (N=83), hypersensitivity pneumonitis (N=10) and healthy control subjects (N=15). RESULTS: In hypersensitivity pneumonitis, natural killer T cell fractions were over 7-fold greater [median (IQR): 5.5% (3.5-8.1) versus 0.7% (0.5-1.2), p<0.0001], and HLA-DR+ fractions of CD8+ lymphocytes were almost two fold greater [median (IQR): 79% (75-82) versus 43% (34-52), p<0.0001] than in sarcoidosis. In healthy control subjects, natural killer T cell fractions of leucocytes and HLA-DR+ fractions of CD8+ lymphocytes were lower [median (IQR): 0.3% (0.3-0.6) and 30% (26-34), p=0.02 and p=0.01 compared to sarcoidosis]. The combined use of these two markers seems to discriminate the diseases very well. CONCLUSION: This study suggests a role for the bronchoalveolar lavage fluid lymphocyte subsets HLA-DR+ CD8+ T cells and natural killer T cells in the diagnostic work up of sarcoidosis and hypersensitivity pneumonitis.

The development and characterization of a 57K single nucleotide polymorphism array for rainbow trout.

In this study, we describe the development and characterization of the first high-density single nucleotide polymorphism (SNP) genotyping array for rainbow trout. The SNP array is publically available from a commercial vendor (Affymetrix). The SNP genotyping quality was high, and validation rate was close to 90%. This is comparable to other farm animals and is much higher than previous smaller scale SNP validation studies in rainbow trout. High quality and integrity of the genotypes are evident from sample reproducibility and from nearly 100% agreement in genotyping results from other methods. The array is very useful for rainbow trout aquaculture populations with more than 40 900 polymorphic markers per population. For wild populations that were confounded by a smaller sample size, the number of polymorphic markers was between 10 577 and 24 330. Comparison between genotypes from individual populations suggests good potential for identifying candidate markers for populations' traceability. Linkage analysis and mapping of the SNPs to the reference genome assembly provide strong evidence for a wide distribution throughout the genome with good representation in all 29 chromosomes. A total of 68% of the genome scaffolds and contigs were anchored through linkage analysis using the SNP array genotypes, including ~20% of the genome assembly that has not been previously anchored to chromosomes.

Activated CD8(+) T cells and NKT cells in BAL fluid improve diagnostic accuracy in sarcoidosis.

PURPOSE: The clinical diagnosis of pulmonary sarcoidosis is based on the presence of noncaseating granulomas in an appropriate clinical setting with either bilateral hilar adenopathy and/or parenchymal infiltrates. Lymphocytosis with an increased CD4/CD8 T cell ratio in bronchoalveolar lavage fluid is supportive. We evaluated the diagnostic accuracy of a predictive binary logistic regression model in sarcoidosis based on sex, age, and bronchoalveolar lavage fluid cell profile with and without the inclusion of HLA-DR(+) CD8(+) T cells and natural killer T-cell fractions. METHODS: A retrospective analysis of differential cell counts and lymphocyte phenotypes by flow cytometry in bronchoalveolar lavage was performed in 183 patients investigated for possible diffuse parenchymal lung disease. A logistic regression model with age, sex, lymphocyte fraction, eosinophils, and CD4/CD8 ratio in bronchoalveolar lavage fluid (basic model) was compared with a final model, which also included fractions of HLA-DR(+) CD8(+) T cells and natural killer T cells. Diagnostic accuracy of the two models was assessed by receiver operating characteristic (ROC) curves. RESULTS: The area under the ROC curve for the basic and final model was 0.898 [95 % confidence interval (CI) 0.852-0.945] and 0.937 (95 % CI 0.902-0.972), respectively, p = 0.008. CONCLUSIONS: Assessment of HLA-DR(+) CD8(+) T cell and natural killer T-cell fractions may improve diagnostic accuracy and further strengthen the importance of bronchoalveolar lavage in the diagnostic workup of sarcoidosis.

BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmon.

A better understanding of the genotype-phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.

A SNP/microsatellite genetic linkage map of the Atlantic cod (Gadus morhua).

A first genetic linkage map of the Atlantic cod (Gadus morhua) was produced, based on segregation data from 12 full-sib families of Norwegian origin. The map contained 174 single nucleotide polymorphism markers and 33 microsatellites, distributed on 25 linkage groups and had a length of 1225 cM. A significant difference in recombination rates between sexes was found, the average ratio of female:male recombination rates being 1.78 +/- 1.62 (SD).

