Peritoneal Cytokine Levels Can Predict Anastomotic Leak on the First Postoperative Day.

BACKGROUND: Accumulating evidence suggests that peritoneal cytokine concentrations may predict anastomotic leak after colorectal surgery, but previous studies have been underpowered. OBJECTIVE: We aimed to test this hypothesis by using a larger prospectively collected data set. DESIGN: This study is an analysis of prospectively collected data. SETTINGS: This study was conducted at 3 public hospitals in Auckland, New Zealand. PATIENTS: Patients undergoing colorectal surgery recruited as part of 3 previous randomized controlled trials were included. MAIN OUTCOME MEASURES: Data on peritoneal and plasma levels of interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α on day 1 after colorectal surgery were reanalyzed to evaluate their predictive value for clinically important anastomotic leak. Area under receiver operating characteristic curve analysis was performed. RESULTS: A total of 206 patients with complete cytokine data were included. The overall anastomotic leak rate was 8.3%. Concentration levels of peritoneal interleukin-6 and interleukin-10 on day 1 after colorectal surgery were predictive of anastomotic leak (area under receiver operating characteristic curve, 0.72 and 0.74; p = 0.006 and 0.004). Plasma cytokine levels of interleukin-6 were higher on day 1 after colorectal surgery in patients who had an anastomotic leak, but this was a poor predictor of anastomotic leak. Levels of other peritoneal and plasma cytokines were not predictive. LIMITATIONS: The study was not powered a priori for anastomotic leak prediction. Although the current data do suggest that peritoneal levels of interleukin-6 and interleukin-10 are predictive of leak, the discriminative value in clinical practice remains unclear. CONCLUSIONS: Peritoneal levels of interleukin-6 and interleukin-10 on day 1 after colorectal surgery can predict clinically important anastomotic leak.

Osteoporosis and apolipoprotein E genotype in older adults: the Rancho Bernardo study.

Recent studies reported an association between apolipoprotein E (ApoE) 4 and osteoporosis. We examined the association of ApoE 4 genotype with bone mineral density (BMD), bone loss and fracture risk in 596 men and 332 community-dwelling women aged 45-95 years. Women were postmenopausal and not using estrogen. At the baseline visit, BMD was measured at the ultradistal and midshaft radius using single photon densitometry, and at the hip and lumbar spine using dual-energy X-ray absorptiometry. Hip and lumbar spine BMD levels were remeasured 4 years later. Self-reported fractures were confirmed by radiology reports in 95% of cases. ApoE allele distribution did not vary by age; 25% of men and 20% of women had one ApoE 4 allele. There were no differences in BMD at the lumbar spine, total hip, ultradistal or midshaft radius in men or women with the ApoE 4 allele compared with men or women without the ApoE 4 allele. After an average 4 year interval, there were also no differences in the annualized percent change in BMD at the hip or lumbar spine in men or women with or without an ApoE 4 allele. One or more clinical fractures were reported by 55 men and 109 women. Fewer, not more, clinical fractures were reported in men and women with an ApoE 4 allele; these differences were not statistically significant (p = 0.21 and p = 0.62, respectively). These data do not support the hypotheses that there is an association between ApoE genotype and BMD, bone loss or osteoporotic fractures in older community-dwelling men or women.

Use of directly coupled ion-exchange liquid chromatography-mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy as a strategy for polar metabolite identification.

Ion-exchange LC-MS and LC-NMR have been successfully used to identify a novel N-acetyl metabolite of a highly polar drug candidate [2-(ethanimidoylamino)ethyl]sulfonyl alanine (GW273629) under development as a therapeutic agent. This has been achieved using a simple HPLC method without the need for complicated and time consuming pre- or post-column derivatisation. Ion-exchange chromatography using simple ionic strength buffer and organic solvent mobile phases, as applied here, should be suitable for the analysis of other charged polar species. Optimisation of the system described could result in the development of a rational generic HPLC approach specifically designed for the characterisation of polar drug molecules and their metabolites.

Genome screen for QTLs contributing to normal variation in bone mineral density and osteoporosis.

