Peripheral and site-specific CD4(+) CD28(null) T cells from rheumatoid arthritis patients show distinct characteristics.

Proinflammatory CD4(+) CD28(null) T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4(+) CD28(null) T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4(+) T cells. Forty-four patients with RA, displaying a distinct CD4(+) CD28(null) T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-γ, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4(+) CD28(null) T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4(+) CD28(+) T cells and produced higher levels of both IFN-γ and TNF after TCR cross-linking. CD4(+) CD28(null) T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4(+) CD28(null) cells from the blood, a significant production was observed in CD4(+) CD28(null) T cells from synovial fluid. CD4(+) CD28(null) T cells were not only found to differ from conventional CD4(+) CD28(+) T cells in the circulation, but we could also demonstrate that synovial CD4(+) CD28(null) T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28(null) population.

Altered immunoregulatory profile during anti-tumour necrosis factor treatment of patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor (TNF) therapy. We set out to investigate the unclear immunoregulatory mechanisms of the treatment. Thirty-four patients with IBD treated with anti-TNF were included. Lymphocytes from peripheral blood and intestinal biopsies were analysed by flow cytometry. Regulation of antigen-stimulated proliferation was analysed by blocking of interleukin (IL)-10, transforming growth factor (TGF)-ß or depletion of CD25(+) cells in peripheral blood mononuclear cell cultures. No changes in CD4(+)CD25(+), CD25(+)TNF-RII(+) or CD4(+)CD25(+) forkhead box protein 3 (FoxP3(+)) T cells could be observed in peripheral blood after, in comparison to before, 6 weeks of treatment. The suppressive ability of CD4(+)CD25(+) cells did not change. There was an initial decrease of CD4(+)CD25(+) cells in intestinal mucosa after 2 weeks of treatment, followed by an increase of these cells from weeks 2 to 6 of treatment (P < 0·05). This was accompanied by an increased percentage of CD69(+) cells among these cells after 6 weeks of treatment compared to before treatment (P < 0·01). There was also an increase of mucosal T helper type1 cells from weeks 2 to 6 (P < 0·05). In addition, CD25(+)TNF-RII(+) cells in the mucosa were decreased after 6 weeks of treatment compared to before treatment (P < 0·05). Before treatment, peripheral blood mononuclear cell baseline proliferation was increased when IL-10 was blocked (P < 0·01), but not after. In CD25(+) cell-depleted cultures proliferation increased after treatment (P < 0·05). Our data indicate that anti-TNF treatment leads to an induction of effector T cells. Anti-TNF therapy has no significant impact on regulatory T cells in IBD, although the composition of regulatory T cell subsets may change during treatment.

Which IO model best simulates the real thing?

OBJECTIVE: To determine which model best simulates the actual IO procedure in children. METHODS: Forty emergency and critical care physicians with significant IO experience (6 or more IO procedures) in real children were recruited at 4 academic centers. Study subjects were provided with a kit containing 15 gauge IO needles and 5 IO models; a plastic IO doll leg (PL), a turkey femur/thigh (TT), a turkey tibia/drumstick (TD), a chicken femur/thigh (CT), and a pork rib (PR). Study participants scored the similarity of the bone model to that of placing an IO in a child (0 = no experience, 1 = perfect simulation, 2 = excellent, but not perfect, 3 = good, 4 = moderate, 5 = poor) and the hardness of the bone (H = too hard, J = just right, S = too soft) for each age group (preterm, newborn, 1-4 months, 5-12 months, 12-36 months, 3-6 years, 6-12 years, and >12 years old) of whom they had previous experience. Mean scores were calculated and compared to determine which model provided the best simulation. RESULTS: After excluding zero scores, overall mean scores for the CT, TD, TT, PL, and PR models were 3.2, 3.3, 3.3, 3.3, and 4.4 (P < 0.001), respectively. The pork rib had the worst score in all age groups, while the other 4 models had roughly similar scores. All models had substantial percentages of the models classified as too hard or too soft, except for the pork rib, which was predominantly too hard. CONCLUSIONS: For IO research and teaching purposes, bone models should be age appropriate. This study suggests that there is great variability in preference with the chicken, turkey, and plastic models.

Correlates of vaccination for hepatitis B among adolescents: results from a parent survey.

