Addition of cultured Schwann cells to tendon autografts and freeze-thawed muscle grafts improves peripheral nerve regeneration

The effects of addition of Schwann cells on peripheral nerve regeneration through a novel graft material-the tendon autograft-and a conventional freeze-thawed muscle graft, were studied in the rat sciatic nerve. Adult Schwann cell cultures were established from predegenerated nerves. The Schwann cells were added to the autologous grafts by coculture (tendon autograft) or injection (freeze-thawed muscle graft). Both graft types supported adherence of the added Schwann cells. Addition of cultured Schwann cells to the two different graft models improved regeneration by increasing the rate of axonal outgrowth as compared with similar grafts without added cells.     

Expression and regulation of small proline-rich protein 2 in allergic inflammation

Asthma is a complex inflammatory pulmonary disorder that is on the rise despite intense ongoing research. We aimed to elucidate novel pathways involved in the pathogenesis of asthma. Employing asthma models induced by different allergens (ovalbumin and Aspergillus fumigatus), we uncovered the involvement of two members of the small proline-rich protein (SPRR) family, SPRR2a and SPRR2b, known to be involved in epithelial differentiation but not allergic disease. In situ hybridization revealed induction of SPRR2 signal in a subset of bronchial epithelial cells and mononuclear cells associated with inflammation after allergen challenge. Allergen-induced SPRR2 mRNA accumulation in the lung occurred in a time-dependent manner, with peak expression 10-96 h after a second ovalbumin challenge. Transgenic overexpression of interleukin (IL)-13 in the lungs resulted in a marked increase of SPRR2 expression, and allergen-induced SPRR2 expression was significantly decreased in IL-13-deficient mice. Studies in gene-targeted mice revealed that allergen-induced SPRR2 was dependent upon STAT6. Finally, we aimed to determine if the induction of SPRR2 by allergen was tissue specific. Notably, SPRR2 was markedly increased in the small intestine after induction of allergic gastrointestinal inflammation. Thus, SPRR2 is an allergen- and IL-13-induced gene in experimental allergic responses that may be involved in disease pathophysiology.     

Experimental infection of pigs with aTaenia species from Korea: parasitological and serological aspects

Belgian Landrace piglets were experimentally infected with eggs of aTaenia sp. of Korean origin. At autopsy, metacestodes were present only in the livers. The proportion of degenerated metacestodes increased from 12%–39% at 5 weeks to 94%–100% at 10 weeks after infection. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised against the excretory-secretory products ofT. saginata metacestodes detected circulating antigen in the sera of the pigs at 1 week post-infection. A good correlation was found between the presence of viable metacestodes and the detection of circulating antigen; the latter disappeared as the metacestodes died off. However, the antibodies were detected only after 3 weeks of infection and onwards until the necropsy of the pigs.Financial support for this study was provided by the Institute for Scientific Research in Industries and Agriculture (IWONL, Brussels)     

IL-4 production by human polymorphonuclear neutrophils

Polymorphonuclear neutrophils (PMN) are phagocytic cells, able to secrete a large range of cytokines, including inflammatory cytokines, chemokines, as well as the Th1 cytokines interferon-gamma (IFN-gamma) and interleukin (IL)-12. Although PMN do not seem to express IL-10 and IL-13, no information exists on the ability of PMN to produce IL-4. Therefore intracellular flow cytometry was performed in the presence or absence of Brefeldin A. Similarly to eosinophils, freshly isolated neutrophils from normal donors contained low amounts of IL-4, which significantly increased upon culture with Brefeldin A (P < 0001). Immunostaining performed on cytospin preparations of normal granulocytes confirmed the presence of intracellular IL-4. Using a highly sensitive ELISA, the levels of IL-4 secreted by cultured PMN and peripheral blood mononuclear cells (PBMC) were compared. PBMC secrete up to 60 times more IL-4 as PMN but, in the presence of calcium ionophore, only PMN showed a slight but significant increase in IL-4 secretion (P < 0.05). In conclusion, we report here the presence within human PMN of intracellular IL-4, which can at least partly be released under calcium ionophore stimulation. The relevance of this production of IL-4 by human PMN is discussed.     

Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii.

The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.     

Effect of recombinant human granulocyte-macrophage colony-stimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation

Recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) has been reported to increase the leukocyte count in subhuman primates subjected to total-body irradiation and in patients with the acquired immunodeficiency syndrome. We administered this substance to 19 patients with breast cancer or melanoma treated with high-dose combination chemotherapy and autologous bone marrow support. Groups of three or four patients were treated with 2.0, 4.0, 8.0, 16.0, or 32.0 micrograms per kilogram of body weight per day of glycosylated rHuGM-CSF by continuous intravenous infusion for 14 days, beginning three hours after bone marrow infusion. Total leukocyte and granulocyte recovery was accelerated in these patients as compared with 24 historical controls matched for age, diagnosis, and treatment. Leukocyte counts (mean +/- SD) obtained 14 days after transplantation were 1511 +/- 1003 per microliter in patients given 2 to 8 micrograms per kilogram per day, 2575 +/- 2304 in those given 16 micrograms, and 3120 +/- 1744 in those given 32 micrograms, as compared with 863 +/- 645 per microliter in the controls. No consistent effect on platelet counts was noted. Toxic effects were generally mild and not clearly dose-related in patients given 2 to 16 micrograms per kilogram per day. Edema, weight gain, or myalgias occurred in all patients given 32 micrograms per kilogram; marked weight gain, generalized edema, pleural effusions, and hypotension developed in two patients, one of whom also had acute renal failure. Our results indicate that rHuGM-CSF can accelerate myeloid recovery after high-dose chemotherapy and autologous bone marrow transplantation, over a range of doses that can be tolerated. In this setting the ability to increase the dose is limited by the development of myalgias and fluid retention.     

Gonadotrophin-releasing hormone agonist dose-dependency of pituitary desensitization during controlled ovarian hyperstimulation in IVF

The aim of this study was to find the minimal effective daily s.c. dose of the gonadotrophin-releasing hormone (GnRH) agonist, triptorelin acetate, that suppresses the GnRH-induced release of luteinizing hormone (LH) at time of human chorionic gonadotrophin (HCG) injection and thereby prevents spontaneous LH surges during in-vitro fertilization (IVF) stimulation cycles. Therefore, a double-blind, prospective and randomized titration study was performed. A total of 48 IVF patients were divided into four groups of 12 patients. Each group received a different dose of triptorelin acetate, namely 5, 15, 50 or 100 microg s.c. daily. Standard ovarian stimulation was carried out using urinary follicle stimulating hormone (FSH) preparations. A 500 microg GnRH test was performed 90 min before the HCG injection in order to measure the degree of pituitary desensitization. Spontaneous LH surges were not detected in any of the groups, although three patients in the 5 microg group had ovulated at the time of ovum retrieval. The pituitary LH response to the GnRH test at time of HCG, expressed as area under the curve (AUC), appeared to be dose-dependent. Thus, a daily s.c. dose of 100 microg triptorelin acetate appears to be too high, since adequate desensitization of the pituitary (i.e. no spontaneous LH surge) can be achieved with doses as low as 15 and 50 microg.      

B lymphocytes secreting IgG linked to latent transforming growth factor- beta prevent primary cytolytic T lymphocyte responses

B lymphocytes secreting IgG linked to latent transforming growth factor (TGF)-beta (IgG-TGF-beta) prevent cytolytic T lymphocyte (CTL) responses to unrelated antigens in mixed lymphocyte cultures (MLC) so long as resting resident macrophages and functional Fc receptors are present. This was shown using IgG-secreting plaque-forming cells (PFC) to sheep erythrocytes (SRBC) obtained from popliteal lymph nodes of mice injected repeatedly in foot pads with SRBC. Remarkably, as few as approximately 300 PFC prevented CTL responses of 5 x 10(5) normal syngeneic spleen cells in MLC. Supranatants of short-term cultures of PFC also prevented CTL responses, and suppression was prevented by eliminating or dissociating IgG and TGF-beta present in supranatants or by antibody against active TGF-beta. Furthermore, the latency- associated peptide of latent TGF-beta was detected in approximately 10% of foci of IgG captured from single PFC, indicating that at least some B lymphocytes secrete IgG-TGF-beta as a complex. Resting resident macrophages (which do not produce latent TGF-beta) and functional Fc receptors were required for suppression, consistent with idea that IgG- TGF-beta is taken up through Fc receptors for IgG and that active TGF- beta, cleaved from latent TGF-beta of the complex, is delivered directly to potentially responding CTL. If CTL responses in man are similarly regulated by B lymphocytes, then an ongoing B cell response in patients with chronic viral infections or bearing immunogenic cancers may prevent effective therapeutic vaccination.      