A major determinant of the risk for osteoporosis is peak bone mineral density (BMD), which is largely determined by genetic factors. We recently reported linkage of peak BMD in a large sample of healthy sister pairs to chromosome 11q12-13. To identify additional loci underlying normal variations in peak BMD, we conducted an autosomal genome screen in 429 Caucasian sister pairs. Multipoint LOD scores were computed for BMD at four skeletal sites. Chromosomal regions with LOD scores above 1.85 were further pursued in an expanded sample of 595 sister pairs (464 Caucasians and 131 African-Americans). The highest LOD score attained in the expanded sample was 3.86 at chromosome 1q21-23 with lumbar spine BMD. Chromosome 5q33-35 gave a LOD score of 2.23 with femoral neck BMD. At chromosome 6p11-12, the 464 Caucasian pairs achieved a LOD score of 2.13 with lumbar spine BMD. Markers within the 11q12-13 region continued to support linkage to femoral neck BMD, although the peak LOD score was decreased to 2.16 in the sample of 595 sibling pairs. Our study is the largest genome screen to date for genes underlying variations in peak BMD and represents an important step toward identifying genes contributing to osteoporosis in the general population.

A genetic linkage map of the baboon (Papio hamadryas) genome based on human microsatellite polymorphisms.

A first-generation genetic linkage map of the baboon (Papio hamadryas) genome was developed for use in biomedical and evolutionary genetics. Pedigreed baboons (n = 694) were selected from the breeding colony maintained by the Southwest Foundation for Biomedical Research. To facilitate comparison with the human genome, the baboon linkage map consists primarily of human microsatellite loci amplified using published human PCR primers. Genotypes for 325 human microsatellites and 6 novel baboon microsatellites were used in linkage analyses performed with the MultiMap expert system. The resulting sex-averaged meiotic recombination map covers all 20 baboon autosomes, with average spacing among loci of 7.2 cM. Direct comparison among homologous (orthologous) loci reveals that, for 7 human autosomes, locus order is conserved between humans and baboons. For the other 15 autosomes, one or more rearrangements distinguish the two genomes. The total centimorgan distances among homologous markers are 28.0% longer in the human genome than in the baboon, suggesting that rates of recombination may be higher in humans. This baboon linkage map is the first reported for any nonhuman primate species and creates opportunities for mapping quantitative trait loci in baboons, as well as for comparative evolutionary analyses of genome structure.

Seroconversion to antibodies against Kaposi's sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi's sarcoma.

BACKGROUND: If Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma, serologic evidence of infection should be present in patients before the disease develops. METHODS: Using an immunoblot assay for two latent nuclear antigens of KSHV, we tested serum samples from homosexual male patients with the acquired immunodeficiency syndrome (AIDS) with and without Kaposi's sarcoma (HIV-infected men with hemophilia), HIV-seronegative blood donors, and HIV-seronegative patients with high titers of antibodies against Epstein-Barr virus (EBV). Serial serum samples obtained from patients with Kaposi's sarcoma before the diagnosis of the disease were tested for evidence of seroconversion. RESULTS: Of 40 patients with Kaposi's sarcoma, 32 (80 percent) were positive for antibodies against KSHV antigens by the immunoblot assay, as compared with only 7 of 40 homosexual men (18 percent) without Kaposi's sarcoma immediately before the onset of AIDS. Of 122 blood donors, 22 EBV-infected patients, and 20 HIV-infected men with hemophilia, none were seropositive. When studied by the immunoblot assay over a period of 13 to 103 months, 21 of the 40 patients with Kaposi's sarcoma (52 percent) seroconverted 6 to 75 months before the clinical appearance of Kaposi's sarcoma. The median duration of antibody seropositivity for KSHV-related latent nuclear antigens before the diagnosis of Kaposi's sarcoma was 33 months. CONCLUSIONS: In most patients with kaposi's sarcoma and AIDS, seroconversion to positivity for antibodies against KSHV-related nuclear antigens occurs before the clinical appearance of Kaposi's sarcoma. This supports the hypothesis that Kaposi's sarcoma results from infection with KSHV.

Batched analysis of genotypes.

Polymorphic microsatellite markers are widely used in molecular analyses. The range of allele sizes and the allele frequencies within a population are important characteristics of the marker. Their determination previously has involved genotyping a large number of individuals. We have developed a technique for defining these characteristics by coamplification of many samples in a DNA pool. Groups of 32 and 42 DNA samples were genotyped and results were compared with those from individual genotype determinations. To improve the accuracy in the estimation of allele frequencies, arithmetic removal of stutter bands was carried out and the consistency of each marker was characterized. This approach was also applied to a group of 94 individuals. All of the work has been done using nonradioactive methods. Potential applications of this technique are in population genetics, high throughput genotyping, and loss of heterozygosity studies.

Vitamin D receptor alleles and rates of bone loss: influences of years since menopause and calcium intake.