OBJECTIVE: To identify correlates of hepatitis B vaccination status in sixth-grade students in the year prior to implementation of a requirement mandating immunization for seventh-grade entry. METHODS: A survey of parents of sixth-graders in 5 schools in San Diego County. Two logistic regression models were tested to predict the outcome variables: initiation and completion of the vaccination series. RESULTS: Factors associated with initiating the series included a recent nonacute medical visit, white race, hearing about the vaccination law from a health care provider, and the availability of a school-based vaccination clinic. Factors associated with completing the series included English as the primary language spoken at home, hearing about the law from a health care provider, a school-based vaccination clinic, and higher socioeconomic status. Health insurance was not significantly related to either outcome. CONCLUSIONS: There was a clear benefit for hepatitis B immunization status to have had a recent nonacute medical visit, to have heard about the law from a health care provider, and to have a school-based vaccination clinic. The factors associated with starting vs completing the vaccination series were not identical. However, both health care provider and school characteristics were related to starting and completing the vaccination series. Thus, a multifaceted strategy may be most appropriate for successful coverage of an adolescent population with a vaccination series such as hepatitis B.

Initial sequencing and analysis of the human genome.

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms.

We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.

An SNP map of the human genome generated by reduced representation shotgun sequencing.

Most genomic variation is attributable to single nucleotide polymorphisms (SNPs), which therefore offer the highest resolution for tracking disease genes and population history. It has been proposed that a dense map of 30,000-500,000 SNPs can be used to scan the human genome for haplotypes associated with common diseases. Here we describe a simple but powerful method, called reduced representation shotgun (RRS) sequencing, for creating SNP maps. RRS re-samples specific subsets of the genome from several individuals, and compares the resulting sequences using a highly accurate SNP detection algorithm. The method can be extended by alignment to available genome sequence, increasing the yield of SNPs and providing map positions. These methods are being used by The SNP Consortium, an international collaboration of academic centres, pharmaceutical companies and a private foundation, to discover and release at least 300,000 human SNPs. We have discovered 47,172 human SNPs by RRS, and in total the Consortium has identified 148,459 SNPs. More broadly, RRS facilitates the rapid, inexpensive construction of SNP maps in biomedically and agriculturally important species. SNPs discovered by RRS also offer unique advantages for large-scale genotyping.

Conjunctival malignant melanoma in a horse.

A case of malignant melanoma originating from the conjunctiva of a horse is reported. The tumor exhibited locally aggressive behavior as evidenced clinically by recurrence following two treatment episodes including surgical excision on each occasion and one application of cryotherapy. The orbit was subsequently exenterated and histologically malignant conjunctival melanoma was confirmed. Histopathologic features included variable pigmentation with amelanotic sites demonstrating marked cellular and nuclear pleomorphism with high numbers of mitotic figures. Cords of neoplastic cells invaded the sclera and cornea. Following exenteration, the horse exhibited no recurrence of the tumor for five years before being lost to follow-up. To our knowledge, this is the first report of primary malignant conjunctival melanoma in a horse.

Toward real-world sequencing by microdevice electrophoresis.

We report results using a microdevice for DNA sequencing using samples from chromosome 17, obtained from the Whitehead Institute Center for Genome Research (WICGR) production line. The device had an effective separation distance of 11.5 cm and a lithographically defined injection width of 150 microm. The four-color raw data were processed, base-called by the sequencing software Trout, and compared to the corresponding ABI 377 sequence from WICGR. With a criteria of 99% accuracy, we achieved average continuous reads of 505 bases in 27 min with 3% linear polyacrylamide (LPA) at 150 V/cm, and 460 bases in 22 min with 4% LPA at 200 V/cm at a temperature of 45 degrees C. In the best case, up to 565 bases could be base-called with the same accuracy in <25 min. In some instances, Trout allowed for accurate base-calling down to a resolution R as low as R = 0.35. This may be due in part to the high signal-to-noise ratio of the microdevice. Unlike many results reported on capillary machines, no additional sample cleanup other than ethanol precipitation was required. In addition, DNA fragment biasing (i.e., discrimination against larger fragments) was reduced significantly through the unique sample injection mechanism of the microfabricated device. This led to increased signal strength for long fragments, which is of great importance for the high performance of the microdevice.

First and second order maternal behavior related afferents of the lateral habenula.

We demonstrated previously that the lateral habenula (Lhb) mediates maternal behavior. Our present goal was to identify the first and second order afferent connections of the Lhb, particularly those relevant for maternal behavior. Using pseudorabies virus (PRV) as a retrograde transneuronal tracer and the retrograde tracer Fluoro-Gold, we identified first order Lhb afferent projections from the lateral preoptic area, bed nucleus of the stria terminalis, nucleus accumbens and ventral tegmental area, each important for the display of maternal behavior. Maternally relevant second order neurons originated from the medial preoptic area and amygdala. Additional regions with first and second order neurons afferent to the Lhb were also identified.

Estrogen implants in the lateral habenular nucleus do not stimulate the onset of maternal behavior in female rats.