In vitro experiments demonstrate that monocytes and dendritic cells are rendered apoptotic by extracorporeal photochemotherapy, but exhibit unaffected surviving and maturing capacity after 30 Gy gamma irradiation.

Extracorporeal photochemotherapy (ECP) has been shown to induce apoptosis in lymphocytes. Until recently the prevailing opinion has been that the monocytes were mainly not affected by this treatment. This study has investigated the effect of ECP and gamma irradiation on monocytes and immature dendritic cells (DC) in vitro and followed the ability of the cells to differentiate and survive post treatment. ECP induced apoptosis in lymphocytes, monocytes and immature DC within 72 h following treatment, in contrast to 30 Gy gamma irradiation, which seemed mainly to affect lymphocytes. The minority of the surviving ECP-treated monocytes presented a reduced ability to differentiate into immature DC within this time frame. We also demonstrated that immature DC after ECP-treatment lost their normal ability to mature on stimulation with lipopolysaccharide. As monocytes and immature DC seem to have a reduced ability to differentiate after ECP-treatment, it is suggested that the therapeutic effect of ECP is caused by in vivo effects of reinfused apoptotic cells, rather than by infusion of monocytes induced to differentiate into immature DC.

Conservation genomics of Atlantic salmon: SNPs associated with QTLs for adaptive traits in parr from four trans-Atlantic backcrosses.

European Atlantic salmon (Salmo salar) differ in skin pigmentation and shape from the North American lineage of Atlantic salmon but the genetic basis of these differences are poorly understood. We created four large (N=300) backcross families by crossing F1 hybrid male siblings to two females from the European and two from the North American aquacultural strains. We recorded 15 morphological landmarks and two skin pigmentation, three growth and three condition traits on parr. The backcross families were genotyped for at least 129 SNPs (single nucleotide polymorphisms) within expressed sequence tags (ESTs) spaced throughout the Atlantic salmon linkage map. The high polymorphism and low rates of crossover in our hybrid sires provided enough statistical power to detect 79 significant associations between SNP markers and quantitative traits after experiment-wide permutation analysis for all families within traits. Linkage group AS22 contained a quantitative trait loci (QTL) for parr mark number; its homolog AS24 contained a large QTL, which explained 26% of the phenotypic variance in parr mark contrast. We found 25 highly significant QTLs for body shape and fin position on seven different linkage groups, and 16 for growth and condition on six different linkage groups. QTL(s) for pectoral fin position, caudal peduncle position, late parr growth and condition index were associated with an SNP on linkage group AS1, which was linked to the sex-determining locus. Our work adds to the evidence that much of the variation in growth rate, shape and skin pigmentation observed among Atlantic salmon parr from different natal streams is genetic.

Extracorporeal photochemotherapy in patients with cutaneous T-cell lymphoma: is clinical response predictable?

BACKGROUND: Extracorporeal photochemotherapy (ECP) has been accepted as a standard therapy in cutaneous T-cell lymphomas (CTCL), a category of lymphomas mainly resistant to conventional therapies. Approximately one half of patients demonstrate a reduction in skin affliction by at least 50% within 12 months of therapy and are categorized as responders to ECP. Predictive criteria for selecting patients who will respond to ECP are lacking. Such criteria would however, be of great benefit. OBJECTIVES: This study compared T-cell clonality and serum levels of soluble interleukin-2 receptor (sIL-2R), lactate dehydrogenase (LD), neopterin, beta2-microglobulin (beta(2)-M) and granzyme B in CTCL patients in order to evaluate their potential usefulness as predictive markers. PATIENTS/METHODS: Serum and T lymphocytes obtained from 16 patients with CTCL receiving ECP treatment were evaluated in an open retrospective study. RESULTS: We found no evident correlation between detected T-cell clonality and response to ECP. The non-responding group had on average a higher level of serum sIL-2R. This difference was significant after 6 and 12 months of therapy, but not pretreatment. An individual reduction in serum sIL-2R, neopterin and beta(2)-M during a 6-month course of ECP was well correlated to clinical remission. CONCLUSIONS: Seven out of 16 patients were classified as responders. Neither T-cell clonality nor any of the serum markers assessed pretreatment could reliably predict the response to ECP treatment. However, the individual relative changes in sIL-2R, neopterin and beta(2)-M during 6 months of ECP treatment coherently displayed correlation to the clinical response, as assessed after 12 months of ECP treatment.

