Genes and gene expression in the brains of human alcoholics

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex (SFC). Propensity to alcoholism is associated with several genes. γ-Aminobutyric acid (GABA)A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the regional presentation of GABAA and glutamate-NMDA (N-methyl-d-aspartate) receptors in SFC. Autopsy tissue was obtained from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and matched controls. ADH1C, DRD2B, EAAT2, and APOE genotypes modulated GABAA-β subunit protein expression in SFC toward a less-effective form of the receptor. Most genotypes did not divide alcoholics and controls on glutamate-NMDA receptor pharmacology, although gender and cirrhosis did. Genotype may affect amino acid transmission locally to influence neuronal vulnerability.

Expression of prostate-specific antigen variant MRNA transcripts in prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA) is important in tumour detection, monitoring disease progression and tumour recurrence. however, PSA is not a cancerspecific marker as levels can also be elevated in benign prostatic disease. A number of different mRNA transcripts of PSA have also been identified in prostatic tissue, but have not been fully characterized (PSA 424, PSA 525, Schulz transcript). Tissue specimens from transurethral resection of the prostate (TURP) or radical prostatectomy were obtained from 17 men with BPH and 15 men with prostate cancer. Total RNA was extracted, and reverse-transcriptionpolymerase chain reaction (RT-PCR) and Southern analysis carried out using transcript-specific primers and probes to determine which mRNA PSA transcripts were expressed. Real-time PCR was performed to determine transcript levels between the two groups using transcript-specific primers and SYBR green fluorescence. Values obtained were normalized to a standard housekeeping gene, B2-microglobulin. Transcripts amplified by RT-PCR and real-time PCR were confirmed by DNA sequencing. Our results show that the transcripts were present in some, but not all, BPH and cancer samples indicating that they are not specific to either BPH or cancer. Analysis of real-time PCR normalized values using a Student’s t -test, shows that there is a significant difference between the two groups for PSA 424, but not wild-type PSA, PSA 525 or the Schulz transcript. Although a larger cohort of samples is needed to further confirm these results, these findings suggest that mRNA levels of PSA 424 may have some utility as a diagnostic or prognostic marker in prostate cancer detection.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

Molecular studies in gorgonian alloimmunity : search for gene homologs of the immunoglobulin gene superfamily in Swiftia exserta

Humoral and cells surface molecules of the mammalian immune system, grouped into the Immunoglobulin Gene Superfamily, share protein structure and gene sequence homologies with molecules found among diverse phylogenetic groups. In histocompatibility studies, the gorgonian coral Swiftia exserta has recently demonstrated specific alloimmunity with memory (Salter-Cid and Bigger, 1991. Biological Bulletin Vol 181). In an attempt to shed light on the origins of this gene family and the evolution of the vertebrate immune response, genomic DNA from Swiftia exserta was isolated, purified, and analyzed by Southern blot hybridization with mouse gene probes corresponding to two molecules of the Immunoglobulin Gene Superfamily, the Thy-1 antigen, and the alpha-3 domain of the MHC Class I histocompatibility marker. Hybridizations were conducted under low to non-stringent conditions to allow binding of mismatched homologs that may exist between the mouse gene probes and the Swiftia DNA. Removal of non-specific binding (sequences less than 70% homologous) occurred in washing steps. Results show that with the probes selected, the method chosen, and the conditions applied, no evidence of sequences of 70% or greater homology to the mouse Thy-1 or MHC Class I alpha-3 genes exist in Swiftia exserta genome.

Roles of CTCF and YY1 in T Cell Receptor Gene Rearrangement And T Cell Development

Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.

We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.

Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.

Association between the oxytocin receptor (OXTR) gene and mesolimbic responses to rewards.

BACKGROUND: There has been significant progress in identifying genes that confer risk for autism spectrum disorders (ASDs). However, the heterogeneity of symptom presentation in ASDs impedes the detection of ASD risk genes. One approach to understanding genetic influences on ASD symptom expression is to evaluate relations between variants of ASD candidate genes and neural endophenotypes in unaffected samples. Allelic variations in the oxytocin receptor (OXTR) gene confer small but significant risk for ASDs for which the underlying mechanisms may involve associations between variability in oxytocin signaling pathways and neural response to rewards. The purpose of this preliminary study was to investigate the influence of allelic variability in the OXTR gene on neural responses to monetary rewards in healthy adults using functional magnetic resonance imaging (fMRI). METHODS: The moderating effects of three single nucleotide polymorphisms (SNPs) (rs1042778, rs2268493 and rs237887) of the OXTR gene on mesolimbic responses to rewards were evaluated using a monetary incentive delay fMRI task. RESULTS: T homozygotes of the rs2268493 SNP demonstrated relatively decreased activation in mesolimbic reward circuitry (including the nucleus accumbens, amygdala, insula, thalamus and prefrontal cortical regions) during the anticipation of rewards but not during the outcome phase of the task. Allelic variation of the rs1042778 and rs237887 SNPs did not moderate mesolimbic activation during either reward anticipation or outcomes. CONCLUSIONS: This preliminary study suggests that the OXTR SNP rs2268493, which has been previously identified as an ASD risk gene, moderates mesolimbic responses during reward anticipation. Given previous findings of decreased mesolimbic activation during reward anticipation in ASD, the present results suggest that OXTR may confer ASD risk via influences on the neural systems that support reward anticipation.

Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis

Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16-25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection.

An Immunogenetic Signature of Ongoing Antigen Interactions in Splenic Marginal Zone Lymphoma Expressing IGHV1-2*04 Receptors.

Purpose: Prompted by the extensive biases in the immunoglobulin (IG) gene repertoire of splenic marginal-zone lymphoma (SMZL), supporting antigen selection in SMZL ontogeny, we sought to investigate whether antigen involvement is also relevant post-transformation.