The natural onset of maternal behavior in the rat is hormonally mediated. Estrogen, progesterone, and prolactin administered to ovariectomized females in amounts and sequences that produce circulating levels similar to those found during pregnancy stimulate the onset of maternal behavior. In fact, maternal behavior can be stimulated by estrogen alone, administered either peripherally or by implant in the central nervous system. The lateral habenula (Lhb), which is a necessary component in the neural circuit that supports maternal behavior, contains a subset of neurons with estrogen receptors. The present study investigated whether estradiol implants directly in the Lhb are sufficient to stimulate maternal behavior. Female rats, hysterectomized and ovariectomized on day 16 of pregnancy, received estrogen implants in the Lhb or, as a positive control, in the medial preoptic area (MPOA). An additional control group received cholesterol implants in the Lhb. All females were tested for pup retrieval, nest building, crouching behavior, locomotor activity, and carrying behavior. Estradiol implants into the Lhb did not stimulate the onset of maternal behavior. Females with estrogen implants in the Lhb scored significantly lower in pup retrieval and crouching behavior compared to females with implants in the MPOA and were not significantly different from females with cholesterol implants in the Lhb. There were also no significant differences in overall activity or carrying behavior among the groups.

Intact neurons of the lateral habenular nucleus are necessary for the nonhormonal, pup-mediated display of maternal behavior in sensitized virgin female rats.

Our research has demonstrated that the lateral habenular nucleus (Lhb) is necessary for the hormonal onset but not the postpartum maintenance of maternal behavior in the rat (K. P. Corodimas, J. S. Rosenblatt, & J. I. Morrell, 1992; K. P. Corodimas, J. S. Rosenblatt, M. E. Canfield, & J. I. Morell, 1993; T. Matthews-Felton, K. P. Corodimas, J. S. Rosenblatt, & J. I. Morell, 1995). To test the role of the Lhb in the nonhormonal onset of maternal behavior, we used the sensitization model in which the continual exposure of females to pups induces maternal behavior. Ovariectomized females received bilateral cytotoxic lesions of neurons of either the Lhb or the dorsal medial cingulate cortex-hippocampus, or they were unoperated. Maternal behavior, activity, and oromotor carrying capability were tested. Complete lesions of the neurons of the Lhb induced significant deficits in pup retrieval and nest building. Sniffing, licking, and crouching behaviors were unaltered. Activity and carrying ability were normal. These results indicate a role for the Lhb that extends to the nonhormonally dependent onset of maternal behavior, but they also indicate a more limited role than in the mediation of the hormonal onset of the behavior.

Measuring the psychosocial impact of urinary incontinence: the York Incontinence Perceptions Scale (YIPS).

OBJECTIVE: The York Incontinence Perceptions Scale (YIPS) was developed to measure the psychosocial aspects of urinary incontinence and its management. DESIGN: Testing of internal consistency and validity of the YIPS. SETTING AND PARTICIPANTS: Subjects were 101 female rural community residents (mean age = 67.4 years) diagnosed with urinary incontinence and participating in a 25-week longitudinal randomized control study testing the efficacy of treating incontinence with a behavioral/educational intervention. MEASUREMENTS: Participants completed the YIPS, a bladder chart monitoring daily incontinence episodes, the Aids to Living Scale, the Incontinence Impact Questionnaire, and single-item ratings of self-perceptions of amount of leakage, continence status, and overall health status. MAIN RESULTS: The YIPS had high internal consistency (a = .78). Positive adjustment on the YIPS was correlated with lower frequency of incontinence (r = -.44), and self-ratings of improvement in amount of leakage (r = .60), improved continence status (r = .38), and overall health status (r = .32). At the end of the 25-week treatment period, the participants in the treatment group reported a more positive adjustment on the YIPS than did participants in the control group (t[99] = 4.78, P < .001), which was concordant with a reduction in the incidence of incontinence in the treatment group (F[1,91] = 6.95, P < .01). CONCLUSIONS: The YIPS is a brief, yet reliable, instrument that addresses such psychosocial issues as coping, control, and acceptance of incontinence.

Metacarpophalangeal pattern profile analysis in Williams syndrome.

Many patients with Williams syndrome (WS) are not diagnosed until they are old enough to demonstrate the characteristic personality and facial changes. A number of these changes are quite subtle and none of them is present in all affected individuals. The cause of WS remains obscure and consequently, there are no cytogenetic, biochemical, or molecular studies to help in the diagnosis of patients in whom the diagnosis is uncertain. We have generated a mean WS metacarpophalangeal pattern profile (MCPP) on 21 clinically diagnosed individuals with WS. This mean syndrome profile shows that WS hands are smaller than average age-matched control hands and that the distal phalanx of the thumb is disproportionately large with respect to the rest of the hand. A mathematical model, which effectively discriminates WS patients from unaffected control individuals, was developed using discriminant analysis of the MCPP data. Of the 21 WS patients classified by this method, only 2 were misclassified as "normal." Similarly, 2 of the 24 control individuals were misclassified as "WS," yielding an over-all successful classification rate of 91%.