A linkage map of Atlantic salmon (Salmo salar) reveals an uncommonly large difference in recombination rate between the sexes.

A genetic linkage map of the Atlantic salmon (Salmo salar) was constructed, using 54 microsatellites and 473 amplified fragment length polymorphism (AFLP) markers. The mapping population consisted of two full-sib families within one paternal half-sib family from the Norwegian breeding population. A mapping strategy was developed that facilitated the construction of separate male and female maps, while retaining all the information contributed by the dominant AFLP markers. By using this strategy, we were able to map a significant number of the AFLP markers for which all informative offspring had two heterozygous parents; these markers then served as bridges between the male and female maps. The female map spanned 901 cM and had 33 linkage groups, while the male spanned 103 cM and had 31 linkage groups. Twenty-five linkage groups were common between the two maps. The construction of the genetic map revealed a large difference in recombination rate between females and males. The ratio of female recombination rate vs. male recombination rate was 8.26, the highest ratio reported for any vertebrate. This map constitutes the first linkage map of Atlantic salmon, one of the most important aquaculture species worldwide.

Aberrant expression of the CD8 antigen in B cell chronic lymphocytic leukaemia.

We report a case of aberrant expression of the T cell antigen CD8 in a patient with B cell chronic lymphocytic leukaemia (B-CLL). A 62-year-old Caucasian female with several enlarged lymph nodes and suspected to have B-CLL was referred to our laboratory for routine immunophenotyping. Peripheral blood cell count showed moderate leucocytosis without other abnormalities. The dual-colour flow cytometric analysis showed a typical B-CLL phenotype (CD45+, CD19+, kappa+, lambda-, CD20+, CD23+, IgM+, HLA-DR+, CD5/CD19+, CD3-). In addition, aberrant expression of the T cell marker CD8 was found, present on approximately 64% of the leukemic cells. This is a rare even, the significance and nature of this aberration has not yet been fully determined. In this case, our patient had a rapid response to treatment with a remarkable reduction in the number of leukemic cells only two weeks after beginning treatment.

Comparative investigation of proliferation markers and their prognostic relevance in human meningiomas.

OBJECTIVE: In human meningiomas, histology alone does not always predict the clinical outcome. Proliferative activity has therefore, become a potential tool in the histopathological grading of these tumors. The aim of this study was to investigate different Ki67 antibodies on meningiomas, to compare their proliferation indices (PI) and with other proliferation markers, such as S-phase fraction and mitotic activity, and to see whether these factors correlate with histological tumor grade. MATERIAL AND METHODS: The study included 43 meningiomas graded according to the criteria of WHO and Jääskeläinen et al. [1985, 1986]. Paraffin sections were used for immunohistochemical detection of Ki67 antigen and flow-cytometric determination of S-phase fraction. RESULTS: The PIs displayed an overall increase with increasing histological grade, however, the range of values for benign, atypical and anaplastic meningiomas were wide, resulting in considerable overlap between the groups. There were for the most significant correlations between the different proliferation markers. CONCLUSIONS: Ki67-equivalent antibodies and S phase fraction have no advantage over counting mitoses to assess the proliferative activity in meningiomas. Thus, mitotic activity justifies its role in meningioma grading.

Antinuclear antibodies (ANA) in Gordon setters with symmetrical lupoid onychodystrophy and black hair follicular dysplasia.

Antinuclear antibodies (ANA) were demonstrated in 3 out of 10 Gordon setters with symmetrical lupoid onychodystrophy and in 5 out of 13 Gordon setters with black hair follicular dysplasia. Two dogs showed both symmetrical lupoid onychodystrophy and black hair follicular dysplasia, and one of these was ANA positive. The results suggest that symmetrical lupoid onychodystrophy and black hair follicular dysplasia in the Gordon setter might be autoimmune diseases that are pathogenetically related, which might indicate a common genetic predisposition.

Occupational and adult lead exposure in Wisconsin.

