A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis.

BACKGROUND: We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS: Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS: MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S): This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

Multi-locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host-adapted strain.

AIMS: Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST)to investigate evolutionary relationships between them. METHODS AND RESULTS: Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. CONCLUSIONS: Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. SIGNIFICANCE AND IMPACT OF THE STUDY: Host-pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.

Risk factors for the occurrence of Escherichia coli virulence genes eae, stx1 and stx2 in wild bird populations.

Shiga toxin-producing Escherichia coli (STEC) can cause serious disease in human beings. Ruminants are considered to be the main reservoir of human STEC infections. However, STEC have also been isolated from other domestic animals, wild mammals and birds. We describe a cross-sectional study of wild birds in northern England to determine the prevalence of E. coli-containing genes that encode Shiga toxins (stx1 and stx2) and intimin (eae), important virulence determinants of STEC associated with human disease. Multivariable logistic regression analysis identified unique risk factors for the occurrence of each virulence gene in wild bird populations. The results of our study indicate that while wild birds are unlikely to be direct sources of STEC infections, they do represent a potential reservoir of virulence genes. This, coupled with their ability to act as long-distance vectors of STEC, means that wild birds have the potential to influence the spread and evolution of STEC.

Next generation sequencing (NGS) to improve the diagnosis and management of patients with disorders of sex development (DSD).

Disorders of sex development (DSDs) are a diverse group of conditions where the chromosomal, gonadal or anatomical sex can be atypical. The highly heterogeneous nature of this group of conditions often makes determining a genetic diagnosis challenging. Prior to next generation sequencing (NGS) technologies, genetic diagnostic tests were only available for a few of the many DSD-associated genes, which consequently had to be tested sequentially. Genetic testing is key in establishing the diagnosis, allowing for personalised management of these patients. Pinpointing the molecular cause of a patient's DSD can significantly impact patient management by informing future development needs, altering management strategies and identifying correct inheritance pattern when counselling family members. We have developed a 30-gene NGS panel, designed to be used as a frontline test for all suspected cases of DSD (both 46,XX and 46,XY cases). We have confirmed a diagnosis in 25 of the 80 patients tested to date. Confirmed diagnoses were linked to mutations in AMH, AMHR2, AR, HSD17B3, HSD3B2, MAMLD1, NR5A1, SRD5A2 and WT1 which have resulted in changes to patient management. The minimum diagnostic yield for patients with 46,XY DSD is 25/73. In 34/80 patients, only benign or likely benign variants were identified, and in 21/80 patients only variants of uncertain significance (VOUS) were identified, resulting in a diagnosis not being confirmed in these individuals. Our data support previous studies that an NGS panel approach is a clinically useful and cost-effective frontline test for patients with DSDs.

Simon metabolites of alpha-tocopherol are not formed via a rate-controlling scission of the 3'C-H bond.

The major in vivo oxidation products of alpha-tocopherol, alpha-T, are the Simon metabolites, 1 and 2. For these compounds to be formed from alpha-T the polyisoprenoid tail of alpha-T must be oxidatively cleaved at the 3' carbon atom. Comparison of the levels of 2R,4'R,8'R-alpha-(3',3'-2H2)-T and 2R,4'R,8'R-alpha-[5,7-(C2H3)2]-T remaining in various tissues of rats which had been preloaded with equal quantities of these two forms of vitamin E following a change to a vitamin E-free diet has shown that there is no statistically significant difference in the rates of loss of these two deuterium-labeled alpha-T's. This demonstrates that the Simon metabolites are not formed by a rate-controlling scission of the 3'C-H bond of alpha-T.

Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells.

Previous studies have shown that thrombospondin (TSP) is an adhesion factor for some human tumor cells. The previous studies have shown further that tumor cells which utilize TSP as an adhesion factor also synthesize it. This study continues the effort to understand how TSP production and expression are regulated in human tumor cells and the consequences of this for the cells. It is shown that differences among cell lines in their capacity to biosynthesize TSP are associated with differences in TSP specific mRNA levels. This indicates that biosynthesis is regulated at the transcriptional level. There is also a direct relationship between TSP biosynthesis and secretion into the culture medium and expression at the cell surface. The cells which are the most biosynthetically active secrete amounts of TSP into the culture medium that are sufficient to elicit a detectable response in the cell-substrate adhesion assay. The kinetics of TSP secretion by these cells are in accord with the kinetics of attachment and spreading of the same cells in the absence of exogenous adhesion factors. These data are consistent with the idea that endogenously produced TSP promotes the adhesion of the cells which synthesize it in an autocrine manner.

