Laser capture microdissection for the investigative pathologist.

An important step in translational research is the validation of molecular findings from in vitro experiments using tissue specimens. However, tissue specimens are complex and contain a multitude of diverse cell populations that interfere with the molecular profiling data of a specific cell type. Laser capture microdissection (LCM) alleviates this issue by providing a valuable tool for the enrichment of a specific cell type within complex tissue samples. However, LCM and molecular analysis from tissue specimens can be complex and challenging due to numerous issues related with the tissue processing and its impact on the integrity of biomolecules in the specimen. The intricate nature of this application highlights the essential role a pathologist plays in translational research by contributing an expertise in histopathology, tissue handling, tissue analysis techniques, and clinical correlation of biological findings. The present review examines key practical aspects in tissue handling, specimen selection, quality control, and sample preparation for LCM and downstream molecular analyses that are a primary objective of the investigative pathologist.

The effect of perceived and actual social support on the mental health of HIV-positive persons.

The purpose of this paper is to compare the ways in which perceived and actual social support affect the mental health of gay men, straight or bisexual men, and women living with HIV/AIDS. Participants included 125 women and 232 men with an HIV-positive or AIDS diagnosis involved in three larger investigations of HIV, disclosure and mental health. Results suggest each sub-group experienced perceived social support as significantly predictive of better mental health while the effect of actual social support was minimal.

An element in intron 1 of the CFTR gene augments intestinal expression in vivo.

The elements controlling the complex developmental and tissue-specific expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene lie outside the basal promoter region and have not been characterized. We previously identified a tissue-specific DNase I hypersensitive site (DHS) in intron 1 (185 + 10 kb) of the CFTR gene. Here we show that removal of the core element abolishes the activity of this DHS in transient transfection assays of reporter/enhancer gene constructs. We then compared expression from a 310 kb yeast artificial chromosome (YAC) that contains the entire CFTR gene with expression from the same YAC from which the DHS element had been deleted. Stable transfection of a human colon carcinoma cell line showed that transcription from the deleted YAC was reduced by approximately 60%. In transgenic mice, deletion of the intron 1 DHS had no effect on expression in the lung, but reduced expression in the intestine by approximately 60%. Thus, the regulatory element associated with the intron 1 DHS is tissue-specific and is required for normal CFTR expression levels in the intestinal epithelium in vivo.

Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation.

A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin-mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation.

Tumor necrosis factor alpha (TNF alpha) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNF alpha are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at -308 in the TNF alpha promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNF alpha production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at -308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNF alpha gene regulation and may be responsible for the association of TNF2 with high TNF alpha phenotype and more severe disease in infections such as malaria and leishmaniasis.

An AP-1 site is involved in the NGF induction of IL-1 alpha in PC12 cells.

The nerve growth factor which induces phenotypic changes in PC12 pheochromocytoma cells also induces the expression of the proinflammatory cytokine interleukin 1 alpha in these cells. We have studied the signal transduction and transcriptional mechanisms involved in this induction of interleukin 1 alpha by nerve growth factor. The nerve growth factor induction of interleukin 1 alpha transcription in PC12 cells is exerted via the TrkA receptor, as demonstrated by inhibition of the nerve growth factor stimulated increases in the interleukin 1 alpha mRNA levels by the TrkA specific alkaloid K-252a. The promoter region(s) involved in induction of interleukin 1 alpha expression by nerve growth factor in PC12 pheochromocytoma cells were studied by deletion mutagenesis in a part of the 5' regulatory region of the human interleukin 1 alpha gene (bases -163 to +64). This promoter region was inserted into the promoterless pBLCAT3 plasmid, using the interleukin 1 alpha 5' fragment as the promoter to drive nerve growth factor inducible expression of the CAT (chloramphenicol acetyl transferase) reporter gene. Four mutants, with deletions of 9-15 bases in the 5' regulatory region of the human interleukin 1 alpha gene, were constructed: three deleted stretches correspond to regions with high sequence similarity to regions in other genes, coding for nerve growth factor-induced proteins, e.g. NGFI-A, NGFI-B, NGFI-C, ERK2 and VGF gene. These deletions, of which some reduced the basal, non-nerve growth factor stimulated expression of the CAT reporter protein, do not prevent the two- to threefold induction by nerve growth factor. The deletion which eliminated a putative AP-1 binding site, immediately upstream of the transcription start site in the interleukin 1 alpha promoter, almost completely prevented the nerve growth factor mediated induction of CAT reporter gene expression, suggesting that in PC12 cells the major site of nerve growth factor regulation of interleukin 1 alpha expression is at this AP-1 site.

A regulatory element in intron 1 of the cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells.

Interaction between HLA-DR and HLA-DP, and between HLA and interleukin 1 alpha in juvenile rheumatoid arthritis indicates heterogeneity of pathogenic mechanisms of the disease.

EOP-JRA is an autoimmune disease that displays associations with DPB1*0201, DR8, DR5, and DR6, as well as an association with IL1A2 (a variant of IL1 alpha gene, not HLA linked). The purpose of this study was to analyze interactions between these genetic factors. We studied 103 EOP-JRA patients, 181 random controls, and 69 DR8-positive controls. We found a positive interaction between DPB1*0201 and the DRB1 alleles encoding DR3, DR5, or DR6, but not DR8. In addition, we found evidence for an interaction between IL1A2 and DR(3, 5, or 6) and DP2, but not DR8. We interpret the data to suggest heterogeneity in the HLA-associated pathogenic mechanisms of EOP-JRA.

