Impact of repeated bouts of eccentric exercise on myogenic gene expression

Evidence indicates that repeated-bouts of eccentric exercise (EE) do not exacerbate the extent of muscle damage indices, as compared to a single-bout. We hypothesized that molecular adaptations, under repeated-bouts of EE, would include suppression of muscle repair inhibitory factors such as myostatin and up-regulation of muscle repair positive regulatory factors such as myogenic regulatory factors (MRFs). Fifteen males were recruited for this study. The exercise group (n = 9) successfully completed six sets of 15 reps of maximum voluntary eccentric contractions, for six consecutive days, using a dynamometer (Multicont-II). Blood and muscle biopsy samples were obtained from each subject 1 week prior to exercise, 2 days post the first training session, and 24 h after the last training session. Gene expression levels were determined using real-time RT-PCR. Blood samples were analyzed for creatine kinase (CK) and lactate-dehydrogenase (LDH) activity. Repeated-bouts of EE induced a large down-regulation of myostatin mRNA (−73%) which persisted throughout the study. The responses of MRFs were mild. At day 3 only myogenin increased significantly (1.9 fold) while MyoD decreased by 45%. Surprisingly, at day 7, despite the presence of muscle damage indices, all MRFs returned to the pre-exercise levels. The results of the present study showed that repeated-bouts of EE, for six consecutive days, dramatically decreased Myostatin mRNA expression but impaired the expression patterns of MRFs such that, with the exception of myogenin that showed a moderate non-sustained increase, MyoD and MYf5 response was minimal. Grants: Funding was provided by the Ministry of Health of the Hungarian Government. Grant number: ETT 388/2003.     

A clinically integrated curriculum in Evidence-based Medicine for just-in-time learning through on-the-job training: The EU-EBM project

Background

Little research has been conducted to investigate role stress experienced by faculty members in medical schools in developing countries. This becomes even more important when the process of reform in medical education has already taken place, such as the case of Iran. The objectives of this study were to investigate and assess the level and source of role-related stress as well as dimensions of conflict among the faculty members of Iranian medical schools. Variables like the length of academic work, academic rank, employment position, and the departments of affiliation were also taken into consideration in order to determine potentially related factors.

Methods

A survey was conducted at three different ranks of public medical schools. The validated Organizational Role Stress Scale was used to investigate the level of role stress and dimensions of role conflict among medical faculty members. The response rate was 66.5%.

Results

The findings show that role stress was experienced in high level among almost all faculty members. All three studied medical schools with different ranks are threatened with relatively the same levels of role stress. Specific differences were found among faculty members from different disciplines, and academic ranks. Also having permanent position and the length of services had significant correlation with the level of role stress. The major role- related stress and forms of conflict among faculty members were role overload, role expectation conflict, inter-role distance, resource inadequacy, role stagnation, and role isolation.

Conclusion

The most role-related stressors and forms of conflict among faculty members include too many tasks and everyday work load; conflicting demands from colleagues and superiors; incompatible demands from their different personal and organizational roles; inadequate resources for appropriate performance; insufficient competency to meet the demands of their role; inadequate autonomy to make decision on different tasks; and a feeling of underutilization. The findings of this study can assist administrators and policy makers to provide an attractive working climate in order to decrease side effects and consequences of role stress and to increase productivity of faculty members. Furthermore, understanding this situation can help to develop coping strategies in order to reduce role-related stress.     

The Unenlarged Lymph Nodes of HIV-1–infected, Asymptomatic Patients with High CD4 T Cell Counts Are Sites for Virus Replication and CD4 T Cell Proliferation. The Impact of Highly Active Antiretroviral Therapy

The efficacy of triple drug therapy for HIV-1 infection encourages its early use to prevent damage to the immune system. We monitored the effects of such therapy on 12 patients with 14–75-mo histories of minimal disease, i.e., CD4⁺ counts constantly >500/μl and little or no lymph node enlargement. In this way, we could first determine the extent of viral replication and immunoarchitectural changes in unenlarged nodes early in disease, and second follow the response to triple therapy in plasma and lymphoid tissue in tandem. As is known for lymph nodes with more advanced disease, the germinal centers showed productively infected T cells, i.e., CD4⁺CD1a⁻CD68⁻ cells labeling intensely for HIV-1 RNA after in situ hybridization. The unenlarged nodes also showed extensive HIV-1 RNA retention on a well-preserved, follicular dendritic cell (FDC) network, and the follicles were abnormal. There were numerous CD8⁺ cells, many expressing TIA-1 granule antigen. Also, in contrast to normal follicles, CD4⁺ T cell proliferation was active, with marked increases in the number of cycling, Ki-67⁺CD4⁺CD45R0⁺ cells. After 28 d and 3 mo of therapy, productively infected T cells decreased dramatically and often were not apparent. The labeling of the FDC network for viral RNA also decreased, but not for gag protein. We conclude that HIV-1 replicates and accumulates in lymphoid organs before damage of the immune system, that at this stage of disease de novo production of T cells occurs in the lymphoid tissue, and that the infection is sensitive to triple drug therapy in both plasma and lymph nodes.     

Differences in T cell subsets between men and women with idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder, occurring predominantly in women. We studied by flow cytofluorimetry the T cell subsets in men and women with ITP and compared them with healthy sex-matched volunteers. In healthy controls, women were found to have higher proportions of T helper/inducer (Th/i) and lower T suppressor/cytotoxic (Ts/c) lymphocytes and consequently higher Th/i:Ts/c ratios than men. Accordingly, in clinical surveys, patients and controls should be matched for sex for proper comparisons. In patients with ITP in its active phase, an imbalance in T cell subsets was found in both sexes. The perturbation was more severe in women who had a marked decrease in number and proportion of Th/i lymphocytes and an increase in the proportion of Ts/c lymphocytes, whereas in men only, the proportion of Th/i lymphocytes was decreased. When patients with active disease were compared to those with ITP in remission, the decrease in Th/i subsets still persisted in both sexes but the Ts/c subset in women had returned to normal proportions. Therefore, the immune imbalance in ITP is more marked in women than men; imbalances in both Th/i and Ts/c are present in women while Ts/c appears not to be involved in men.     