Lead is a versatile metal with many industrial applications. It is among the oldest recognized occupational health hazards. Lead poisoning has been a reportable disease in Wisconsin since 1911. Although reportable, it was not until Wisconsin established an Occupational and Environmental Health Epidemiology program in 1979 that modern reporting levels were adopted, physician and laboratory reporting promoted and publicized, and elevated blood lead report tracking initiated. With the federal funding from the National Institute of Occupational Safety and Health (NIOSH), a comprehensive adult blood lead surveillance program was created in 1987. Eleven years of surveillance trend data reveal a Wisconsin success story. Most Wisconsin industries have made substantial strides toward reducing occupational lead exposure. The improvement is reflected in the reduced number of elevated blood lead levels in Wisconsin's adult blood lead surveillance data. However, Wisconsin must remain vigilant as new and re-emerging lead exposures continue to be identified through adult blood lead surveillance. Wisconsin will also need to continue with its occupational lead exposure reduction efforts if it is to achieve the Federal Healthy People 2010 goals and objectives to have no adult blood lead level greater than 25 micrograms/dL.

Bronchoscopy and bronchoalveolar lavage findings in cross-country skiers with and without "ski asthma".

Bronchial hyperresponsiveness to methacholine with asthma-like symptoms ("ski asthma") is frequent in elite cross-country skiers. To further the understanding of "ski asthma", 10 nonasthmatic, nonatopic controls and 30 adolescent elite skiers were investigated by bronchoscopy and bronchoalveolar lavage (BAL). Nine skiers were atopic without allergy symptoms. Compared with controls, the macroscopic inflammatory index in the proximal airways in skiers was three-fold greater (median (interquartile range) 3.0 (2.0-5.0) versus 1.0 (0.8-2.3), p=0.008). In the BAL fluid, skiers had significantly greater total cell (p<0.05) and percentage lymphocyte (p<0.01) and mast cell counts (p<0.05). Neutrophil and eosinophil counts were not significantly different and eosinophil cationic protein was not detected. Tumour necrosis factor-alpha and myeloperoxidase were detected in 12 (40%) and six (20%) skiers, respectively. In skiers with ski asthma, the inflammatory index was greater than in nonasthmatic skiers. Lymphocyte subtypes and activation markers, and concentration of albumin, fibronectin and hyaluronan were not different from those in controls. Cross-country skiers have a minor to moderate degree of macroscopic inflammation in the proximal airways at bronchoscopy and a bronchoalveolar lavage fluid profile which differs in several respects from healthy controls. Skiers with ski asthma tend to show even higher degrees of bronchial inflammation.

Metal working fluid-associated hypersensitivity pneumonitis: an outbreak investigation and case-control study.

Occupational exposure to bacterial or fungal antigens has been associated with hypersensivity pneumonitis (HP), an immunologically-mediated pulmonary disease. Between August 1995 and April 1996, 34 employees working in machining and assembly areas of an engine manufacturing plant were clinically diagnosed with HP. Of these, 20 employees met an epidemiologic case definition. In a case-control study, no exposure variables, including duration and intensity of metal working fluid (MWF) exposure, were statistically associated with an increased risk of disease. Neither cases nor controls demonstrated precipitin reactivity against unused samples of the seven MWF and two biocides used in the plant. HP cases had a significantly higher prevalence of positive precipitin reactions to used oil soluble and synthetic MWFs. Reactivity to used but not unused MWF suggests a biocontaminant, probably bacteria or fungi, is the causative antigen in the development of HP in this setting.

Expression of the receptor tyrosine kinase Ret on the plasma membrane is dependent on calcium.

Mutations in the Ret receptor tyrosine kinase are responsible for a variety of human syndromes, including multiple endocrine neoplasia 2 and Hirschsprung's disease. Ret is expressed as a 150-kDa precursor form in the endoplasmic reticulum and a 170-kDa mature form at the plasma membrane. Here we show that expression of p170(ret) is dependent on calcium. Depletion of extracellular calcium completely blocks p170(ret) expression, which is not caused by a decrease in half-life of p170(ret) at the plasma membrane but by a defect in processing of p150(ret) into p170(ret). This processing defect can be mimicked by treating the cells with thapsigargin, a drug that releases calcium from internal stores, indicating that reduction in luminal calcium is responsible for the processing defect. We propose that a relatively high concentration of luminal calcium is necessary for the proper folding of Ret in the endoplasmic reticulum.

