Evaluation of epigenetic methylation biomarkers for the detection of colorectal cancer using droplet digital PCR.

Colorectal cancer (CRC) is the third most common cancer worldwide. Screening programs allow early diagnosis and have improved the clinical management of this disease. Aberrant DNA methylation is increasingly being explored as potential biomarkers for many types of cancers. In this study we investigate the methylation of ten target genes in 105 CRC and paired normal adjacent colonic tissue samples using a MethylLight droplet digital PCR (ML-ddPCR) assay. Receiver operator characteristic (ROC) curves were used to determine the diagnostic performance of all target genes individually and in combination. All 515 different combinations of genes showed significantly higher levels of methylation in CRC tissue. The combination of multiple target genes into a single test generally resulted in greater diagnostic accuracy when compared to single target genes. Our data confirms that ML-ddPCR is able to reliably detect significant differences in DNA methylation between CRC tissue and normal adjacent colonic tissue in a specific selection of target genes.

Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I.

Mice homozygous for the lpr gene have a defect in fas (CD95), a cell surface receptor that belongs to the tumor necrosis factor receptor family and that mediates apoptosis. This genetic abnormality results in lymphoproliferation characterized by the accumulation of CD4-CD8- (double negative [DN]) T cells, autoantibody production, and background strain-dependent, end-organ disease. Our previous results suggested that major histocompatibility complex (MHC) class I may be involved in the development of DN cells. To test this hypothesis, we derived C57BL/6-lpr/lpr (B6/lpr) mice that were deficient for the beta 2-microglobulin gene (beta 2m lpr) and had no detectable class I expression. At 6 mo of age, compared with B6/lpr littermates with normal class I genes, these mice showed greatly reduced lymphadenopathy, mostly due to a dramatic decrease in the number of DN cells. Significant changes in the percentage of other T cell subsets were noted, but only gamma/delta+ T cells showed a marked increase in both percentage and absolute numbers. Analysis of T cell receptor V beta expression of the remaining DN T cells in beta 2m -lpr mice showed a shift to a CD4-like repertoire from a CD8-like repertoire in control B6/lpr mice, indicating that a small MHC class II selected DN population was unmasked in lpr mice lacking class I. We also found that the production of immunoglobulin G (IgG) autoantibodies (antichromatin and anti-single stranded DNA), total IgG and IgG2a, but not total IgM or IgM rheumatoid factor, was significantly reduced in the beta 2m -lpr mice. This work suggests that >90% of DN T cells in lpr mice are derived from the CD8 lineage and are selected on class I. However, a T cell subset selected on class II and T cells expressing gamma/delta are also affected by the lpr defect and become minor components of the aberrant DN population.

A 60-year-old male with bloody diarrhea and altered mental status secondary to a suspected intravascular crotalid envenomation.

We present the case of a 60-year-old male who presented to our emergency department approximately 2 hours after a suspected intravascular pit viper envenomation to the area of the left ankle. By his arrival to our facility he was profoundly confused and hypotensive with bloody diarrhea, coagulopathy and multi-organ dysfunction including renal insufficiency, non-ST elevation myocardial infarction, lactic acidosis and hemoconcentration. His only cutaneous sign of envenomation was a small (1-2 cm) area of ecchymosis adjacent to the Achilles tendon on the lateral aspect of the ankle. He was given aggressive crystalloid resuscitation for circulatory support and received 20 vials of Cro-fab (10 vials 2 hours apart) and 2 units of FFP in the Emergency Department. The patient ultimately had resolution of his coagulopathy and was discharged on hospital day five with no long term sequela.

Genetic linkage of IgG autoantibody production in relation to lupus nephritis in New Zealand hybrid mice.

F1 hybrids of New Zealand black (NZB) and New Zealand white (NZW) mice are a model of human systemic lupus erythematosus. These mice develop a severe immune com-plex-mediated nephritis, in which antinuclear autoantibodies are believed to play the major role. We used a genetic analysis of (NZB x NZW)F1 x NZW backcross mice to provide insight into whether different autoantibodies are subject to separate genetic influences and to determine which autoantibodies are most important in the development of lupus-like nephritis. The results showed one set of loci that coordinately regulated serum levels of IgG antibodies to double-stranded DNA, single-stranded DNA, total histones, and chromatin, which overlapped with loci that were linked to the production of autoantibodies to the viral glycoprotein, gp70. Loci linked with anti-gp70 compared with antinuclear antibodies demonstrated the strongest linkage with renal disease, suggesting that autoantibodies to gp70 are the major pathogenic antibodies in this model of lupus nephritis. Interestingly, a distal chromosome 4 locus, Nba1, was linked with nephritis but not with any of the autoantibodies measured, suggesting that it contributes to renal disease at a checkpoint distal to autoantibody production.

Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts.

Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.

The intragraft CD8+ T cell response in renal allograft rejection in the mouse.

To identify the role of donor class I alloantigens in regulating the CD8+ T cell response to a kidney allograft, we analyzed and compared the CD8+ infiltrate in kidney transplants from MHC class I-deficient (class I-) mouse donors and class I+ controls. One week after transplantation, there was a prominent CD8+ infiltrate in control allografts, whereas CD8+ T cells were virtually absent in grafts from class I- donors. In class I+ allografts, infiltrating CD8+ cells utilized a wide range of T cell receptor (TCR) Vbeta families and their Vbeta usage was similar to that of the systemic CD8+ population. However, there was a modest but significant overrepresentation of cells bearing Vbeta8 in the graft compared with the spleen due to an expansion of CD8+ Vbeta8.3+ cells. This could be detected as early as 1 week and became more pronounced by 3 weeks after transplantation. In 3-week allografts, only 52% of CD8+ cells expressed alphabetaTCR. Among T cells isolated from class I+ grafts, the CD8+ Vbeta8+ cells demonstrated allospecific responses that were numerically larger than responses of the CD8+ Vbeta8- population. In contrast to the early (1 week) time point, significant numbers of CD8+ cells could be isolated from class I- grafts by 3 weeks after transplantation and their Vbeta repertoire resembled that seen in controls. While increasing numbers of CD8+ Vbeta8+ were present in the class I- grafts at 3 weeks, this increase was not statistically significant. Thus, expression of class I alloantigens on a kidney graft plays an important role in regulating the rate of accumulation of CD8+ T cells in rejecting kidney grafts. However, the TCR Vbeta repertoire of the CD8+ T cell infiltrate is largely determined by factors that are independent of normal class I expression on the graft.

Type I interferon signaling is involved in the spontaneous development of lupus-like disease in B6.Nba2 and (B6.Nba2 x NZW)F(1) mice.

Several studies have described a role for type I interferons (IFNalphabeta) in the initiation and/or prolongation of autoimmune diseases. Most pronounced has been the association of disease activity with what is now known as 'the interferon signature' of gene expression in peripheral blood mononuclear cells from lupus patients. In correlation, studies have shown that inhibition of IFNalphabeta signaling abrogates disease in various mouse models of lupus. New Zealand black (NZB) and B6.Nba2 congenic mice spontaneously develop elevated levels of serum anti-nuclear autoantibodies (ANAs). Nevertheless, neither of these strains develop fatal renal disease. The female F1 offspring of NZB or B6.Nba2 crossed with New Zealand white (NZW) mice do, however, develop kidney disease. We have previously shown that increases in endogenous IFNalphabeta levels in (B6.Nba2 x NZW)F1 mice leads to accelerated development of renal disease in an IFNalphabeta-dependent manner. We now show that B6.Nba2 and (B6.Nba2 x NZW)F1 mice deficient for the IFNalphabeta-receptor fail to develop ANA and renal disease, although the mice have substantial immune complex deposition in the glomeruli. Thus, endogenous IFNalphabeta might influence disease by affecting B-cell activation and differentiation, as well as the kidneys' susceptibility to damage, the latter perhaps through induction of a local inflammatory milieu.

Familial visceral neuropathy: a defined entity?

Familial visceral neuropathy (FVN) is a heterogeneous group of disorders due to abnormalities of the myenteric plexus. FVN with neuronal intranuclear inclusions is one particular form of FVN with a variable phenotype that includes achalasia, gastro-esophageal reflux, intestinal dysmotility and pseudo-obstruction, dysarthria, peripheral neuropathy and pupillary defects, and the presence of intranuclear inclusions within the neurons of the enteric nervous system. We present a four-generation family in which 10 individuals (7 of whom have been examined) are affected with FVN. The family was previously reported as familial esophageal achalasia, an autosomal recessive condition (MIM200400). At that time, several individuals in a single sibship were affected and there were no manifestations in either parent. Since that report, two individuals have had affected children and the mother has developed symptoms and has abnormalities on electromyography, thus enabling us to reclassify the family. This family provides further evidence of autosomal dominant inheritance, with marked variation in expression.

p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes.

