Stool DNA testing for the detection of colorectal neoplasia in patients with inflammatory bowel disease.

BACKGROUND: Current approaches to the detection of colorectal neoplasia associated with inflammatory bowel disease (IBD-CRN) are suboptimal. AIM: To test the feasibility of using stool assay of exfoliated DNA markers to detect IBD-CRN. METHODS: This investigation comprised tissue and stool studies. In the tissue study, gene sequencing and methylation assays were performed on candidate genes using tissue DNA from 25 IBD-CRNs and from 25 IBD mucosae without CRN. Mutations on p53, APC, KRAS, BRAF or PIK3CA genes were insufficiently informative, but several aberrantly methylated genes were highly discriminant. In the stool study, we evaluated candidate methylated genes (vimentin, EYA4, BMP3, NDRG4) in a prospective blinded study on buffered stools from 19 cases with known IBD-CRN and 35 age- and sex-matched IBD controls without CRN. From stool-extracted DNA, target genes were assayed using quantitative allele-specific real-time target and signal amplification method. RESULTS: IBD-CRN cases included 17 with ulcerative colitis (UC) and two with Crohn's disease (CD); nine had cancer and 10 had dysplasia. Controls included 25 with UC and 10 with CD. Individually, BMP3, vimentin, EYA4 and NDRG4 markers showed high discrimination in stools with respective areas under the ROC curve of 0.91, 0.91, 0.85 and 0.84 for total IBD-CRN and of 0.97, 0.97, 0.95 and 0.85 for cancer. At 89% specificity, the combination of BMP3 and mNDRG4 detected 9/9 (100%) of CRC and 80% of dysplasia, 4/4 (100%) of high grade and 4/6 (67%) of low grade. CONCLUSION: These findings demonstrate the feasibility of stool DNA testing for non-invasive detection of colorectal neoplasia associated with inflammatory bowel disease.

An immunofiltration device for the simultaneous detection of non-treponemal and treponemal antibodies in patients with syphilis.

OBJECTIVE: The development of a rapid immunofiltration (flow-through) test for the simultaneous detection of non-treponemal and treponemal antibodies in the serum of patients with syphilis. METHODS: The assay is rapid, inexpensive, and requires limited expertise in interpreting the results. The test is based on the principle of immunofiltration, with two antigens and control material spotted on the membrane of a through-flow device. A positive test is characterised by the appearance of three red/magenta spots within 2-10 min. RESULTS: A total of 376 banked serum samples obtained from the Georgia Public Health Laboratory was examined by the flow-through test, the rapid plasma reagin (RPR) test and the Treponema pallidum passive particle agglutination assay (TPPA). The sensitivity and specificity of the non-treponemal spot were 96.5% and 97.7%, respectively, when compared with the RPR test, and the sensitivity and specificity of the treponemal test spot were 97.3% and 99.1% when compared with the TPPA test. In addition, the test yielded equivalent results to those obtained in comparator tests when 104 sera from cases of syphilis of known stage, 49 sera from diseases other than syphilis and 23 sera known to exhibit biological false-positive reactions were tested in parallel. CONCLUSIONS: These results indicate that the dual treponemal and non-treponemal assay could be used as a screen and confirmatory test for the serological diagnosis of syphilis in remote or resource-poor settings where there is a need to provide counselling and treatment at the initial consultation.

MHC Class II alleles in ulcerative colitis-associated colorectal cancer.

OBJECTIVES: Patients with ulcerative colitis are at risk for colorectal cancer (CRC). Although prior studies have shown a link between HLA genotypes and ulcerative colitis (UC) susceptibility, none have investigated HLA genotypes and UC-CRC. We therefore investigated HLA-DR/DQ alleles in UC-CRC cases and UC-controls. Furthermore, since methylation of the Class II transactivator (CIITA) gene may silence HLA expression in tumours, we correlated HLA allele frequencies with CIITA gene methylation and HLA-DR expression. METHODS: Cases and controls were matched for duration/extent of ulcerative colitis, age, ethnicity and gender, but not for primary sclerosing cholangitis (PSC). DNA was extracted from archived tissue blocks from 114 UC-CRC cases and 114 UC-controls. HLA-DR/DQ genotyping was performed using sequence-specific-oligonucleotide polymerase chain reaction (SSO-PCR). CIITA methylation was determined using methylation-specific PCR. HLA-DR immunohistochemistry was done following standard protocols. RESULTS: UC-CRC cases were more likely than UC-controls to carry the DR17 or DR13 alleles (p<0.0001 or p = 0.02, respectively). Although CIITA methylation did not vary significantly between cases and controls, DR17 and DQ2 were associated with CIITA methylation (p = 0.04 and 0.02, respectively). UC-controls more frequently carried the DR7, DR1 or DQ5 alleles (p = 0.002, 0.05 or 0.01, respectively). After adjusting for PSC, DR17 remained significantly associated with an increased risk for UC-CRC while DR7 and DQ5 remained protective. CONCLUSIONS: We report a significant association between specific HLA alleles and either the risk for (DR17) or protection from (DR7, DQ5) UC-CRC. This suggests a possible genetic predisposition for increased UC-CRC risk. In addition, DQ2 and DR17 were associated with CIITA methylation.