A genetic marker for the 1,25-dihydroxyvitamin D receptor (VDR) is reported to account for much of the heritable component of bone density. It is not known whether VDR genotype influences bone accretion or loss, or how it is related to calcium metabolism. The VDR genotype was determined in 229 healthy postmenopausal women who previously participated in a calcium trial. VDR alleles were designated according to presence (b) or absence (B) of the BsmI restriction enzyme cutting site. There were 83 bb, 102 Bb, and 44 BB individuals. Two-thirds of the women took 500 mg of calcium supplement (mean calcium intake = 892 mg/day) and one-third a placebo (mean = 376 mg/day). Bone mineral density (BMD) at the femoral neck, spine, and radius were measured by dual- and single-photon absorptiometry at baseline and after 1 and 2 years. Among women more than 10 years postmenopausal, those with the BB genotype had the lowest femoral neck BMD. Rates of bone loss over 2 years were greater in the BB group at all sites (e.g., at the femoral neck, bb, 0.45 +/- 0.43; Bb, -0.01 +/- 0.40; BB, -0.99 +/- 0.50%/year; BB vs. bb, p = 0.01), and this trend was found both in women < 10 years since menopause (e.g., at the radius, bb, 0.43 +/- 0.47; Bb, -0.37 +/- 0.42; BB, -1.20 +/- 0.59% per year; BB vs. bb, p = 0.02) and those > or = 10 years (radius, bb, -0.71 +/- 0.41; Bb, 0.08 +/- 0.39; BB, -1.41 +/- 0.49% per year; BB vs. Bb, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence and clinical correlations of MLL gene rearrangements in AML-M4/5.

Rearrangements of the human trithorax gene (MLL, HRX, Htrx-1, All-1) were studied by Southern blotting in blast cells stored at presentation from 65 adults with de novo acute myelomonocytic (AML-M4) and acute monocytic leukemia (AML-M5). MLL rearrangements were demonstrated in 15 (23%) cases, including eight patients in whom karyotype analysis had failed to detect abnormalities of chromosome band 11q23. The patients with MLL rearrangements did not differ significantly from those with germline configurations in terms of the sex and age of the patients, the presence of lymphadenopathy, hepatosplenomegaly, or central nervous system involvement, and the absolute blast count at diagnosis. Kaplan-Meier analysis of the treated patients demonstrated no difference in survival for patients with MLL rearrangements compared with those without rearrangements. Therefore, in contrast to infantile acute leukemia, in adults with AML-M4 and AML-M5, MLL rearrangements do not identify a subgroup of patients with different clinical features or prognosis.

Cloning and characterization of the t(X;11) breakpoint from a leukemic cell line identify a new member of the forkhead gene family.

Chromosome translocations involving 11q23 are associated with a number of different types of leukemia. These translocations fuse a gene encoding a putative transcription factor, HTRXI, to genes on other chromosomes. We report cloning and sequencing the t(X;11) breakpoint region from a cell line established from an infant with acute lymphocytic leukemia. The gene AFXI, on the X chromosome, is expressed in a variety of cell types. Sequence analysis indicates a high degree of homology between AFXI and the forkhead family of transcription factors. The high degree of identity within the forkhead region and the lack of homology outside that region suggest that AFXI represents a novel forkhead family member. It is predicted that a chimeric fusion protein with altered DNA binding activity will be the result of the translocation.

Effects of reward and nonreward on frustration and attention in attention deficit disorder.

The effects of reward schedule (100%, 50%, and 30%) and termination of rewards (extinction) on 30 attention deficit disorder with hyperactivity (ADD-H) and 30 normal children were studied using measures of frustration (speed/strength of lever pulling) and attention (reaction time to a light signal). ADD-Hs pulled harder on the lever than controls during extinction and on the lowest (30%) partial schedule, providing empirical evidence that they respond with greater frustration than normals when expected rewards fail to appear. The groups did not differ on the attentional measure on 100% reward. However, the partial schedules appeared to have an alerting or motivating effect on the controls, so that they responded more quickly and consistently than ADD-Hs on the partial schedules. Findings are discussed with reference to opposing theories regarding the nature of the abnormal response of ADD-Hs to reward.

Human trithorax gene rearrangements in therapy-related acute leukaemia after etoposide treatment.

Molecular analysis of leukaemic blasts from 9 patients with secondary myeloid leukaemia reveals rearrangements of the human trithorax gene (Htrx-1) in three patients, including one in whom abnormalities of chromosome 11 band q23 were not detected by conventional cytogenetics. All three patients had been treated with epipodophyllotoxins, whilst none of the six without rearrangements had received these agents. The patients with rearrangements also presented with different clinical features. These findings support the separation of secondary leukaemia into two classes, and correlate rearrangements of the Htrx-1 gene with a group of secondary leukaemias that follow specific cancer treatment regimens.

Highly polymorphic tetramer repeat (GATA)n on human chromosome 11p15.3.

