Increased expression of leukocyte Ig-like receptor-1 and activating role of UL18 in the response to cytomegalovirus infection

NK and T cells are important for combating CMV infection. Some NK and T cells express leukocyte Ig-like receptor-1 (LIR-1), an inhibitory receptor recognizing MHC class I and the CMV-encoded homolog UL18. We previously demonstrated an early increase in LIR-1-expressing blood lymphocytes in lung-transplanted patients later developing CMV disease. We now show that NK and T cells account for the observed LIR-1 augmentation. Coincubation of PBMC from CMV-seropositive donors with virus-infected lung fibroblasts led to a T cell-dependent secretion of IFN-gamma, produced mainly by LIR-1(+) T cells and by NK cells. Cytokine production during coculture with fibroblasts infected with virus containing the UL18 gene was augmented compared with the UL18 deletion virus, suggesting a stimulatory role for UL18. However, purified UL18Fc proteins inhibited IFN-gamma production of LIR-1(+) T cells. We propose that cytokine production in the transplant induces NK and T cells to express LIR-1, which may predispose to CMV disease by MHC/LIR-1-mediated suppression. Although the UL18/LIR-1 interaction could inhibit T cell responses, this unlikely plays a role in response to infected cells. Instead, our data point to an activating role for viral UL18 during infection, where indirect intracellular effects cannot be excluded.     

Depression in Persons with Diabetes by Age and Antidiabetic Treatment: A Cross-Sectional Analysis with Data from the Hordaland Health Study

BackgroundPersons with diabetes have increased risk of depression, however, studies addressing whether the risk varies by age and type of antidiabetic treatment have yielded conflicting results. The aim of this study was to investigate if the association between diabetes and depression varied by type of antidiabetic treatment in a large community based sample of middle-aged (40–47 years) and older adults (70–72 years).MethodsData from 21845 participants in the Hordaland Health Study (HUSK) were analyzed in a cross-sectional design. Diabetes was assessed by self-report and classified as un-medicated, treated by oral antidiabetic agents or by insulin. Depression was defined as a score ≥8 on the depression subscale of the Hospital Anxiety and Depression Scale and/or self-reported use of antidepressant agents. Associations between diabetes and depression were estimated using logistic regression.ResultsPersons in their forties with diabetes had a doubled prevalence of depression (OR: 1.96 (95% C.I.: 1.35, 2.83)) compared to persons without diabetes, while a lower and non-significant association was found among persons in their seventies. Persons in their forties with orally treated diabetes had about three times higher prevalence of depression (OR: 2.92 (95% C.I.: 1.48, 5.77)) after adjustment for gender, BMI, physical activity, alcohol consumption and education, compared to non-diabetic persons in the same age-group. No association between depression and insulin or un-medicated diabetes was found.ConclusionsClinicians should be aware that persons in their forties with orally treated diabetes are at a marked increased risk of depression.     

Two-level factorial screening for influence of temperature, pH, and aeration on production of Serratia marcescens nuclease.

Two high-nuclease-yielding mutants of Serratia marcescens, derived by chemical mutagenesis (W280, W355), and two strains with the pBR322 plasmid 403-SD2, carrying a nuclease gene and a chloramphenicol resistance gene [Escherichia coli CSH50(403-SD2) and S. marcescens CH30(403-SD2)] were investigated for nuclease production in a factorial shake flask experiment, with temperature (30 and 37 degrees C), pH (with or without CaCO3 tablets), and aeration (with or without baffles) as variable conditions. Yields varied 10-fold depending on the conditions investigated.     