Apoptotic cell death of human interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are specialized mesenchyme-derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c-Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase-3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double-labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 +/- 0.2% of ICC were positive for activated caspase-3 and in the circular muscle layer 2.1 +/- 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.

Childhood agricultural and adult occupational exposures to organic dusts in a population-based case-control study of systemic lupus erythematosus.

Organic dust exposure can influence the development and symptoms of immune-related diseases such as atopy and asthma, but has rarely been examined in relation to systemic autoimmunity. The present analyses explore the association of lifetime farm and occupational organic dust exposures with systemic lupus erythematosus (SLE) in recently diagnosed patients (n = 265) compared with controls (n = 355) frequency matched by age, sex and state. Questionnaire data included childhood farm residence, childhood and adult experience with specific crops, and adult work in textiles, hog or poultry processing and paper or furniture manufacture. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression models including age, sex, state, race, education and silica exposure. Overall childhood or adult farm contact and childhood farm residence were not associated with SLE. Farm contact with livestock was inversely associated with SLE (OR = 0.55, 95% CI 0.35, 0.88). This effect was most pronounced among those with childhood farm residence and both childhood and adult livestock exposure (OR = 0.19; 95% CI 0.06, 0.63), but was difficult to separate from adult exposure to grains or corn. Other adult occupational exposures were not associated with SLE risk overall, regardless of childhood farm residence or livestock exposure, although an inverse association was seen among non-smokers (OR = 0.59; 95% CI 0.33, 1.1), particularly for textile work (OR = 0.34; 95% CI 0.19, 0.64). These exploratory findings support the development of studies to specifically investigate the effects of organic dust exposure on SLE risk, with particular attention to exposure assessment and characterization of demographics, smoking and other occupational exposures.

An experimental investigation of nitrogen gas produced during denitrification.

In situ denitrification relies on indigenous microorganisms to reduce nitrate to N(2) gas. However, when initial nitrate concentrations are large, produced gas volumes also can be very large, potentially resulting in reduced water saturation and hydraulic conductivity in the treatment zone. In this study, we investigated the fate of N(2) and other gases produced during denitrification in a laboratory flow cell containing packed sediment. Denitrifying activity was stimulated by additions of nitrate and ethanol. Microbial activity was monitored by measuring nitrate, nitrite, and ethanol concentrations; gas saturations were measured during the experiment using a gamma imaging system. Biomass was measured using phospholipid fatty acid analysis of sediment samples. Bioenergetic calculations calibrated to measured nitrate consumed and biomass produced predicted that 1.2 L N(2) gas/L water should have been produced following the addition of 100 mM nitrate. However, the maximum measured gas saturation was only 23%, indicating substantial gas loss from the sediment pack. Temporal gamma images and visual observations confirm that small gas bubbles formed in the sediment pack coalesced into larger bubbles and migrated upward through gas-filled channels to the sediment pack surface. Although gas saturations increased, there was no significant change in sediment pack hydraulic conductivity. These results suggest that in permeable reactive barriers used for in situ denitrification, gas production will not necessarily lead to unlimited gas accumulation in the pore space and that the effects of gas production on water saturation and hydraulic conductivity may be relatively minor.

Use of synthetic cardiolipin and lecithin in the antigen used by the venereal disease research laboratory test for serodiagnosis of syphilis.

