Human T-cell subset changes during culture

When human peripheral blood lymphocytes (PBL) are cultured with either concanavalin A (Con A)-treated or control autologous T lymphocytes, the mitogenic responses of the PBL co-cultured with Con A-treated cells are much lower. We have investigated the cell surface receptor changes during culture of T cells with and without mitogen in an attempt to explain this differential regulatory phenomenon. We present data here which show that human T cells cultured in complete medium alone gain helper cells with time. Con A-treated T cells are known to lose helper cells during culture. Erythrocyte rosette-purified T cells were cultured with or without Con A for 84 h and the numbers of cells with receptors for the Fc regions of either IgM (T mu) or IgG (T gamma) were enumerated daily. T mu cells have been associated with helper activity while T gamma cells have predominantly suppressor activity. Treatment with 10 micrograms/ml of Con A decreased T mu by approximately 50%. Untreated cells, however, showed significant increases in T mu (44 +/- 30.5% in twelve individuals). The great variance in T mu increases is due to the fact that individuals having higher initial T mu values showed smaller increases. These changes probably represent the gain or loss of receptors because total cell numbers did not change. There was no significant change in the number of T gamma cells in either control or Con A-treated cultures during the same 84 h period. In co-culture experiments in which the responses of fresh autologous PBL were determined, 60-h control T-cell cultures enhanced the mitogen responses of the fresh cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen effects on object memory and cholinergic receptors in young and old female mice.

We investigated whether object recognition memory is modulated by estrogen in young (5 month) and aged (24 month) female C57Bl/6J mice, and if cholinergic muscarinic receptors might contribute to this response. Mice that were ovariectomized, or ovariectomized plus estradiol-treated three weeks before behavioral testing or quantitative autoradiography were compared to intact mice. Memory for a previously encountered object deteriorated significantly between 3 and 6h after initial exposure, regardless of animal age. In both young and aged mice, estradiol-treated mice showed significantly greater recall than did ovariectomized mice. In both age groups, the apparent number of [(3)H]pirenzepine/M(1)-like and [(3)H]AFDX384/M(2)-like muscarinic receptor binding sites was reduced in the basal forebrain as well as its projection areas following ovariectomy, but this decrease was not alleviated by estrogen. Aging poorly affected object memory, but reduced muscarinic binding in some cortical subregions and in the caudate nucleus. These findings suggest that estrogen effects on memory in C57Bl/6J mice are not due to changes in the number of muscarinic receptors.     

CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype.

PURPOSE: The study's purpose was to understand the molecular basis for different clinical phenotypes of the 5T variant, a tract of 5 thymidines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts processing of CFTR mRNA and reduces synthesis from the corresponding CFTR alleles. METHOD: We analyzed the polymorphic TG dinucleotide repeat adjacent to the 5T variant in intron 8 and the codon 470 in exon 10. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, Delta F508. Almost all Delta F508 mutation alleles occur in a 10TG-9T-470M haplotype. Therefore, it is possible to determine the haplotype of the 5T variant in trans. RESULTS: Of the 74 samples analyzed, 41 (55%) were 11TG-5T-470M, 31 (42%) were 12TG-5T-470V, and 2 (3%) were 13TG-5T-470M. Of the 49 cases for which we had clinical information, 17.6% of females (6/34) and 66.7% of males (10/15) showed symptoms resembling atypical cystic fibrosis. The haplotype with the highest penetrance in females (42% or 5/12) and more than 80% (5/6) in males is 12TG-5T-470V. We also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5T-470V haplotype. CONCLUSION: Overall, the 5T variant has a milder clinical consequence than previously estimated in females. The clinical presentations of the 5T variant are associated with the 5T-12TG-470M haplotype.     

Organ specific apoptosis following polymicrobial intraperitoneal infection

Background Leukocyte apoptosis allows safe removal of potentially harmful cells and facilitates resolution of inflammation. We hypothesized that the number of apoptotic cells changes in a disproportionate fashion in parenchymal organs in response to intra-abdominal infection. Materials and methods The percentage of apoptotic cells in the liver, spleen, lung, and peripheral blood was evaluated following cecal ligation and puncture (CLP) in mice. Tissue myeloperoxidase (MPO) levels were measured as an index of neutrophil extravasation. Results Liver & spleen MPO continually increased, while lung MPO remained low after CLP. In parallel to the increase in MPO, liver & spleen apoptosis continually increased throughout the 9-day follow-up period, whereas lung apoptosis remained unchanged. Conclusions The distribution of apoptotic cells during intraperitoneal infection occurs in an organ specific manner, with significant increases in the spleen and liver. This distribution likely reflected the clearance of apoptotic cells as the inflammatory focus became contained. Supported by the American Association for the Surgery of Trauma, John H. Davis Research Scholarship, and by the Veterans Administration Merit Review Project 0005. Received 7 October 2005; returned for revision 22 November 2005; accepted by G. Wallace 23 December 2005     

