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

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

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

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

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

Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.

<p>PCR results with isolated LNCaP cell DNA after co-culture with PBMCs from CFS patients (lane 1–5) and healthy donors (lanes 6–10). Five representative samples out of 10 co-cultures for each group are shown. As control, LNCaP cells were infected with XMRV-containing supernatant from 22Rv1 cells (lane 12). A water-only control (no template control, NTC) was run in lane 11. Results of the GAPDH PCR with the same samples are shown in the lower panel. M  = 100 bp marker.</p

Serological assays.

<p>ELISAs with recombinant XMRV proteins were used to detect specific humoral responses. The cut-off was calculated as the mean of all sera from healthy controls plus three times the standard deviation. (A) Results of an XMRV Env antibody ELISA with sera from 36 CFS patients and 17 healthy controls (upper panel) and 112 MS patients and 10 healthy controls (lower panel) identified after unblinding. Sera were incubated at a dilution of 1∶200. (B) Titration of positive control goat sera versus recombinant Gag protein captured by the monoclonal anti-MLV Gag antibody R187. (C) Capture ELISA for the detection of XMRV anti-Gag antibodies in CFS (upper panel) and MS (lower panel) patient sera and healthy controls (gray bars in both panels). Human sera were diluted 1∶200 in blocking buffer.</p

Human PBMC cultures are susceptible to productive infection by XMRV.

<p>(A) Detection of proviral XMRV sequences by nested PCR in DNAs of PBMC from 5 CSF patients (lanes 1–5) and 5 healthy donors (HD, lanes 6–10) infected for a week with DNase I treated (left hand panel) or DNase I treated and heat-inactivated (right hand panel) supernatants from the XMRV producing 22Rv1 cell line. The no template control (NTC) is in lane 11. DNA prepared from 22Rv1 cells was used as a positive control (lane 12, left panel). Results of a single round PCR for GAPDH are depicted underneath. (B) On the day of DNA isolation, supernatants from the infected PBMC cultures were used to test for virus transmission to LNCaP indicator cells. The results of a nested XMRV PCR with DNA prepared from the exposed LNCaP cells a week after incubation with the PBMC supernatants are shown (lanes 1–10). The control set up was the same as described above (lanes 11 and 12). M  = 100 bp marker. The entire experiment has been repeated twice with similar results.</p

Diagnostic PCR of activated PBMC from CFS and MS patients and healthy donors.

<p>Representative results of nested XMRV PCRs with template DNAs from activated PBMCs after 7 days of culture. Samples from 5 CFS patients (lanes 1–5), 5 MS patients (lanes 6–10) and 5 healthy donors (HD, lanes 11–15) are shown. 200 ng of human genomic DNA spiked with 14 pg of 22Rv1 DNA (genome equivalent of approximately two cells) were used as positive control (lane 16). Results of corresponding single round PCRs for GAPDH as control for DNA integrity and absence of PCR inhibitors are shown in the lower panel. M  = 100 bp marker.</p

Baseline data for control subjects and patients with CHF.

<p>Baseline data for control subjects and patients with CHF.</p