The Venereal Disease Research Laboratory (VDRL) test is a microflocculation test for syphilis that uses an antigen containing cardiolipin, lecithin, and cholesterol. For more than 50 years, the preparation of natural cardiolipin and lecithin for this test has been based on the Pangborn method which involves isolating and purifying these components from beef hearts. This process is tedious and time-consuming and results in a variable purity range. In our studies, we found that a VDRL antigen using synthetic tetramyristoyl cardiolipin and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (lecithin) was as specific in detecting syphilis as a VDRL antigen made with natural components. In 85% of the cases, we obtained an endpoint titer of 1/2 or 1 dilution more than a titer obtained with a VDRL antigen made with natural components. The use of these pure synthetic compounds, with a purity of 99%, would offer advantages in the standardization and stability of the VDRL antigen. Because this antigen is the basic ingredient in the preparation of nontreponemal reagents such as the rapid plasma reagin, toluidine red unheated serum test, and the unheated serum reagin, the use of this synthetic VDRL antigen should also increase the reactivity of these reagents.

Births: final data for 1998.

OBJECTIVES: This report presents 1998 data on U.S. births according to a wide variety of characteristics. Data are presented for maternal demographic characteristics including age, live-birth order, race, Hispanic origin, marital status, and educational attainment; maternal lifestyle and health characteristics (medical risk factors, weight gain, and tobacco and alcohol use); medical care utilization by pregnant women (prenatal care, obstetric procedures, complications of labor and/or delivery, attendant at birth, and method of delivery); and infant health characteristics (period of gestation, birthweight, Apgar score, abnormal conditions, congenital anomalies, and multiple births). Also presented are birth and fertility rates by age, live-birth order, race, Hispanic origin, and marital status. Selected data by mother's State of residence are shown including teenage birth rates and total fertility rates, as well as data on month and day of birth, sex ratio, and age of father. Trends in fertility patterns and maternal and infant characteristics are described and interpreted. METHODS: Descriptive tabulations of data reported on the birth certificates of the 3.94 million births that occurred in 1998 are presented. RESULTS: Birth and fertility rates increased in 1998 by about 1 percent, the first increase since 1990. Birth rates for teenagers fell 2-5 percent. Rates for women in their twenties increased 1-2 percent each, whereas rates for women in their thirties rose 2-4 percent. All measures of childbearing by unmarried women increased in 1998; the number of births rose 3 percent, the birth rate increased about 1 percent while the percent of births that were to unmarried women rose to 32.8 percent. Smoking by pregnant women overall dropped again in 1998, but continued to increase among teenagers. Improvements in prenatal care utilization continued. The cesarean delivery rate increased for the second year after declining for 7 consecutive years. The proportion of multiple births continued to rise; higher order multiple births (e.g., triplets, quadruplets) rose by 13 percent in 1998, following a 14 percent rise from 1996 to 1997. Key measures of birth outcome--the percents of low birthweight and preterm births--increased. These changes are in large part the result of increases in multiple births.

Trends in the attendant, place, and timing of births, and in the use of obstetric interventions: United States, 1989-97.

OBJECTIVES: This report presents recent trends in the circumstances surrounding live births in the United States. Specifically, this report will examine the changes in the attendant and place of birth as well as changes in the day and month of birth. Trends in the use of four obstetric procedures (electronic fetal monitoring, ultrasound, induction of labor, and stimulation of labor) are examined as well as trends in cesarean births, vaginal births after a previous cesarean, and births delivered by forceps and vacuum extraction. METHODS: Descriptive tabulations were calculated for each year between 1989 and 1997 using data reported on birth certificates. RESULTS: While the vast majority of births in 1997 were attended by physicians, 92 percent, this has declined steadily as the percent of births attended by midwives has slowly increased to account for 7 percent of all births. About 99 percent of births were in hospitals, basically unchanged from 1989, but the percent of out-of-hospital births that were in residences increased whereas those in freestanding birthing centers declined. While births were more common on weekdays than on weekends in 1989, they have become even more concentrated on weekdays since 1989. The most popular months to give birth continue to be July, August, and September. The percent of mothers receiving electronic fetal monitoring, ultrasound, induction, and stimulation all increased over the period with the most dramatic increase being the doubling of the use of induction. Between 1989 and 1996, the rate of cesarean births dropped by 9 percent whereas the rate of vaginal birth after a previous cesarean (VBAC) increased by 50 percent. However, the trends appear to have changed between 1996 and 1997--the cesarean rate increased slightly while the VBAC rate declined by 3 percent. There is wide variation by State in both of these rates. The percent of births that were delivered by forceps consistently declined during the period whereas the use of vacuum extraction consistently increased.

Trends in twin and triplet births: 1980-97.

