MALDI imaging mass spectrometry: discrimination of pathophysiological regions in traumatized skeletal muscle by characteristic peptide signatures.

Due to formation of fibrosis and the loss of contractile muscle tissue, severe muscle injuries often result in insufficient healing marked by a significant reduction of muscle force and motor activity. Our previous studies demonstrated that the local transplantation of mesenchymal stromal cells into an injured skeletal muscle of the rat improves the functional outcome of the healing process. Since, due to the lack of sufficient markers, the accurate discrimination of pathophysiological regions in injured skeletal muscle is inadequate, underlying mechanisms of the beneficial effects of mesenchymal stromal cell transplantation on primary trauma and trauma adjacent muscle area remain elusive. For discrimination of these pathophysiological regions, formalin-fixed injured skeletal muscle tissue was analyzed by MALDI imaging MS. By using two computational evaluation strategies, a supervised approach (ClinProTools) and unsupervised segmentation (SCiLS Lab), characteristic m/z species could be assigned to primary trauma and trauma adjacent muscle regions. Using "bottom-up" MS for protein identification and validation of results by immunohistochemistry, we could identify two proteins, skeletal muscle alpha actin and carbonic anhydrase III, which discriminate between the secondary damage on adjacent tissue and the primary traumatized muscle area. Our results underscore the high potential of MALDI imaging MS to describe the spatial characteristics of pathophysiological changes in muscle.

Unraveling local tissue changes within severely injured skeletal muscles in response to MSC-based intervention using MALDI Imaging mass spectrometry.

Pre-clinical and clinical studies are now beginning to demonstrate the high potential of cell therapies in enhancing muscle regeneration. We previously demonstrated functional benefit after the transplantation of autologous bone marrow mesenchymal stromal cells (MSC-TX) into a severe muscle crush trauma model. Despite our increasing understanding of the molecular and cellular mechanisms underlying MSC's regenerative function, little is known about the local molecular alterations and their spatial distribution within the tissue after MSC-TX. Here, we used MALDI imaging mass spectrometry (MALDI-IMS) in combination with multivariate statistical strategies to uncover previously unknown peptide alterations within severely injured skeletal muscles. Our analysis revealed that very early molecular alterations in response to MSC-TX occur largely in the region adjacent to the trauma and only to a small extent in the actual trauma region. Using "bottom up" mass spectrometry, we subsequently identified the proteins corresponding to the differentially expressed peptide intensity distributions in the specific muscle regions and used immunohistochemistry to validate our results. These findings extend our current understanding about the early molecular processes of muscle healing and highlights the critical role of trauma adjacent tissue during the early therapeutic response upon treatment with MSC.

Deficient EBV-specific B- and T-cell response in patients with chronic fatigue syndrome.

Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4(+) and CD8(+) T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.

Intra-Arterial MSC Transplantation Restores Functional Capacity After Skeletal Muscle Trauma.

INTRODUCTION: Skeletal muscle trauma leads to severe functional deficits, which cannot be addressed by current treatment options. Our group could show the efficacy of local transplantation of mesenchymal stroma cells (MSCs) for the treatment of injured muscles. While local application of MSCs has proven to be effective, we hypothesized that a selective intra-arterial transplantation would lead to a better distribution of the cells and so improved physiological recovery of muscle function. MATERIALS AND METHODOLOGY: 18 female Sprague Dawley rats received an open crush trauma of the left soleus muscle. Autologous MSC were transduced using dsCOP-GFP and 2.5 x 10(6)cells were transplanted into the femoral artery of 9 animals one week after trauma. Control animals (n=9) received a saline injection. Cell tracking, analysis of tissue fibrosis and muscle force measurements were performed after 3 weeks. RESULTS: Systemic MSC-therapy improved the muscle force significantly compared to control (fast twitch: 82.4%, tetany: 61.6%, p = 0.02). The histological analysis showed no differences in the quantity of fibrotic tissue. Histological examination revealed no cells in the traumatized muscle tissue 21 days after transplantation. CONCLUSIONS: The present study demonstrated an effect of systemically administered MSCs in the treatment of skeletal muscle injuries. For possible future therapeutic approaches a systemic application of MSCs seems to present an alternative to a local administration. Such systemic treatment would be preferable since it allows functional improvement and possible cellular concentration at injury sites that are not easily accessible.

No evidence for XMRV in German CFS and MS patients with fatigue despite the ability of the virus to infect human blood cells in vitro.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms. METHODOLOGY: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production. CONCLUSION: None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.

Leukocyte redistribution: effects of beta blockers in patients with chronic heart failure.

BACKGROUND: Overproduction of pro-inflammatory cytokines is a well established factor in the progression of chronic heart failure (CHF). Changes in cellular immunity have not been widely studied, and the impact of standard medication is uncertain. Here we investigate whether a leukocyte redistribution occurs in CHF and whether this effect is influenced by beta-blocker therapy. METHODOLOGY: We prospectively studied 75 patients with systolic CHF (age: 68+/-11 years, left ventricular ejection fraction 32+/-11%, New York Heart Association class 2.5+/-0.7) and 20 age-matched healthy control subjects (age: 63+/-10 years). We measured the response of cells to endotoxin exposure in vitro, analysed subsets of lymphocytes using flow cytometry, and assessed plasma levels of the pro-inflammatory markers interleukin 1, 6, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptors 1 and 2. PRINCIPAL FINDINGS: While no differences in the number of leukocytes were noted between patients with CHF and healthy controls, we detected relative lymphopenia in patients with CHF (p<0.001 vs. control), mostly driven by reductions in T helper cells and B cells (both p<0.05). The number of neutrophils was increased (p<0.01). These effects were pronounced in patients who were beta-blocker naïve (32% of all patients with CHF). Increased plasma levels of soluble tumor necrosis receptor-1 correlated with the relative number of lymphocyte subsets. CONCLUSIONS: In patients with CHF, we detected a redistribution of leukocyte subsets, i.e. an increase in neutrophils with relative lymphopenia. These effects were pronounced in patients who were beta-blocker naïve. The underlying mechanism remains to be elucidated.