Isolation and subfractionation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation on Percoll

The use of Percoll for isolation and subfractionation of PBMC and T-lymphocytes by discontinuous and continuous density gradient centrifugation is described: PBMC were isolated from human peripheral blood by discontinuous density gradient centrifugation on Percoll. The use of Percoll instead of Ficoll-Isopaque has the advantage that Percoll, in contrast to Ficoll-Isopaque, does not alter the density of monocytes. Therefore, a better separation of lymphocytes and monocytes was achieved after subsequent continuous density gradient centrifugation on Percoll. E-RFC were isolated by discontinuous density gradient centrifugation after a first low speed centrifugation step banding lymphocytes and SRBC on a Percoll-Ficoll cushion, and a subsequent high speed centrifugation step separating high density rosettes and SRBC from low density non-E-RFC. The advantage of this procedure is the short time of performance and that there is no need to resuspend the lymphocyte/SRBC pellet. PBMC, nph.PBMC T-lymphocytes were further subfractionated by continuous density gradient centrifugation on Percoll. The method described here resulted in a good separation of lymphocytes and monocytes. However, to obtain lymphocyte fractions with minute numbers of contaminating monocytes, a depletion of monocytes prior to further subfractionation of the lymphocytes by continuous density gradient centrifugation is recommended. A marker analysis of T-lymphocytes subfractionated by continuous density gradient centrifugation on Percoll shows that high density T-lymphocytes are enriched in ANAE positive lymphocytes of type 1 and depleted of ANAE positive lymphocytes of type 2. Low density T-lymphocytes are enriched in ANAE type 2 cells and depleted of ANAE type 1 cells. On the other hand, no considerable differences were found when analyzing the T-cells from different fractions for differentiation antigens by means of monoclonal antibodies (anti Lyt 3, OKT4, and OKT8). The results may indicate that subfractionation of T-lymphocytes by continuous density gradient centrifugation on Percoll provided T-cells in different functional states rather than T-cells of distinct subclasses.     

Psychosocial work characteristics as risk factors for the onset of fatigue and psychological distress: prospective results from the Maastricht Cohort Study

BACKGROUND: Prolonged fatigue has recently attracted attention in occupational (mental) health research since it may lead to sickness absenteeism and work disability. To date, little is known about the role of psychosocial work characteristics in the aetiology of fatigue. In this study we examined prospectively a wide range of psychosocial work characteristics as possible risk factors for the onset of fatigue and psychological distress in the working population. METHODS: This study is based on 8833 employees, participating in the Maastricht Cohort Study of 'Fatigue at Work'. A wide range of psychosocial work characteristics, measured at baseline, was used to predict the onset of fatigue and psychological distress 1 year later. Fatigue was measured with the Checklist Individual Strength; the General Health Questionnaire was used to measure psychological distress. RESULTS: The cumulative incidence of fatigue during 1 year follow-up was 9.7% (N = 492) in men, and 13.5% (N = 241) in women. Psychological demands at work as well as physical and emotional demands increased the risk for fatigue in men, whereas decision latitude in men and co-worker social support in women were protective against fatigue. These prospective associations remained significant after adjustments for potential confounders and baseline fatigue. As regards psychological distress, no association was found with decision latitude, while conflicts at work increased the risk of psychological distress. CONCLUSIONS: Psychosocial work characteristics were significant predictors for the onset of fatigue in the working population. The prospective associations suggest some differential effects in the aetiology of fatigue and psychological distress. Good interpersonal relationships at work and high decision authority were demonstrated to be relevant aspects that should be targeted for prevention.