This report presents data from U.S. birth certificates on the numbers and rates of twin and triplet and other higher order multiple births for 1980-97. Over the study period, the number of twin births rose 52 percent (from 68,339 to 104,137) and triplet and other higher order multiple births (heretofore referred to as "triplet/+") climbed 404 percent (from 1,337 to 6,737 births). Comparable but less pronounced rises were observed in twin and triplet/+ birth rates. Growth in twin and triplet/+ birth rates was most marked among women aged 30 years and over. Between 1980-82 and 1995-97, the twin rate rose 63 percent for women aged 40-44 years, and soared nearly 1,000 percent for women 45-49 years. (As one result, there were more twins born to women 45-49 years of age in 1997, than during the entire decade of the 1980's.) The triplet/+ birth rate rose nearly 400 percent for women in their thirties and exploded by more than 1,000 percent for women in their forties. The extraordinary rise in multiple births resulted in a shift in age-specific patterns, and the highest twin and triplet/+ birth rates now are for women 45-49 years of age. Historical differences in twinning rates between non-Hispanic white and black mothers have been largely eliminated (28.8 per 1,000 non-Hispanic white compared with 30.0 for black women). Non-Hispanic white women were more than twice as likely as non-Hispanic black or Hispanic women to have a triplet/+ birth. Rates of low birthweight, very low birthweight, and infant mortality were 4 to 33 times higher for twins and triplet/+ compared with singleton births. The risk for these adverse outcomes was lowest for twins and triplet/+ born to women 35-44 years of age. Twin birth rates for Massachusetts and Connecticut were at least 25 percent higher than the U.S. rate; triplet/+ rates for Nebraska and New Jersey were twice the national level.

Outcome of MDR-TB patients, 1983-1993. Prolonged survival with appropriate therapy.

We analyzed the clinical and laboratory findings and outcome of 173 patients hospitalized at our institution from 1983 to 1994 with multidrug-resistant tuberculosis (MDR-TB) and evaluated outcome. The 173 patients (mean age 40 +/- 1 yr) were predominantly male (92%), African American or Hispanic (80%), and mostly undomiciled. Over half (52%) were known to be HIV-infected. HIV-positive MDR-TB patients had significantly more pulmonary and constitutional symptoms, more extrapulmonary disease, and fewer cavitary lesions on chest radiographs. Fifty-five percent of the patients in the cohort have died; mortality was significantly greater for HIV-positive than HIV-negative (72% versus 20%, p < 0.01). The median duration of survival of MDR-TB patients was 22 +/- 1 mo. Overall, extrapulmonary involvement was a risk factor for shorter survival, while a cavitary lesion on initial chest film and institution of appropriate treatment were positive predictors of survival. In HIV+ patients, only appropriate therapy was associated with prolonged survival (median of 14.1 mo). Interestingly, there was a trend toward better outcome in the first half of the decade reviewed. We conclude that although mortality from MDR-TB is high in both HIV-positive and HIV-negative patients, institution of appropriate therapy is the factor most strongly associated with a favorable outcome. Development of new diagnostic and therapeutic strategies for MDR-TB are urgently needed.

The effect of whole body hyperthermia on the immune cell activity of cancer patients.

The effect of moderate whole body hyperthermia (WBH) on the immunological responses of cancer patients was studied by monitoring their peripheral blood mononuclear cells (PBMC) in vitro. WBH of 2 degrees C above normal body temperature was induced in patients by utilizing a 433 MHz microwave system with a series of 6-9 external antennae. A total of three treatments were given with a frequency of one per week. Blood samples were drawn before treatment, when the target temperature was attained, and one hour after heating was discontinued. Changes in the number of PBMC, the mitogenic response, natural killer cell activity, and the production of Interleukin-1, Interleukin-2, and Interferon (IL-1, IL-2, IF) were measured in vitro. Mitogenic responses were increased 2-3 fold when the temperature was raised. Although there was an increase in the number of PBMC, during the course of heating, repeated treatment resulted in a selective and transient reduction in the number of PBMC. Nevertheless, the PBMC were capable of producing a higher level of cytokines and attained an enhanced ability to destroy target cells in vitro. The results indicate that, by inducing a fever-like condition, the immunological responses are enhanced in vitro.

Characterization of a T-cell subset prevalent in immunoregulatory disorders in humans.