Impedance control and internal model use during the initial stage of adaptation to novel dynamics in humans

This study investigated the neuromuscular mechanisms underlying the initial stage of adaptation to novel dynamics. A destabilizing velocity-dependent force field (VF) was introduced for sets of three consecutive trials. Between sets a random number of 4–8 null field trials were interposed, where the VF was inactivated. This prevented subjects from learning the novel dynamics, making it possible to repeatedly recreate the initial adaptive response. We were able to investigate detailed changes in neural control between the first, second and third VF trials. We identified two feedforward control mechanisms, which were initiated on the second VF trial and resulted in a 50% reduction in the hand path error. Responses to disturbances encountered on the first VF trial were feedback in nature, i.e. reflexes and voluntary correction of errors. However, on the second VF trial, muscle activation patterns were modified in anticipation of the effects of the force field. Feedforward cocontraction of all muscles was used to increase the viscoelastic impedance of the arm. While stiffening the arm, subjects also exerted a lateral force to counteract the perturbing effect of the force field. These anticipatory actions indicate that the central nervous system responds rapidly to counteract hitherto unfamiliar disturbances by a combination of increased viscoelastic impedance and formation of a crude internal dynamics model.     

A 10-Mb paracentric inversion of chromosome arm 2p inactivates MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred

Genomic deletions of the MSH2 gene are a frequent cause of hereditary nonpolyposis colorectal cancer (HNPCC), a common hereditary predisposition to the development of tumors in several organs including the gastrointestinal and urinary tracts and endometrium. The mutation spectrum at the MSH2 gene is extremely heterogeneous because it includes nonsense and missense point mutations, small insertions and deletions leading to frameshifts, and larger genomic deletions, the latter representing approximately 25% of the total mutation burden. Here, we report the identification and molecular characterization of the first paracentric inversion of the MSH2 locus known to cause HNPCC. Southern blot analysis and inverse PCR showed that the centromeric and telomeric breakpoints of the paracentric inversion map within intron 7 and to a contig 10 Mb 3' of MSH2, respectively. Pathogenicity of the paracentric inversion was demonstrated by conversion analysis. The patient's lymphocytes were employed to generate somatic cell hybrids to analyze the expression of the inverted MSH2 allele in an Msh2-deficient rodent cellular background. The inversion was shown to abolish MSH2 expression by both northern and western analysis. This study confirms that Southern blot analysis still represents a useful and informative tool to screen for and identify complex genomic rearrangements in HNPCC. Moreover, monoallelic expression analysis represents an attractive approach to demonstrate pathogenicity of unusual mutations in autosomal dominant hereditary conditions.     

Effects of neutrophil, natural killer cell, and macrophage depletion on murine Clostridium piliforme infection.

Clostridium piliforme infection (Tyzzer's disease) induces enterohepatic disease in many domestic and laboratory animals. Murine susceptibility to Tyzzer's disease varies with host strain, age, and immune status However, little is known about the role of the immune system in control of this disease. To investigate the role of host immunity in Tyzzer's disease, mice were depleted of either neutrophils, natural killer cells, or macrophages by antibody administration or chemotherapy. After depletion, DBA/2 mice, which are naturally susceptible to C. piliforme, or naturally resistant C57BL/6 mice were inoculated intravenously with C. piliforme. Animals were euthanized 3 days postinoculation and evaluated for gross and histologic lesions and hepatic bacterial load. In juvenile DBA/2 or C57BL/6 mice, depletion of either neutrophils or natural killer cells increased severity of disease. In adult mice, depletion of natural killer cells significantly increased severity of Tyzzer's disease in the resistant (C57BL/6) but not in the susceptible (DBA/2) strain. Macrophage depletion did not alter the course of infection in either mouse strain. These studies indicate an important role for neutrophils and natural killer cells in the pathogenesis of murine Tyzzer's disease. The role of macrophages in murine C. piliforme infection will require further evaluation.     