Magnetic resonance angiography of the central chest veins. A new gold standard?

PURPOSE: The systemic chest veins may be difficult to show comprehensively by contrast venography, especially if there is limited venous access or contraindications to intravenous contrast. As an alternative, can magnetic resonance angiography (MRA) reliably detect occluded chest veins and predict suitable sites for central venous access? PATIENTS AND METHODS: Eighty-four patients were examined using breath-hold time-of-flight MRA and three-dimensional image reconstruction. Thirty-three were evaluated to identify possible central venous access. Fifty-seven patients were examined to diagnose and stage central venous occlusion. RESULTS: The associated diagnoses were malignancy 46, parenteral nutrition 21, hemodialysis 6, chemotherapy 4, and other long-term venous access 7. Of the 28 patients in whom MRA predicted a patent site for central venous access, satisfactory access was achieved. In two patients, cannulation of veins shown to be occluded on MRA was attempted unsuccessfully. Correlation with contrast venography was available in 17. There was agreement with MRA concerning the level of occluded veins in all cases. Contrast venography did not show all patent veins, including some accessed during surgical line placement. CONCLUSION: Compared with surgical line placement or contrast venography, MRA of the systemic chest veins is accurate. Patent and occluded chest veins are reliably defined, including potential sites for central line placement, in a way that is not possible with other techniques. MRA may be the new "gold standard" for defining systemic venous anatomy in the chest.

Regulation of cytokine mRNA expression in lipopolysaccharide-stimulated human macrophages.

One of the responses of the human macrophage to lipopolysaccharide (LPS) is the production of a number of cytokines. The regulation of these cytokines is still not clearly understood. To study this regulation, mRNA levels of interleukin 1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF-alpha), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-8/neutrophil chemotactic factor were determined in 10-day-old differentiated macrophages following stimulation with a low dose of LPS (0.001 to 10 ng/mL) with use of the polymerase chain reaction. Increased levels of mRNA for IL-8 were detectable after exposure to a very low dose of LPS (0.001 ng/mL) and levels of IL-1 beta and TNF-alpha were detectable only after stimulation with doses of 0.01 ng/mL. The mRNA for IL-8 was detected 30 minutes after the addition of LPS, while those for IL-1 beta and TNF-alpha were only measurable at 1 hour. The mRNAs for IL-1 alpha, IL-6, and GM-CSF were detectable only with a higher dose of lipopolysaccharide and only after a longer exposure time. In addition, the messages for IL-6 and GM-CSF were measurable for a short time, while those of IL-8 and of IL-1 beta were detectable for a longer time. The secretion of TNF-alpha and GM-CSF tightly followed gene activation, and that of IL-6 and IL-8 steadily increased even after the mRNA level of these cytokines returned to baseline. Secretion of IL-1 alpha and IL-1 beta was hardly detected, although their gene activation was obvious. These data indicate that cytokine mRNA levels following lipopolysaccharide stimulation are highly regulated. Individual cytokines show variable patterns of response. These responses are both dose and time dependent and are not necessarily associated with the secretion of protein.

Concentration-response curves for ethylene-oxide-induced heritable translocations and dominant lethal mutations.

Male mice were subjected to repeated inhalation exposures to different concentrations (165, 204, 250, or 300 ppm) of ethylene oxide (EtO) during an 8.5-week period. Transmitted clastogenic effects of these exposures were measured in terms of induction of dominant lethal mutations and heritable translocations. The concentration-response curves for both endpoints are not linear but are markedly concave upward. Significant increases in dominant lethals were detected at all concentrations, except the lowest one. In comparison, the incidences of heritable translocations were significantly increased at all concentrations.

No evidence found for induction of dominant lethal mutations and heritable translocations in male mice by calcium cyclamate.

Calcium cyclamate, an artificial sweetener, was studied for its effectiveness in inducing transmissible chromosomal aberrations in germ cells of male mice. Both the dominant-lethal and the heritable translocation tests were carried out following daily treatment (on weekdays) of males by oral intubation with the maximum tolerated dose for 6 weeks. Calcium cyclamate is negative in both tests; therefore, there is no evidence of induced chromosome breakage and exchange.