In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53, but the molecular basis for this co-operation remains unresolved. Here we show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a p53 and p21Cip1-dependent cycle arrest and to terminal differentiation. Raf activation in keratinocytes lacking p53 or p21Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21Cip1 function is necessary to disable growth-inhibitory Raf/MAP kinase signalling. Activation of oncogenes, including Ras, has been reported to stabilize and activate p53 via induction of the tumour suppressor p19ARF. However, the response to Raf in p19ARFI-/- keratinocytes was indistinguishable from wild-type controls. Thus, p19ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms.

Possible role of insertion sequence IS257 in dissemination and expression of high- and low-level trimethoprim resistance in staphylococci.

The transposon-like structure Tn4003 and related elements were found to encode high- and low-level trimethoprim resistance (Tpr) in Staphylococcus aureus and coagulase-negative staphylococci. By using transcriptional fusions in Escherichia coli, the variation in resistance levels was found to correlate with the transcriptional activity of the region presumed to carry the promoter for the operon containing the Tpr dihydrofolate reductase gene, dfrA, encoded by these elements. The reduced transcriptional activities exhibited by elements encoding low-level Tpr appear to be a consequence of deletions adjacent to the copy of IS257 which normally encodes the -35 sequences of these promoters. The data obtained not only support the involvement of IS257 in the transcription of the proposed thyE-dfrA-orf-140 operon of Tn4003 but may also implicate this insertion sequence in the mechanisms resulting in the variation in Tpr levels observed in staphylococci.

Biased liver T cell receptor V beta repertoire in a murine graft-versus-host disease model.

Murine graft-vs-host disease (GVHD) results in destruction of small bile ducts in the liver. We analyzed the TCR V beta repertoire of lymphocytes isolated from the livers and spleens of individual B10.D2 into irradiated BALB/c GVHD mice by means of two-color immunofluorescence. Each mouse showed an increase in at least one V beta population in the liver and spleen, but the expanded V beta populations were heterogeneous and variable among individual GVHD mice. Overall, the repertoire of liver CD4 cells was biased toward V beta 2 and 3 expression with 65 and 88% of mice, respectively, showing an increase in these subsets. The splenic CD4 cell repertoire was biased toward V beta 3 and 4 expression (50% of mice each). The repertoire of CD8 cells was less biased with 20 to 35% of mice showing expansions of V beta 3+, 4+, 5+, 6+, 8.1+, 8.2+, and 8.3+ T cells in both the liver and spleen. V beta 2+ CD4 cells were increased preferentially in the liver compared with the spleen. These results indicate that the infiltrating liver and splenic T cells are polyclonal and suggest that donor T cells recognize multiple host non-MHC Ags in this GVHD model. Alloantigens recognized by V beta 2+ CD4 cells appear to be selective for the liver. Expansion of V beta 3+ CD4 cells may reflect recognition of the host Mls-3 superantigen.

Combination contraceptive effects on monthly cycle of plasma aldosterone, renin activity and renin substrate.

A post-ovulatory peak of fasting supine plasma aldosterone (PA) preceded or accompanied by an increase in plasma renin activity (PRA) was previously reported. These studies have now been extended in 4 additional normal menstruating women and 4 women taking oestrogen-progestogen oral contraceptive pills (OCP), all studied daily for an entire cycle. Distinct luteal phase increases in PRA were seen in the 4 normals, with 2 also demonstrating a rise in PA. Plasma renin substrate (PRS) was usually unvarying throughout the control cycles. The women taking OCP, on the other hand, all had PA and PRA peaks that were apparent by the fourth or fifth day of taking "the pill". All 4 of the treated women had elevated PRS levels but only one woman showed an increase which preceded the elevation of PRA and PA. Plasma cortisol levels were usually above the normal range in the women taking OCP. This study thus indicates that factors other than oestrogen-induced increased substrate production may be responsible for the PRA and PA rise during OCP treatment. Such factors might be the natri-uretic effects of oestrogens and progestogens or a direct effect on renin secretion by one of these steroids.

Random amplified polymorphic DNA and plasmid analyses used in investigation of an outbreak of multiresistant Klebsiella pneumoniae.

