Vincent Kindler

The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection

Granuloma formation in the liver of mice infected with BCG coincides with local TNF synthesis. Injection of rabbit anti-TNF antibody, after 1 or 2 weeks of infection, dramatically interferes with the development of granulomas (both in number and size, large epithelioid cells failing to appear) and subsequent mycobacterial elimination. Furthermore, fully developed BCG granulomas, after 3 weeks of infection, rapidly regress after anti-TNF treatment. Antibody treatment also prevents or suppresses accumulation of TNF mRNA and protein, which resumes after disappearance of the antibody. Peritoneal macrophages exposed to TNF transiently accumulate TNF mRNA, and show an enhanced increase in TNF mRNA in response to gamma interferon. We propose that TNF released from macrophages in the microenvironment of developing granulomas is involved in a process of autoamplification: acting in an autocrine or paracrine way, it enhances its own synthesis and release, thus favoring further macrophage accumulation and differentiation leading to bacterial elimination.

A Critical Role for p38 Mitogen-Activated Protein Kinase in the Maturation of Human Blood-Derived Dendritic Cells Induced by Lipopolysaccharide, TNF-α, and Contact Sensitizers

We investigated the involvement of mitogen-activated protein kinases (MAPKs) in the maturation of CD83− dendritic cells (DC) derived from human blood monocytes. Maturating agents such as LPS and TNF-α induced the phosphorylation of members of the three families of MAPK (extracellular signal-regulated kinase l/2, p46/54 c-Jun N-terminal kinase, and p38 MAPK). SB203580, an inhibitor of the p38 MAPK, but not the extracellular signal-regulated kinase l/2 pathway blocker PD98059, inhibited the up-regulation of CD1a, CD40, CD80, CD86, HLA-DR, and the DC maturation marker CD83 induced by LPS and TNF-α. In addition, SB203580 inhibited the enhancement of the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake induced by LPS and TNF-α. Likewise, SB203580 partially prevented the up-regulation of IL-1α, IL-1β, IL-lRa, and TNF-α mRNA upon stimulation with LPS and TNF-α, as well as the release of bioactive TNF-α induced by LPS. DC maturation induced by the contact sensitizers 2,4-dinitrofluorobenzene and NiSO4, as seen by the up-regulation of CD80, CD86, and CD83, was also coupled to the phosphorylation of p38 MAPK, and was inhibited by SB203580. The irritants SDS and benzalkonium chloride that do not induce DC maturation did not trigger p38 MAPK phosphorylation. Together, these data indicate that phosphorylation of p38 MAPK is critical for the maturation of immature DC. These results also suggest that p38 MAPK phosphorylation in DC may become useful for the identification of potential skin contact sensitizers.

EWS-FLI-1 Expression Triggers a Ewing's Sarcoma Initiation Program in Primary Human Mesenchymal Stem Cells

Ewings sarcoma family tumors (ESFT) express the EWS-FLI-1 fusion gene generated by the chromosomal translocation t(11;22)(q24;q12). Expression of the EWS-FLI-1 fusion protein in a permissive cellular environment is believed to play a key role in ESFT pathogenesis. However, EWS-FLI-1 induces growth arrest or apoptosis in differentiated primary cells, and the identity of permissive primary human cells that can support its expression and function has until now remained elusive. Here we show that expression of EWS-FLI-1 in human mesenchymal stem cells (hMSC) is not only stably maintained without inhibiting proliferation but also induces a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWS-FLI-1 in hMSCs may recapitulate the initial steps of Ewings sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWS-FLI-1 in hMSCs, we found the polycomb group gene EZH2, which we show to play a critical role in Ewings sarcoma growth. These observations are consistent with our recent findings using mouse mesenchymal progenitor cells and provide compelling evidence that hMSCs are candidate cells of origin of ESFT.

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells.

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene‐targeting therapies since they differentiate into various cell‐lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non‐hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45+ leukocytes. After culture, leukocytes were replaced by a homogenous layer of adherent CD45− CD14− CD34− CD11b− CD90+ HLA‐ABC+ cells. Culture doubling time (mean = 4 days, range 1.6–6.7 days) was not correlated with patient age (27–81 years, n = 16). Amplified cultures supported long‐term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 106‐fold) were not age‐related. All 14 clones analyzed (from five patients, ages 27–78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM‐derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC‐based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC. J. Cell. Physiol. 198: 110–118, 2004.

IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

Background The EWS-FLI-1 fusion protein is associated with 85–90% of Ewings sarcoma family tumors (ESFT), the remaining 10–15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. Methodology/Principal Findings To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3–5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Conclusion/Significance Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

Autologous bone marrow mononuclear cell transplantation in patients with decompensated alcoholic liver disease: a randomized controlled trial.

Objective Impaired liver regeneration is associated with a poor outcome in patients with decompensated alcoholic liver disease (ALD). We assessed whether autologous bone marrow mononuclear cell transplantation (BMMCT) improved liver function in decompensated ALD. Design 58 patients (mean age 54 yrs; mean MELD score 19, all with cirrhosis, 81% with alcoholic steatohepatitis at baseline liver biopsy) were randomized early after hospital admission to standard medical therapy (SMT) alone (n = 30), including steroids in patients with a Maddrey’s score ≥32, or combined with G-CSF injections and autologous BMMCT into the hepatic artery (n = 28). Bone marrow cells were harvested, isolated and reinfused the same day. The primary endpoint was a ≥3 points decrease in the MELD score at 3 months, corresponding to a clinically relevant improvement in liver function. Liver biopsy was repeated at week 4 to assess changes in Ki67+/CK7+ hepatic progenitor cells (HPC) compartment. Results Both study groups were comparable at baseline. After 3 months, 2 and 4 patients died in the BMMCT and SMT groups, respectively. Adverse events were equally distributed between groups. Moderate alcohol relapse occurred in 31% of patients. The MELD score improved in parallel in both groups during follow-up with 18 patients (64%) from the BMMCT group and 18 patients (53%) from the SMT group reaching the primary endpoint (p = 0.43 (OR 1.6, CI 0.49–5.4) in an intention to treat analysis. Comparing liver biopsy at 4 weeks to baseline, steatosis improved (p<0.001), and proliferating HPC tended to decrease in both groups (−35 and −33%, respectively). Conclusion Autologous BMMCT, compared to SMT is a safe procedure but did not result in an expanded HPC compartment or improved liver function. These data suggest either insufficient regenerative stimulation after BMMCT or resistance to liver regenerative drive in patients with decompensated alcoholic cirrhosis. Trial Registration Controlled-Trials.com ISRCTN83972743.

Human Bone Marrow Stromal Cells and Skin Fibroblasts Inhibit Natural Killer Cell Proliferation and Cytotoxic Activity

Mesenchymal stromal cells (MSCs) are potent immunomodulators that have successfully been used to circumvent various types of inflammations, including steroid-resistant graft-versus-host disease. Although initially believed to be restricted to multipotent MSCs, this immunoregulatory function is shared with differentiated cells from the mesenchymal lineage such as skin fibroblasts (SFs). Mesenchymal cell-induced immunoregulation is so potent that it may allow the reactivation of dormant malignancies, a fact that would preclude using such cells as therapeutic agents. Because NK cells are pivotal effectors controlling tumor cell containment we investigated the effect of allogenic MSCs and SFs on NK cell function in vitro. When NK cells were incubated with IL-15 and MSCs or SFs for 6 days, their proliferation and cytotoxic activity were significantly decreased compared to NK cells cultured with IL-15 alone or with human venous endothelial cells. Cytotoxic activity inhibition reached 86% when assayed on MHC-I+ allogenic primary hematopoietic blasts, and was associated with a significant decrease in cytolytic granule exocytosis and in perforin release. Stromal cell-mediated inhibition was effective only if cell–cell proximity was long lasting: when NK cells were activated with IL-15 in the absence of MSCs and assayed for cytotoxicity in their presence no inhibition occurred. MSC inhibition was ultimately mediated by a soluble factor generated upon incubation with NK cells activated by IL-15 or IL-2. The indoleamine 2,3 dioxygenase was activated in MSCs and SFs because L-kynurenine was detected in inhibitory supernatants, but its blockade did not restore NK cell functions. The profound inhibition of cytotoxic activity directed against allogenic hematopoietic blasts exerted by MSCs and SFs on NK cells may be a concern. Should this occur in vivo it may induce the inability of NK cells to control residual or dormant malignant diseases after infusion of therapeutic MSCs.