Ras-dependent activation of the small GTPase Ral.

The small GTPase Ral is a Ras-like GTPase [1] that has been implicated in growth-factor-induced and Ras-induced DNA synthesis [2-4], and Ras-induced oncogenic transformation [3,5]. Recently, we and others found that three different Ral guanine nucleotide exchange factors (Ral GEFs) - Ral GDS, Rgl and Rlf - bind specifically to the GTP-bound form of several Ras-like GTPases [6-9]. Although oncogenic Ras is able to activate these Ral GEFs [2,5,10], it is unknown whether growth factors can induce the activation of Ral and, if so, which small GTPase is involved in this process. Here, we show that stimulation of various growth factor receptors, including receptor tyrosine kinases and serpentine receptors, results in rapid activation of Ral. This activation correlates with the activation of Ras, and dominant-negative Ras completely inhibits Ral activation induced by insulin and epidermal growth factor (EGF). From these results, we conclude that Ral activation is a direct downstream effect of growth-factor-induced Ras activation.

Distributions of HLA class II alleles in autoantibody subsets among Norwegian patients with systemic lupus erythematosus.

In order to find potential correlations between HLA class II alleles and anti-SS-A, -SS-B, -Sm and anti-snRNP responses among Norwegian patients with systemic lupus erythematosus (SLE), HLA-DRB1, -DRB3*0101, -DQA1 and -DQB1 alleles were determined by DNA typing 50 patients and 108 controls. HLA distributions were analysed in the following autoantibody subgroups: anti-SS-A with -SS-B, anti-SS-A without -SS-B, anti-snRNP without -Sm, anti-SS-A without -snRNP and anti-snRNP without -SS-A. The autoantibodies were detected by EIA (enzyme immunuassay). Patients with anti-SS-A and -SS-B had significantly increased frequencies of DRB1*03, DRB3*0101, DQA1*0501, DQB1*0201 (in linkage disequilibrium) versus controls and versus patients without anti-SS-A and -SS-B. No differences in HLA distribution were found when the group with anti-SS-A alone was compared to the group with anti-SS-A and concomitant -SS-B. Comparing the groups with and without anti-SS-A and -SS-B, the highest RR were found for the alleles DRB1*03, DRB3*0101, DQB1*0501, DQB1*0201 (in linkage disequilibrium) with RR: 16.8, 5.0, 19.6, 10.3, respectively, P < 0.05). RR for DQw2/DQw6 heterozygotes was 3.5 (Ns.), and RR for cases having DQ alpha molecules with glutamine in position 34 and DQ beta molecules with leucine in position 26 on both chains was 6.3 (P < 0.05). No HLA associations were observed in the group with anti-snRNP without concomitant -Sm or without concomitant -SS-A. These results show that production of anti-SS-A and -SS-B is associated to the HLA alleles DRB1*03, DRB3*0101, DQA1*0501, DQB1*0201, and that this haplotype shows stronger correlation to these responses than DQw2/DQw6 heterozygosity or HLA molecules having glutamine in position 34 (DQ alpha) and leucine in position 26 (DQ beta). The failure to observe any correlation with DRBI*15,16 (DR2) in the group with anti-SS-A alone may demonstrate ethnic differences concerning this response. The failure to identify any HLA associations for the anti-snRNP response may reflect the heterogeneity of the molecules that constitute this antigen.

Can maternal antiphospholipid antibodies predict the birth of a small-for-gestational age child?

BACKGROUND: The aim of this study was to examine the relationship between the maternal level of antiphospholipid antibodies (aPA) measured by anticardiolipin antibodies (aCL) and fetal growth retardation (SGA). METHODS: A nested case control design was carried out in a prospective cohort study of 1552 para I and para II women. The study group consisted of all 138 women who gave birth to a SGA-child (defined as birthweight < 10th percentile). A control group of 276 women was randomly selected from mothers of non-SGA children. Levels of aPA were measured in banked sera drawn from the women in the 33rd week of pregnancy and compared between cases and controls. RESULTS: There were 3 (2.5%) sera with aPA above 97.5 percentile among the cases and 3 (1.2%) among the controls. This difference was not statistically significant. CONCLUSION: Antiphospholipid antibody measurements obtained at 33 weeks of gestation cannot be used to assess the risk of birth of a small for gestational age infant among parous women.