Multiresistant Klebsiella pneumoniae strains with plasmid-borne extended-spectrum beta-lactamases (ESBL) are increasingly frequent nosocomial pathogens. A major outbreak of clinical infections, mainly involving patients in the Newborn Services Unit with limited spread to adult patients, occurred at our hospital. This epidemic was investigated by typing the isolates phenotypically and with random amplified polymorphic DNA analysis (RAPD) and plasmid analysis. Forty-eight isolates, consisting of 44 consecutive clinical isolates and 4 selected surveillance isolates, were studied. A single decamer primer was used for the RAPD, and this was effective in demonstrating that the majority of isolates (45 of 48) had the same profile. Three other isolates had different RAPD patterns identifying them as nonepidemic strains. Plasmids were extracted by alkaline lysis with Magic-miniprep kits from 10 isolates selected to represent the epidemic and nonepidemic strains. This method produced small (< 20-kb) plasmids; larger ESBL-carrying plasmids were not produced, but the small plasmids nonetheless allowed strain differentiation. Antibiotic susceptibility patterns alone were not reliable as strain indicators, since some isolates with the RAPD pattern characteristic of the epidemic strains did not express ESBL and therefore were susceptible to extended-spectrum cephalosporins. The investigation showed the predominance of a single epidemic strain that was transmitted between patients in the Newborn Services Unit. RAPD was the best of the methods used for detecting strain differences, and its speed and ability to type a wide variety of species suggest that it will be an increasingly useful molecular epidemiologic tool.

Selection of the T cell receptor repertoire in Lpr mice.

The development of double-negative (CD4-, CD8-) T cells and other T cells subsets in lymphoproliferation (lpr) mice continues to be poorly defined. Recent studies indicate that lpr is a mutation of a receptor mediating apoptosis. It has thus been hypothesized that T cell development in the thymus should be abnormally affected. In this study, we analyzed the TCR V beta repertoire of double-negative T cells as well as CD4+ and CD8+ single-positive subsets in various lpr and matched non-lpr strains. Particular comparisons were made to determine the influence of different class I and class II molecules on repertoire formation. The data demonstrate that positive and negative selection of the CD4+ and CD8+ subsets are normal in lpr mice when compared with non-lpr congenic mice. Surprisingly, the result also suggest that double-negative T cells are mostly selected on class I MHC molecules in a pattern similar to the CD8+ population, and that T cells positively selected on class II MHC antigens may be absent from the double-negative population. In all lpr strains, we also found an increased percentage of double-negative V beta 8.3+ cells out of proportion to levels in the CD4+ or CD8+ subsets. Longitudinal studies and studies in thymectomized animals showed that this increase reflects a peripheral process selectively affecting V beta 8.3+ double-negative T cells. Together, these repertoire data provide new insight into the effect of the lpr genetic defect on T cell development and the derivation of double-negative T cells. Despite the role of Fas in apoptosis and the abnormal expression of this gene in lpr mice, the present results support the hypothesis that thymic events are relatively normal in lpr mice, and that the double-negative T cells are mostly class I MHC selected and expanded by abnormal peripheral processes.

Genetic amniocentesis: gestation-specific pregnancy outcome and comparison of outcome following early and traditional amniocentesis.

Amniocentesis remains the most common prenatal diagnostic invasive procedure for fetal karyotyping. During counselling prior to this procedure miscarriage rates are often quoted as a single figure. In this review of 2924 amniocenteses, we report that miscarriage rates vary with the gestational age at which the procedure is performed. The total miscarriage rate was 1.0 per cent after early amniocenteses (11 + 0-14 + 6 weeks) and 1.2 per cent after traditional mid-trimester amniocenteses (15 + 0-18 + 6 weeks). The rate was greatest (3.1 per cent) for amniocenteses performed after 18 + 6 weeks' gestation. The cumulative miscarriage risk increased from 0.03 per cent one week after the procedure to plateau at 1.1 per cent five weeks after the procedure. The preterm and still-birth rates following amniocenteses were similar in early and traditional mid-trimester amniocenteses but were significantly higher when amniocenteses were performed after 19 weeks' gestation. Although the incidence of talipes equinovarus was higher after early amniocentesis compared with traditional mid-trimester amniocenteses (1.4 per cent versus 0.2 per cent), none of the affected infants required corrective surgery. We conclude that counselling for this procedure should be tailored to each unit's unintended fetal loss rate based on cumulative rates. Such figures should be available to parents to assist them in their decision-making.