In vitro activated human T lymphocytes very efficiently attach to allogenic multipotent mesenchymal stromal cells and transmigrate under them

The regulatory effect of human multipotent mesenchymal stromal cells (MSC) on allogenic T lymphocytes is extremely powerful and of important clinical relevance, but the mechanisms underlying this process are not fully elucidated. We report here that T lymphocytes activated with a sub‐mitogenic stimulus such as phytohemaglutinin alone (PHA), or with mitogenic stimuli such as PHA + interleukin‐2 (P‐IL2), or immobilized anti‐CD3 + anti‐CD28 mAb (a3‐28), tightly bound allogenic MSC and transmigrated within 4 h under them, where they remained for approximately 60 h. Allogenic MSC induced T cell proliferation in cultures containing sub‐mitogenic PHA concentrations, and inhibited the mitogenic effect of P‐IL2 or a3‐28. Anti‐γ‐IFN mAb or L‐tryptophan complementation partially restored proliferation in P‐IL2 and a3‐28 cultures, whereby γ‐IFN‐synthesizing CD3+ cells were detectable. MSC‐lymphocyte contact hindrance using transwells abrogated proliferation in PHA cultures, restored it integrally in P‐IL2 cultures, and partially in a3‐28 cultures. These data suggest that MSC‐induced T lymphocyte regulation results from the combination of various processes. Allogenic cell–cell contact, as demonstrated by the PHA co‐cultures is per se stimulatory, whereas γ‐IFN synthesized by activated T lymphocytes, which activates indolamine 2,3‐dioxygenase in MSC, and L‐tryptophan depletion, which is induced by this enzyme, are inhibitory. Transmigration is nevertheless pivotal for the establishment of the inhibition by these mediators because it targets lymphocytes under the stroma in small extracellular spaces surrounded by MSC, where L‐tryptophan is efficiently destroyed, leading to T lymphocyte proliferation arrest. In conclusion lymphocyte transmigration under allogenic MSC potentiates the inhibitory effect of soluble mediators generated by these cells. J. Cell. Physiol. 214: 588–594, 2008.

Impact of selection of cord blood units from the United States and swiss registries on the cost of banking operations

Background: Over the last 2 decades, cord blood (CB) has become an important source of blood stem cells. Clinical experience has shown that CB is a viable source for blood stem cells in the field of unrelated hematopoietic blood stem cell transplantation. Methods: Studies of CB units (CBUs) stored and ordered from the US (National Marrow Donor Program (NMDP) and Swiss (Swiss Blood Stem Cells (SBSC)) CB registries were conducted to assess whether these CBUs met the needs of transplantation patients, as evidenced by units being selected for transplantation. These data were compared to international banking and selection data (Bone Marrow Donors Worldwide (BMDW), World Marrow Donor Association (WMDA)). Further analysis was conducted on whether current CB banking practices were economically viable given the units being selected from the registries for transplant. It should be mentioned that our analysis focused on usage, deliberately omitting any information about clinical outcomes of CB transplantation. Results: A disproportionate number of units with high total nucleated cell (TNC) counts are selected, compared to the distribution of units by TNC available. Therefore, the decision to use a low threshold for banking purposes cannot be supported by economic analysis and may limit the economic viability of future public CB banking. Conclusions: We suggest significantly raising the TNC level used to determine a bankable unit. A level of 125 × 107 TNCs, maybe even 150 × 107 TNCs, might be a viable banking threshold. This would improve the return on inventory investments while meeting transplantation needs based on current selection criteria.

Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells

Genetically engineered dendritic cells (DC) presenting specific antigens to T cells may be of great interest for immunotherapy. For this reason, the production of transgene‐expressing DC derived from CD34+ cells transduced either shortly after ex vivo purification or during their differentiation into DC were evaluated.