Simian immunodeficiency virus-specific cytotoxic T lymphocytes and cell-associated viral RNA levels in distinct lymphoid compartments of SIVmac-infected rhesus monkeys

Major histocompatibility class I-peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8(+) T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8(+) T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8(+) T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8(+) T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8(+) T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell-associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8(+) T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion. (Blood. 2000;96:1474-1479)     

MiR‐34a deficiency accelerates medulloblastoma formation in vivo

Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR‐34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR‐34a in vivo. We generated mice with a constitutive deletion of the miR‐34a gene. These mice were devoid of mir‐34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR‐34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re‐expression of miR‐34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR‐34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR‐34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR‐34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR‐34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR‐34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re‐express miR‐34a in tumors could, therefore, represent an efficient therapeutic option.     

Post-surgical assessment of excised tissue from patients with Buruli ulcer disease: progression of infection in macroscopically healthy tissue

OBJECTIVE: The current standard of treatment of Buruli ulcer disease (BUD) is surgical excision of lesions. Excision size is determined macroscopically assuming the complete removal of all infected tissue. However, dissemination of infection beyond the excision margins into apparently healthy tissue, possibly associated with recurrences, cannot be excluded in this way. To assess the central to peripheral progression of Mycobacterium ulcerans infection and the mycobacterial infiltration of excision margins, excised tissue was examined for signs of infection. METHODS: 20 BUD lesions were excised in general anaesthesia including all necrotic and subcutaneous adipose tissue down to the fascia and at an average of 40 mm into the macroscopically unaffected tissue beyond the border of the lesion. Tissue samples were subjected to PCR and histopathology. RESULTS: Although the bacillary load decreased from central to peripheral, M. ulcerans infection was detected throughout all examined tissue specimens including the peripheral segments as well as excision margins of all patients. During the post-operative hospitalization period (averaging 2 months) no local recurrences were observed. CONCLUSION: Available data suggest a correlation of surgical techniques with local recurrences. The results of this study indicate the unnoticed early progression of mycobacterial infection into macroscopically healthy tissue. Thus, the removal of all infected tissue cannot always be verified visually by the surgeon. Provided that long-term follow up of patients with positive excision margins will establish the clinical relevance of these findings, on-site laboratory assessment of excised tissue in combination with follow up may contribute to reduce recurrence rates.     

Defective platelet-fibrinogen interaction in hereditary canine thrombopathia

A unique, intrinsic, hereditary canine platelet disorder attributable to abnormal fibrinogen receptor availability is described. Thrombopathic platelets from 13 severely affected basset hounds failed to aggregate in response to all agonists tested except thrombin. Normal platelet interaction with the various stimuli was inferred on the basis of their ability to elicit unimpaired shape change in thrombopathic platelets. No quantitative differences in major platelet membrane glycoproteins, intraplatelet fibrinogen, adenine nucleotides, or serotonin uptake were detected. Dense granule secretion was impaired. The ultrastructural appearance of thrombopathic platelets was normal. Fibrinogen-platelet interaction was evaluated by reacting platelet-rich plasma (PRP) with fibrinogen coupled to polymeric acrylonitrile beads and scoring the extent of stimulus-induced agglutination. The aggregatory responses of normal and thrombopathic platelets were closely correlated with fibrinogen receptor availability. In contrast to human platelets, epinephrine-stimulated canine platelets did not interact with immobilized fibrinogen, and arachidonate generally induced only weak agglutination. Thrombopathic platelets agglutinated fibrinogen beads at reduced rates when stimulated with physiologic doses of thrombin and high-dose calcium ionophore, A23187. Our data suggest that thrombin-mediated induction of canine platelet fibrinogen receptors may proceed by pathway(s) alternate to those shared by other platelet agonists, and/or that secreted granule constituents may act synergistically with thrombin to overcome inhibition of signal-response- coupled reactions mediating the interaction of fibrinogen with its receptor. This congenital platelet defect provides further evidence, in a species other than human, for the pivotal role of fibrinogen receptor induction in platelet aggregation.     

Molecular genetic rearrangements distinguish pre- and post-bone marrow transplantation lymphoproliferative processes

Chronic myelocytic leukemia (CML) may display a lymphoproliferative phase (lymphoid blast crisis) that is generally of B cell phenotype. Since lymphoproliferative disorders may occur following bone marrow transplantation (BMT), it may be difficult to distinguish posttransplant relapse of CML lymphoid blast crisis from de novo lymphoproliferation. Lymphoid blast crisis cells from a patient with CML displayed immunoglobulin heavy chain gene (C mu) rearrangement before BMT. Following BMT the patient developed a lymphoproliferative disorder involving multiple organs. Clonal rearrangement of C mu was demonstrated in several involved tissues. The rearranged C mu restriction fragment was distinct from that displayed before BMT. Additionally, rearrangement of the breakpoint cluster region (bcr) was demonstrated in the pretransplant blast crisis sample, but not in the posttransplant lymphoproliferation samples, thus confirming that these lymphoproliferative disorders were distinct. Molecular genetic techniques offer powerful diagnostic tools for monitoring the course of patients with CML undergoing BMT.