Cholesterylene, a newly recognized tissue lipid, found at high levels in the cornea.

In the course of measuring the concentration of cholesterol in an opacified dog cornea by gas-chromatography, relatively large amounts of an unidentified non-saponifiable lipid were recognized. When the unknown lipid was subjected to gas chromatographic-mass spectral analysis it displayed a major ion at m/z 368 M+. and was identified as cholesta-3,5-diene, cholesterylene, by computer match with mass spectral-registry data. Cholesterylene was then shown to be present in the corneas of normal dogs, cows and humans, accounting for 20-25% of the total steroid-sterol in dog corneas and 5-10% in cow and human. Cholesterylene, which can be considered as an extremely nonpolar dehydration product of cholesterol, has not previously been recognized in animal tissues. Although the source of corneal cholesterylene is unknown, preliminary results suggest non-enzymatic formation from cholesterol.

Testing life-cycle theory by computer simulation--I. Introduction of genetical structure.

Computer simulation was used to relax assumptions of analytical life-cycle theory about the eventual outcome of evolution in a constant environment. The computer simulation models, of diploid one-locus genetic systems, are described in detail. Good agreement was obtained between the analytical and simulation outcomes, except in some cases of discrepancy between the male and female life cycles.

Testing life-cycle theory by computer simulation--II. Bet-hedging revisited.

Analytical and computer models were used to reexamine bet-hedging, the reduction in fecundity that is evolutionarily advantageous in conditions of greater variation in juvenile survivorship or less variation in adult survivorship. The computer simulation models represent diploid one-locus genetic systems with semidominance. Schaffer's (1974) predictions proved remarkably robust when variations were symmetrical, and a simple modification allowed successful prediction for the asymmetries that occurred in the computer simulations when variations were large.

Endometrial biopsy technique and uterine pathologic findings in llamas.

Uterine tissue specimens from 90 llamas with history of infertility were examined and graded microscopically; 83 specimens were obtained by endometrial biopsy and 7 specimens were obtained at necropsy. Fifteen llamas (16.7%) had a normal uterus, which was graded 1A. Twenty-three llamas (25.6%) had minor uterine abnormalities that were graded 1B. Forty-five llamas (50.0%) had appreciable endometritis that was graded 2A. Three llamas (3.3%) had endometritis with gland fibrosis that was graded 2B. Two llamas (2.2%) had notable uterine gland fibrosis that was graded 3A. Two llamas (2.2%) had uterine neoplasia that was graded 3B. Thirty-six llamas had follow-up evaluation; 22 became pregnant, most after some form of treatment. Uterine biopsy in llamas was readily performed without complications and was valuable in identifying a wide range of uterine diseases, most of which were amenable to treatment.

An energetics model of an aquatic predator and its application to life-history optima.

A bioenergetics simulation model of the growth and life history of the aquatic predator Nephelopsis obscura Verrill was developed and validated using both experimentation and sensitivity analysis. Sensitivity analysis demonstrated that the model's internal feedbacks resulted in stability similar to homeostatic biological mechanisms. The experimental validation showed the model very accurately predicts growth at 10°C and 15°C but is slightly biased at 20°C. Simulation output was also consistent with the observed data on Nephelopsis from the site from which the simulation input data were obtained and indicated that Nephelopsis growth is more sensitive to prey variation among years than to temperature variation. Although built using data from a population at one extreme of the spectrum observed in life history and growth, the model was able to emulate the growth of Nephelopsis throughout its range. Thus, the variability in size and life history observed in the field can be explained as the result of a plastic phenotype responding to different habitat conditions.

Competition and coexistance between Glossiphonia complanata and Helobdella stagnalis (Glossiphoniidae: Hirudinoidea).

The prey utilization of Glossiphonia complanata and Helobdella stagnalis from Bruce Lake, Alberta, Canada was examined quantitatively using serological techniques. While considerable prey range overlap occurred between the species, niche overlap was low during the winter, peaked in March and declined through the summer. Temporal differences in feeding and intraspecific weight class differences in prey utilization were found in both species but more distinctly in G. complanata. It is suggested that co-existance of G. complanata and H. stagnalis is based on adjustments of weight class food resource partitioning on a temporal basis.