Tests for dominant-lethal effects of 1,2-dibromo-3-chloropropane (DBCP) in male and female mice.

DBCP was studied for dominant-lethal effects in male and female mice and for total reproductive effects in females. In males it was administered either intraperitoneally or subcutaneously while in females it was given only by the former route. No DBCP-related response was observed in either males or females indicating its ineffectiveness in inducing chromosomal aberrations or cytotoxicity in mouse germ cells. These findings differ markedly from the observations made in rats by other investigators. Thus, the probable existence of a species difference in germ cell response to DBCP has been strengthened by the availability of the present results. It should be noted, however, that only two stocks of male mice have been studied so far for dominant-lethal and germ cell cytotoxicity effects.

Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death.

When previously mated female mice were exposed to inhaled ethylene oxide at the time of fertilization of their eggs or during early pronuclear stage of the zygote (before DNA synthesis), a high incidence of mortality among conceptuses and of congenital abnormalities among both the dead and the surviving fetuses was observed. The developmental stage at which death occurred ranged from near the time of implantation to day 17 of gestation when examination of the uterine contents was performed. In comparison, midgestation and late fetal deaths were absent or minimal when the females were exposed either before mating or when conceptuses were in later zygotic stages (pronuclear DNA synthesis) or had reached the early two-cell stage. The random types of congenital abnormality observed and the remarkable stage-dependent sensitivity suggest a genetic basis for the response. The effects differ, both from genetic damages induced in premating germ cells, which lead only to death near the time of implantation, and from teratogenic damage, which leads to malformations only when exposure of embryos occurs during the period of major organogenesis.

Mutagen-induced fetal anomalies and death following treatment of females within hours after mating.

In an earlier study (Generoso et al., 1987), it was observed that the mutagen, ethylene oxide (EtO), produced remarkable increases in the incidence of developmental abnormalities and death of fetuses when early zygotic stages were exposed. This is a major finding in experimental induction of embryopathy, implicating genetic damage to the zygotes as the likely cause. In the subsequent study reported here, 3 other mutagens--ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), and triethylene melamine (TEM), were studied for embryopathic effects following exposure of dictyate oocytes, prefertilization oviducal eggs and sperm, early pronuclear zygotes, zygotes undergoing pronuclear DNA synthesis, and two-cell embryos. All 4 mutagens produced developmental abnormalities among living fetuses following exposure of early pronuclear zygotes (the only stage studied for this endpoint in this report). With respect to stage specificity and gestational timing of death of conceptuses, EMS and EtO on one hand and ENU and TEM on the other, are very similar to one another. EMS, like EtO, produced a high incidence of midgestation and late fetal deaths only in prefertilization oviducal eggs and sperm and in early pronuclear eggs. In contrast, ENU and TEM produced high losses of conceptuses in all postmating stages studied but death occurred primarily prior to or around the time of implantation. Thus, the frequency of induction and the expression of embryopathy, which ranged from early embryonic preimplantation and late fetal deaths to subtle fetal anomalies, are dependent upon the stage exposed and the mutagen used.

Difference between two hybrid stocks of mice in the incidence of congenital abnormalities following X-ray exposure of stem-cell spermatogonia.

Unbalanced (duplication/deficiency) sperm from balanced reciprocal translocations induced in spermatogonial stem cells of mice generally lead to embryonic lethality around the time of implantation. In a recent study (Generoso et al., 1985), it was found that the incidence of X-ray-induced embryonic lethality differed markedly between two hybrid stocks of irradiated male mice. A parallel difference in the frequencies of reciprocal translocations was observed cytologically in the meiocytes of irradiated males. In the present report, which is an adjunct to the study by Generoso et al. (1985), it was determined whether or not similar differences between the two stocks exist for congenital defects resulting from genetic damage to stem-cell spermatogonia. The results indicate not only an association between the frequencies of induced reciprocal translocations and congenital abnormalities, but also a parallel greater frequency of induced malformations in the (C3H X 101)F1 stock versus the (SEC X C57BL)F1 stock of males.

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole.

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.

Difference in the response of two hybrid stocks of mice to X-ray induction of chromosome aberrations in spermatogonial stem cells.