T cells from individuals with certain autoimmune diseases (rheumatoid arthritis, graft-versus-host disease, acquired immunodeficiency syndrome) express high levels of a cell surface sialoglycoprotein with a molecular weight of 140 kDa (gp140). Although a low frequency of gp 140+ T cells was detected in the blood of normal individuals, upon stimulation with autologous EBV-transformed B cells (AMLR), the frequency of expression of gp140 was increased threefold. To further characterize gp 140+ T cells, rosetting techniques with ox erythrocytes coated with monoclonal anti-gp 140 antibody were used to isolate T-cell subsets for phenotypic, cell cycle, and functional analysis. The majority of gp140+ T cells expressed cytotoxic/suppressor (CD8+) phenotype in both normal and AMLR-activated states. Unstimulated gp140+ T cells had significantly greater nucleic acid content, as measured by acridine orange and flow cytometry, than gp140- T cells. Surprisingly, the gp140+ T-cell subset had a less proliferative response in vitro to pokeweed or phytohemaglutinin mitogens. These results suggest that gp140+ T cells in normal individuals and in patients with autoimmune diseases may have been activated previously in vivo and that they are relatively resistant to reactivation in vitro.

Characterization of a membrane surface glycoprotein associated with T-cell activation.

A cell surface antigen (gp140) was previously shown to exist on T cell subsets as well as on monocytes and macrophages in normal peripheral blood. Elevated expression of this antigen was associated with immune system disorders, acute lymphocytic leukemias, and in vitro activation of T cells. The antigen could be identified with monoclonal antibody (MAb) T305. Gp140 was a biosynthetic product of T cells because it could be labeled with [3H]leucine or [3H] glucosamine. Biochemical studies of gp140 used high performance liquid chromatography with nitrocellulose blotting to isolate aliquots suitable for 125I radiolabeling and immunoprecipitation to demonstrate: a) a reduction in m.w. of gp140 KD to 90 KD after deglycosylation by trifluoromethanesulfonic acid, b) alteration of isoelectric point from 4.1 to 5.7 after neuraminidase treatments, c) absence of N-linked sugars based on resistance to endoglycosidase F, d) resistance to trypsin and chymotrypsin digestion but susceptibility to pronase, and e) presence of sialic acid and lactosaminoglycan as O-linked sugars. Gp140 could be labeled with the periodate/NaB[3H]4 technique, indicating its similarity to a class of sialoglycoproteins previously described on activated T-cells in mouse and man. The antigenic epitope recognized by MAb T305 contains sialic acid linked (2----3) to galactose; however, periodate oxidation of the exocyclic ring of sialic acid did not affect binding by MAb T305. In an attempt to determine the functional role of gp140, we tested the ability of MAb T305 to block: a) proliferation of peripheral blood lymphocytes to mitogens, b) response to interleukin 2 (IL 2) of an IL 2 dependent T cell line, and c) growth of a T-ALL derived cell line. No inhibition of proliferation or growth was noted. Although the function of gp140 remains unknown, its association with lymphocyte activation and certain disease states suggests that it may provide a target for modulation of the immune response. These studies characterize the structural features of gp140 and further define the epitope recognized by MAb T305.

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes.

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.

Studies of immune responses during recovery from Sindbis virus encephalitis in selectively reconstituted, thymectomized, lethally irradiated mice.

Recovery from acute Sindbis virus encephalitis was studied in selectively reconstituted, thymectomized, irradiated mice. Intact mice cleared virus from the brain by day 10 and developed inflammation along with immunoglobulin M (IgM) and IgG antibodies 4 to 6 days after infection. Unreconstituted mice died by day 11, with virus still present but decreasing, no detectable antibody, and no evidence of inflammation. Mice reconstituted with bone marrow cells alone produced only IgM antibody and no inflammation, but cleared virus by day 14. Mice reconstituted with sensitized T cells alone cleared virus by day 10, produced very small amounts of antibody, and developed a prompt inflammatory response.

Studies on purification of antibodies against tumor-specific antigens.

The in vitro cytotoxic effect of anti Sarcoma I sera tumor cells in the presence of an exogenous source of complement was studied. The immune sera were obtained from xenogeneic and allogeneic animals, rabbits and mice, after injections with viable Sarcoma I tumor cells. Antibodies against mouse specific antigens were removed from the rabbit serum, by extensive absorption with mouse erythrocytes and lymphocytes. The absorbed serum was cytotoxic for Sarcoma I tumor cells and did not show any cytotoxicity for A/Jax lymphocytes. The antiserum was purified by an immunoabsorbent column using G-100 Sephadex conjugated with Sarcoma I tumor cell stroma. The eluted, purified antibodies were cytotoxic in vitro. Immunoelectrophoresis and immunodiffusion tests showed the presence of immunoglobulin in the purified antibody preparation. There was a hundred-fold increase of the specific activity of the purified antibodies as compared to the non-purified serum.