Vasopressin antagonist in nucleus tractus solitarius/vagal area reduces pressor and tachycardia responses to paraventricular nucleus stimulation in rats

In urethane-anesthetized rats, injections of 50 pmol of arginine-vasopressin (AVP) or thyrotropin-releasing hormone (TRH) into a lateral cerebral ventricle (i.c.v.) elicit short-latency increases in blood pressure. i.c.v. injection of 50 pmol of the AVP antagonist, d(CH2)5Tyr(Me)AVP, but not of the vehicle (artificial cerebrospinal fluid; a CSF), abolished the pressor action of i.c.v. AVP. The AVP antagonist did not antagonize the TRH-induced pressor responses. In another group of rats, a monopolar stainless-steel electrode was positioned stereotaxically in the paraventricular nucleus (PVN) and pressor responses were elicited by electrical stimulation of the PVN. Micro-injection of 1 nmol of the AVP antagonist, but not of aCSF alone, into the nucleus tractus solitarius/vagal area (NTS/VA), reduced PVN-stimulated pressor responses to 26 +/- 6% of control and stimulation-induced tachycardia to 37.3 +/- 9.0% of control. These studies indicate that the pressor and heart-rate responses to PVN stimulation may be mediated, in part, via AVP receptors in the NTS/VA.     

Production of experimental ulcerative colitis in gnotobiotic guinea pigs with simplified microflora.

Conventional guinea pigs provided with a solution of 5% (wt/vol) degraded carrageenan as the sole source of oral fluids developed ulcerations of their ceca and large intestines within 30 days. Similar lesions were not detected in germfree guinea pigs treated in an identical manner, suggesting that an intestinal microflora was necessary for development of intestinal lesions. To simplify the bacterial flora required for production of cecal ulcerations, 10 pools consisting of 10 bacterial strains each were isolated from the cecal microflora of carrageenan-treated animals. Groups of germfree guinea pigs were associated with 2 of the 10 pools by orogastric intubation and observed for development of disease. One-half of each group was treated with carrageenan. The two bacterial pools were characterized by the presence of cytopathic effects for WI-38 and Vero cells, increased chemotactic activity, and increased concentrations of long-chain fatty acids. The results indicated that animals associated with those two pools developed cecal ulcerations during carrageenan treatment. Preliminary results also indicated that cecal ulcerations developed in germfree animals mono-associated with a strain of Bacteroides vulgatus isolated from one of the pools, regardless of whether carrageenan was administered, suggesting a bacterial involvement in disease development in the absence of carrageenan treatment.     

Inability to activate muscles maximally during cocontraction and the effect on joint stiffness

In order to determine the maximum joint stiffness that could be produced by cocontraction of wrist flexor and extensor muscles, experiments were conducted in which healthy human subjects stabilized a wrist manipulandum that was made mechanically unstable by using positive position feedback to create a load with the characteristics of a negative spring. To determine a subject's limit of stability, the negative stiffness of the manipulandum was increased by increments until the subject could no longer reliably stabilize the manipulandum in a 1° target window. Static wrist stiffness was measured by applying a 3° rampand-hold displacement of the manipulandum, which stretched the wrist flexor muscles. As the load stiffness was made more and more negative, subjects responded by increasing the level of cocontraction of flexor and extensor muscles to increase the stiffness of the wrist. The stiffness measured at a subject's limit of stability was taken as the maximum stiffness that the subject could achieve by cocontraction of wrist flexor and extensor muscles. In almost all cases, this value was as large or larger than that measured when the subject was asked to cocontract maximally to stiffen the wrist in the absence of any load. Static wrist stiffness was also measured when subjects reciprocally activated flexor or extensor muscles to hold the manipulandum in the target window against a load generated by a stretched spring. We found a strong linear correlation between wrist stiffness and flexor torque over the range of torques used in this study (20–80% maximal voluntary contraction). The maximum stiffness achieved by cocontraction of wrist flexor and extensor muscles was less than 50% of the maximum value predicted from the joint stiffness measured during matched reciprocal activation of flexor and extensor muscles. EMG recorded from either wrist flexor or extensor muscles during maximal cocontraction confirmed that this reduced stiffness was due to lower levels of activation during cocontraction of flexor and extensor muscles than during reciprocal contraction.