Ionizing radiation induces balanced reciprocal translocations in spermatogonial stem cells of mice. From cells carrying these rearrangements, which can be scored cytologically in the diakinesis-metaphase I stage, balanced normal, balanced translocated and unbalanced (duplication/deficiency) sperm can be produced. The relationship between expected (calculated from cytological data) and observed frequencies of embryonic lethality (presumably as a result of unbalanced sperm fertilizing the egg) following exposure of spermatogonial stem cells to X-rays was studied in two hybrid stocks. A marked difference in the incidence of induced embryonic lethality was found between the two stocks. Similarly, a difference in the cytological frequencies of translocations was also found, although smaller than that observed for embryonic lethality. Thus, it appears that the difference between the two stocks in the frequencies of embryonic lethality may be attributable both to processes occurring prior to metaphase I and to a difference in the rate of transmission of unbalanced chromosome constitutions.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Female-specific dominant lethal effects in mice.

For some chemicals, induction of presumed dominant lethal mutations has been observed only in female mice and not in males. In those cases, questions arise as to (1) whether the increased embryonic mortality is due to genetic effects of the chemicals in the oocyte or, (2) is caused indirectly through maternal toxicity, and, if genetic, (3) the basis for the sex difference. These questions were studied using the compounds adriamycin and platinol. Neither compound induces dominant lethals in male germ cells, but both increased early embryonic mortality when females were treated prior to mating (treatment of maturing oocytes). Reciprocal zygote transfer experiments ruled out, either entirely or for the large part, maternal toxicity as the cause, and cytogenetic analysis of first-cleavage metaphases revealed high incidences of chromosomal aberrations. The results of both of these experiments thus provide evidence that the early embryonic mortality resulted from genetic effects induced in oocytes. Most interestingly, each compound produced unexpected types of chromosomal aberrations. Adriamycin produced deletions, rings, and presumed chromosome-type rearrangements. Platinol, on the other hand, produced a few chromatid-type aberrations, but the bulk of aberrations were characterized by disorganization of the chromatin, minute fragments, and thread-like chromatin bridges between fragments and chromosomes or between two or more chromosomes. The latter type of cytogenetic damage was observed primarily in the centromeric region. It is hypothesized that the female-specific dominant lethal effects of the two compounds are associated with the diffused state of the maturing oocyte chromosomes.

Sjögren's syndrome and serous otitis media.

Extrasalivary lymphoid abnormalities in Sjögren's syndrome are well described. Patients have been found to have lymphoid infiltration of many organ systems. The nature of the lymphoid abnormalities constitutes a spectrum ranging from benign to malignant disease. A case of an elderly patient with long standing Sjögren's syndrome is presented because of the unusual manifestation of a benign lymphoid mass in the nasopharynx producing unilateral eustachian tube obstruction and serous otitis media. Serous otitis media associated with Sjögren's syndrome is recognized, but to our knowledge in previous cases an extrasalivary lymphoid abnormality has not been implicated as the underlying pathology.

Effect of skin pigmentation on pulse oximetry accuracy in the emergency department.

OBJECTIVE: To determine whether pulse oximeter (PO) accuracy and signal quality are affected by level of skin pigmentation. METHODS: Observational study in a community hospital ED. Consecutive adult patients undergoing arterial blood gas determination were enrolled into the study. Skin pigmentation was determined by comparison with standardized color swatches under controlled lighting; assigned values were used to stratify patients into 3 groups (light, intermediate, and dark) using predetermined criteria. Simultaneous with arterial blood sampling, staff recorded PO reading of O2 saturation using the Nellcor D-25 oximeter. PO values were compared with criterion standard values measured using a 4-wavelength spectrophotometer or co-oximeter. PO signal quality also was recorded. Bias (the mean difference between PO and co-oximeter-measured values of hemoglobin saturation) and precision (the standard deviation of the bias) were calculated. Groups were compared using one-way ANOVA, Bartlett's test for variances, and chi2 test. RESULTS: O2 saturation data were obtained for 284 patients. Bias values did not differ between the 3 skin pigment groups (p = 0.79). Precision was of borderline significance (p = 0.05), but there was no dose-response relation between skin pigmentation and precision. Study personnel reported suboptimal PO function most often among patients in the dark group (p = 0.003), but this finding was of no clinical significance. PO signal failure was rare (<1% of all patients). CONCLUSIONS: Although several prior studies suggest the contrary, this study found that skin pigmentation does not affect the bias or precision of pulse oximetry. Furthermore, skin pigmentation has no clinically significant effect on PO signal quality.