A genetic association between juvenile rheumatoid arthritis and a novel interleukin-1 alpha polymorphism.

OBJECTIVE: The genetic factors that predispose to the development of juvenile rheumatoid arthritis (JRA) and its complications are not completely understood. The cytokine interleukin-1 (IL-1) has been implicated in the pathogenesis of JRA and other inflammatory diseases. This study was performed to test whether polymorphisms of the IL-1 alpha gene might be associated with JRA. METHODS: We sequenced the 5' regulatory region (containing the promoter) of the human IL-1 alpha gene in 18 normal subjects. This revealed a C (IL-1A1) to T (IL-1A2) transition polymorphism at position -889. We studied the frequencies of both alleles in patients with JRA (n = 269) and controls (n = 99). RESULTS: An increased gene carriage of IL-1A2 was found in patients with early-onset, pauciarticular JRA (EOPA-JRA; n = 103) compared with controls (0.66 versus 0.49; P = 0.01, odds ratio [OR] = 2.1). Within this subset of JRA, the association with IL-1A2 was particularly strong in the patients in whom chronic iridocyclitis developed (n = 28) compared with those without chronic iridocyclitis (0.89 versus 0.57; P = 0.002, OR = 6.2). Within the group of EOPA-JRA patients, IL-1A2 was also associated with elevation of the erythrocyte sedimentation rate (P < 0.0025). CONCLUSION: This is the first report of a cytokine gene association with JRA, and we conclude that IL-1 alpha itself, or a gene for which the IL-1 alpha polymorphism is a marker, may contribute to the pathogenesis of EOPA-JRA and the ocular complications found in this group.

Novel genetic association between ulcerative colitis and the anti-inflammatory cytokine interleukin-1 receptor antagonist.

BACKGROUND/AIMS: Ulcerative colitis and Crohn's disease have well-recognized familial tendencies, but the genetic basis of this clinical observation remains unknown. The cytokine interleukin-1 receptor antagonist is a potent anti-inflammatory protein that can prevent immune-mediated bowel inflammation in animals. We have previously characterized a polymorphism within the gene for this cytokine and others in the genes for the proinflammatory cytokines interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor alpha. The aim of this study was to determine whether inflammatory bowel disease was associated with particular alleles of these polymorphic cytokine genes. METHODS: The allelic frequencies of these polymorphic cytokine genes were determined in patients with ulcerative colitis (n = 113), Crohn's disease (n = 78), and healthy controls (n = 261). RESULTS: Allele 2 of interleukin-1 receptor antagonist was significantly over-represented in the ulcerative colitis patients: 35% versus 24% in controls (P = 0.007). Carriage of at least one copy of this allele gave an odds ratio of 2.0 for ulcerative colitis compared with controls. This association with allele 2 of interleukin 1 receptor antagonist was greatest in patients with total colitis and was not seen in Crohn's disease. There were no associations between UC and any of the other cytokine genes examined. CONCLUSIONS: This observation provides evidence that interleukin-1 receptor antagonist may have a role in determining the genetic susceptibility to and pathogenesis of ulcerative colitis.

Interleukin 1 in rheumatoid arthritis: potentiation of immune responses within the joint.

Specific immunoassays were used to measure IL-1 peptides in the serum and synovial fluid of patients with rheumatoid arthritis (RA) and in the serum of age-matched healthy controls. Patients with RA had raised levels of both IL-1 beta and IL-1 alpha in their sera compared to controls. Synovial fluid levels of IL-1 beta significantly correlated with immunoreactive IL-2 and soluble IL-2 receptor (sIL-2R). In addition, incubation of synovial fluid MNC with human recombinant (hr) IL-1 caused a dose-dependent increase in the level of sIL-2R in the cell supernatant. Finally, production of IL-1 beta and IL-6 from RA peripheral blood (PB) and synovial fluid (SF) MNC was examined. PBMNC spontaneously produced low levels of IL-1 beta and IL-6 that were augmented by the addition of hr IL-1 alpha. In contrast, SFMNC spontaneously produced high levels of IL-1 beta but only low levels of IL-6, again this production was augmented by the addition of hr IL-1 alpha. Taken together, the data suggests that IL-1 potentiates immune responses within the joint.

Benzethonium chloride method for proteins adapted to centrifugal analysis.

A benzethonium chloride method for measuring protein concentrations in several types of body fluids has been adapted to the Cobas Bio centrifugal analyzer. The ability of this analyzer to add two reagents stepwise, measuring absorbances after each addition, is essential for proper blanking of urine specimens. The method has excellent curve stability and precision and yields results that correlate well with those of four other protein methods.

An evaluation of the temperature control system of the Centrifichem 400.

We evaluate the manufacturer's temperature calibration procedure for the Union Carbide Centrifichem 400 using a procedure developed in our laboratories. We substituted a Yellow Springs, Inc., Model 45 CU Cuvette Thermometer with a thermistor probe for the mercury thermometers. This dramatically shortened the time required for temperature measurements by eliminating the time required for the mercury thermometers to equilibrate. The cuvette temperature measurements with the YSI 45 CU accurately reflect the analyzer equilibrium temperature only when the YSI 45 CU thermistor probe is in the same cuvette as the analyzer thermistor. We also evaluated the accuracy and precision of the temperature control system and the time required to reach thermal equilibrium. We found that temperature equilibration occurs in less than a minute and has a range of less the +/- 0.1 degree C from the mean temperature. Our procedure for monitoring temperature equilibration gave improved accuracy and precision.