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Summary Specialized transducing phages asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli, The clockwise order of genes on the chromosome is found to be: bglB, (pst, glmS), (uncA, uncB), criC, asn, trkD, rbs, rrnC, ilv.Abbreviations CCC covalently closed circular - MD 10⁶ Daltons; mw, molecular weight - R. restriction endonuclease - LFT, HFT low (high) frequency transducing - a.p.i. average phage input - m.o.i. multiplicity of infection     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

Feeding chronology of Atlantic salmon parr in subarctic rivers: consistency of nocturnal feeding

During the ice-free season in three subarctic rivers in northern Norway and Finland, Atlantic salmon parr Salmo salar exhibited significant diel fluctuations in stomach content weights in August and September, the largest weights being recorded consistently at 0300 hours. Accordingly, the feeding rate of the parr was highest during the night and lowest in daytime, whereas during the other periods of the ice-free season, no pronounced diel fluctuations in food intake occurred. The consistent nocturnal feeding periodicity in early autumn in subarctic rivers may be associated with benthic feeding and an increased availability of benthic prey at night.     

DNA fingerprints from hypervariable mitochondrial genotypes

Conventional surveys of restriction-fragment polymorphisms in mitochondrial DNA of menhaden fish (Brevoortia tyrannus/patronus complex) and chuckwalla lizards (Sauromalus obesus) revealed exceptionally high levels of genetic variation, attributable to differences in mtDNA size as well as in restriction sites. The observed probabilities that any two randomly drawn individuals differed detectably in mtDNA genotype were 0.998 and 0.983 in the two species, respectively. Thus, the variable gel profiles provided unique mtDNA "fingerprints" for most conspecific animals assayed. mtDNA fingerprints differ from nuclear DNA fingerprints in several empirical respects and should find special application in the genetic assessment of maternity.      

Evaluation of complex inheritance involving the most common Bardet-Biedl syndrome locus (BBS1)

Bardet-Biedl syndrome (BBS) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. Patients with BBS are also at increased risk for diabetes mellitus, hypertension, and congenital heart disease. BBS is known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although these loci were all mapped on the basis of an autosomal recessive mode of inheritance, it has recently been suggested-on the basis of mutation analysis of the identified BBS2, BBS4, and BBS6 genes-that BBS displays a complex mode of inheritance in which, in some families, three mutations at two loci are necessary to manifest the disease phenotype. We recently identified BBS1, the gene most commonly involved in Bardet-Biedl syndrome. The identification of this gene allows for further evaluation of complex inheritance. In the present study we evaluate the involvement of the BBS1 gene in a cohort of 129 probands with BBS and report 10 novel BBS1 mutations. We demonstrate that a common BBS1 missense mutation accounts for approximately 80% of all BBS1 mutations and is found on a similar genetic background across populations. We show that the BBS1 gene is highly conserved between mice and humans. Finally, we demonstrate that BBS1 is inherited in an autosomal recessive manner and is rarely, if ever, involved in complex inheritance.     

Application of immunohistochemistry in stereology for quantitative assessment of neural cell populations illustrated in the göttingen minipig

Background

Stereology is the study of estimating geometric quantities. When successfully applied, the combination of immunohistochemistry (IHC) and stereology eliminates intra- and interobserver variability for cell type identification.

Methodology/Principal Findings

We propose a method to validate existing antibody based cell type markers for stereological application. Comparison was made on the 100-days-old Göttingen minipig (G-mini) neocortex between estimates of total neuron number derived from Giemsa staining using morphological criteria and immunohistochemistry-based cell counting with NeuN. The mean total neuron numbers estimated by the two staining methods were not significantly different. Estimated quantities, including glial cell number, neocortical volume, cell densities and glial-to-neuron ratio were also presented. Additionally, we assessed other commonly used glial markers and discussed how to evaluate the advantages and disadvantages of these markers for stereological estimation of cell number.

Conclusion/Significance

The concordance in quantitative estimates of total neuron number derived from NeuN- and Giemsa-stained sections provides evidence for the sensitivity and specificity of NeuN as a neuronal marker in the G-mini. Although time-consuming, quantitative validation of IHC should always be considered in stereological studies if there is doubt of the sensitivity, specificity, or reproducibility of cell type markers. Inaccurate staining may cause both over- and underestimation of the total cell number and inflict considerable limitation when analyzing the results.