A tetrameric repeat sequence (GATA)n was identified by random DNA sequencing of chromosome 11-specific cosmid clones and located at 11p15.3 by fluorescence in situ hybridization. Oligonucleotide primers flanking the repetitive unit were used to amplify the DNA using the polymerase chain reaction (PCR) and this repetitive element was shown to be highly polymorphic and inherited in typical Mendelian fashion. Analysis of amplification products containing the repetitive element from 45 unrelated Caucasian individuals and five families showed at least five alleles at this locus, ranging from 227 to 249 bases in length. This polymorphism may serve as a useful PCR-detectable genetic marker for 11p15.3, a landmark for disease gene isolation, and a locus for identity testing.

Regional mapping of the gene encoding dihydroorotate dehydrogenase, an enzyme involved in UMP synthesis, electron transport, and superoxide generation, to human chromosome region 16q22.

De novo UMP synthesis is a critical metabolic pathway for nucleic acid synthesis and for a variety of metabolic pathways. The pathway is a target for many widely used cancer chemotherapy agents, several of which are pyrimidine analogs. Humans and cattle have been described with mutations in UMP synthesis that lead to serious inborn errors of metabolism. Dihydroorotate dehydrogenase (EC 1.3.3.1) (DHODH) carries out the fourth committed step in the pathway and may also be important for mitochondrial electron transport and oxygen radical metabolism. We report here that the gene encoding this enzyme in humans is located in the chromosomal region 16q22. With the mapping of DHODH, the mapping of all the steps of UMP synthesis is complete. All three genes involved map to different human chromosomes. This information is important in consideration of regulation of UMP synthesis in mammals, including humans.

Structure and expression of the human trithorax-like gene 1 involved in acute leukemias.

The human trithorax-like gene 1 (Htrx1 gene) is disrupted in 11q23 translocations that are associated with acute leukemias. Sequencing of a partial human cDNA revealed an open reading frame encoding 1012 amino acids with extensive homology to the Drosophila trithorax protein, particularly in the zinc finger-like domains. Htrx1 gene appears to be unique in the human genome and has been conserved during evolution. Use of the human cDNA as a probe demonstrates that this gene is interrupted in both infant and adult acute myeloid (AML) and lymphoid (ALL) leukemia patients with 11q23 translocations. The structure of the Htrx1 gene around the breakpoints shows that this part of the human gene is interrupted by nine introns. As a result of the rearrangement, zinc finger domains are translocated in both ALL and AML patients. Expression studies reveal that the Htrx1 gene differentially expresses three transcripts within the normal lymphocyte cell lineage.

Ayurvedic agents produce differential effects on murine and human melanoma cells in vitro.

This study was performed to evaluate the relative efficacy of Maharishi Amrit Kalash ambrosia (MAK-5) and Maharishi Amrit Kalash nectar (MAK-4) on murine (B-16) and human (SK-Mel) melanoma cells in culture. Ethanol extract (EE) of MAK-5 (EE-MAK-5) induced morphological differentiation (enlargement of soma and nuclei and formation of long dendritic processes) and growth inhibition in mouse melanoma cells, whereas EE-MAK-5 inhibited only growth in human melanoma cells. Murine melanoma cells were more sensitive (about 3 times) than human melanoma cells in culture to EE-MAK-5; the aqueous extract (AE) of MAK-5 (AE-MAK-5) was ineffective in both cells. Boiling EE-MAK-5 for 10 minutes or exposing it to light at room temperature for 72 hours did not alter growth-inhibiting potency. Ethanol extract of another herbal agent, MAK-4 (EE-MAK-4), inhibited growth in human melanoma cells but not in mouse melanoma cells. AE-MAK-4 was ineffective for both cells. These results suggest that murine and human melanoma cells respond differently to MAK-5 and MAK-4 and that human melanoma growth-inhibiting agents are present in both EE-MAK-5 and EE-MAK-4.

Detection of single-base mutations in a mixed population of cells: a comparison of SSCP and direct sequencing.

Single-base-pair changes in several genes have been shown to be important in carcinogenesis. We have compared the sensitivity of two commonly used techniques for detection of single-base-pair changes. Defined populations of cells were prepared by mixing wild-type Chinese hamster ovary cells with cells carrying a known mutation in the CTP synthetase gene. RNA was extracted and analyzed for the mutation by single-strand conformational polymorphism and direct sequence analysis of polymerase chain reaction products. We found that both techniques were able to detect the mutation when it was present in 25% of the cells, but that direct sequencing was slightly more sensitive and able to detect the mutation when it was present in only 20% of the population.

A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias.

Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.