Experimental Design: We conducted a large-scale subcloning study of the IG rearrangements of 40 SMZL cases aimed at assessing intraclonal diversification (ID) due to ongoing somatic hypermutation (SHM).

Results: ID was identified in 17 of 21 (81%) rearrangements using the immunoglobulin heavy variable (IGHV)1-2*04 gene versus 8 of 19 (40%) rearrangements utilizing other IGHV genes (P= 0.001). ID was also evident in most analyzed IG light chain gene rearrangements, albeit was more limited compared with IG heavy chains. Identical sequence changes were shared by subclones from different patients utilizing the IGHV1-2*04 gene, confirming restricted ongoing SHM profiles. Non-IGHV1-2*04 cases displayed both a lower number of ongoing SHMs and a lack of shared mutations (per group of cases utilizing the same IGHV gene).

Conclusions: These findings support ongoing antigen involvement in a sizable portion of SMZL and further argue that IGHV1-2*04 SMZL may represent a distinct molecular subtype of the disease.

Molecular characterization of complete and incomplete immunoglobulin heavy chain gene rearrangements in hairy cell leukemia.

PURPOSE: We analyzed patients with hairy cell leukemia (HCL) to achieve a better understanding of the differentiation stage reached by HCL cells and to define the key role of the diversification of cell surface makers, especially CD25 expression. PATIENTS AND METHODS: We analyzed 38 previously untreated patients with HCL to characterize their complete (VDJ(H)) and incomplete (DJ(H)) immunoglobulin (Ig) heavy chain (IgH) rearrangements, including somatic hypermutation pattern and gene segment use. RESULTS: A correlation between immunophenotypic profile and molecular data was seen. All 38 cases showed monoclonal amplifications: VDJ(H) in 97%, DJ(H) in 42%, and both in 39%. Segments from the D(H)3 family were used more in complete compared with incomplete rearrangements (45% vs. 12%; P <.005). Furthermore, comparison between molecular and immunophenotypic characteristics disclosed differences in the expression of CD25 antigen; CD25(-) cases, a phenotype associated with HCL variant, showed complete homology to the germline in 3 of 5 cases (60%), whereas this characteristic was never observed in CD25(+) cases (P <.005). Moreover, V(H)4-34, V(H)1-08, and J(H)3 segments appeared in 2, 1, and 2 CD25(-) cases, respectively, whereas they were absent in all CD25(+) cases. CONCLUSION: These results support that HCL is a heterogeneous entity including subgroups with different molecular characteristics, which reinforces the need for additional studies with a larger number of patients to clarify the real role of gene rearrangements in HCL.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Is There More to This Case than Mere Pulmonary Alveolar Proteinosis? A Clinical Case Presentation

Introduction: Pulmonary alveolar proteinosis (PAP) is a rare disease, associated with excess accumulation of surfactant proteins and lipids in the alveoli. Clinical presentation: We report the case of a 46-year-old woman with a combined presentation of PAP, myelodysplasia and recurrent miscarriages. Conclusions: The concomitant presentation of the above might be compatible with a mutation of the haematopoietic transcription factor gene GATA2.

Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia.

Background: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent. Objective: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection. Patient and Methods: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene). Results: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result. Conclusion: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.

Acute Psychosis as Major Clinical Presentation of Legionnaires’ Disease

We report a case of a 61-year-old woman who presented with acute psychosis as a major manifestation of Legionnaires’ disease in the absence of other neuropsychiatric symptoms. Clinical history revealed dry cough and nausea. Observation showed fever and auscultation crackles in the lower lobe of the right lung. Laboratory testing demonstrated elevated C-reactive protein and lung chest radiograph showed patchy peribronchial and right lower lobe consolidation. Soon after admission, she started producing purulent sputum. Epidemiological data suggested Legionella pneumophila as possible cause of the clinical picture that was confirmed by urinary antigen detection and polymerase chain reaction of the sputum. She was treated with levofloxacin 750 mg/day for 10 days with complete remission of pulmonary and psychiatric symptoms. She has not had further psychotic symptoms.

Association of TNF-α Gene Variants With Clinical Manifestation of Cystic Fibrosis Patients of Iranian Azeri Turkish Ethnicity

Background: Cystic fibrosis (CF), a life-limiting autosomal recessive disorder, is considered a monogenic disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. According to several studies, mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene alone is insufficient to predict the phenotypic manifestations observed in cystic fibrosis (CF) patients. In addition, some patients with a milder CF phenotype do not carry any pathogenic mutation. Tumor Necrosis Factor-alpha (TNF-α) contributes to the pathophysiology of CF by causing cachexia. There is a reverse association between TNF-α concentration in patient's sputum and their pulmonary function. Objectives: To assess the effect of non-CFTR genes on the clinical phenotype of CF, two polymorphic sites (-1031T/C and -308G/A) of the TNF-α gene, as a modifier, were studied. Patients and Methods: Focusing on the lung and gastrointestinal involvement as well as the poor growth, we first investigated the role of TNF-α gene in the clinical manifestation of CF. Furthermore, based on the hypothesis that the cumulative effect of specific alleles of multiple CF modifier genes, such as TNF-α, may create the final phenotype, we also investigated the potential role of TNF-α in non-classic CF patients without a known pathogenic mutation. In all, 80 CF patients and 157 healthy control subjects of Azeri Turkish ethnicity were studied by the PCR–RFLP method. The chi-square test with Yates' correction and Fisher's exact test were used for statistical analysis. Results: The allele and genotype distribution of the investigated polymorphisms, and their associated haplotypes were similar in all groups. Conclusions: There was no evidence that supported the association of TNF-α gene polymorphisms with non-classic CF disease or the clinical presentation of classic CF.