Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers.

BACKGROUND: While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers. METHODS: The angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial. RESULTS: Hypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed. CONCLUSIONS: The present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784.

Allogeneic ABCB5+ mesenchymal stem cells for treatment-refractory chronic venous ulcers: a phase I/IIa clinical trial.

A significant number of chronic venous ulcers (CVUs) fail to heal despite of guideline-conform standard of care. Skin-derived ABCB5+ mesenchymal stem cells (MSCs) can dampen the sustained IL-1ß-driven inflammation present in chronic wounds. Based on their wound healing-facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5+ MSCs have emerged as a potential candidate for cell-based advanced therapy of non-healing CVUs. In the present interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVU had emerged as standard therapy-resistant received one or two topical applications of 1×106 allogeneic ABCB5+ MSCs/cm2 wound area in addition to standard treatment. Out of 83 treatment-emergent adverse events, only three were judged related to the cell product; they were mild or moderate and recovered without sequelae. Wound size markedly decreased from baseline to week 12, resulting in a median wound size reduction of 76% (full analysis set, N=31), 78% (per-protocol set, N=27) and 87% (subset of responders; n=21). In conclusion, the study treatment was well tolerated and safe. The treatment elicited a profound wound size reduction within 12 weeks, identifying ABCB5+ MSCs as a potential candidate for adjunctive therapy of otherwise incurable CVUs. These results justify the conduct of a larger, randomized, controlled trial to confirm clinical efficacy.

Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data.

BACKGROUND AIM: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation. METHODS: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers. RESULTS: As of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32-100%) at 12 weeks and early relief of pain. CONCLUSIONS: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.

Ureaplasma Species Differentially Modulate Pro- and Anti-Inflammatory Cytokine Responses in Newborn and Adult Human Monocytes Pushing the State Toward Pro-Inflammation.

Background:Ureaplasma species have been associated with chorioamnionitis and preterm birth and have been implicated in the pathogenesis of neonatal short and long-term morbidity. However, being mostly commensal bacteria, controversy remains on the pro-inflammatory capacity of Ureaplasma. Discussions are ongoing on the incidence and impact of prenatal, perinatal, and postnatal infection. The present study addressed the impact of Ureaplasma isolates on monocyte-driven inflammation. Methods: Cord blood monocytes of term neonates and adult monocytes, either native or LPS-primed, were cultured with Ureaplasma urealyticum (U. urealyticum) serovar 8 (Uu8) and Ureaplasma parvum serovar 3 (Up3). Using qRT-PCR, cytokine flow cytometry, and multi-analyte immunoassay, we assessed mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-8, IL-12p40, IL-10, and IL-1 receptor antagonist (IL-1ra) as well as Toll-like receptor (TLR) 2 and TLR4. Results: Uu8 and Up3 induced mRNA expression and protein release of TNF-α, IL-1ß and IL-8 in term neonatal and adult monocytes (p < 0.01 and p < 0.05). Intracellular protein expression of TNF-α, IL-1ß and IL-8 in Ureaplasma-stimulated cells paralleled those results. Ureaplasma-induced cytokine levels did not significantly differ from LPS-mediated levels except for lower intracellular IL-1ß in adult monocytes (Uu8: p < 0.05). Remarkably, ureaplasmas did not induce IL-12p40 response and promoted lower amounts of anti-inflammatory IL-10 and IL-1ra than LPS, provoking a cytokine imbalance more in favor of pro-inflammation (IL-1ß/IL-10, IL-8/IL-10 and IL-8/IL-1ra: p < 0.01, vs. LPS). In contrast to LPS, both isolates induced TLR2 mRNA in neonatal and adult cells (p < 0.001 and p < 0.05) and suppressed TLR4 mRNA in adult monocytes (p < 0.05). Upon co-stimulation, Uu8 and Up3 inhibited LPS-induced intracellular IL-1ß (p < 0.001 and p < 0.05) and IL-8 in adult monocytes (p < 0.01), while LPS-induced neonatal cytokines were maintained or aggravated (p < 0.05). Conclusion: Our data demonstrate a considerable pro-inflammatory capacity of Ureaplasma isolates in human monocytes. Stimulating pro-inflammatory cytokine responses while hardly inducing immunomodulatory and anti-inflammatory cytokines, ureaplasmas might push monocyte immune responses toward pro-inflammation. Inhibition of LPS-induced cytokines in adult monocytes in contrast to sustained inflammation in term neonatal monocytes indicates a differential modulation of host immune responses to a second stimulus. Modification of TLR2 and TLR4 expression may shape host susceptibility to inflammation.

Local cytokine changes in complex regional pain syndrome type I (CRPS I) resolve after 6 months.

There is evidence that inflammatory processes are involved in at least the early phase of complex regional pain syndrome (CRPS). We compared a panel of pro- and antiinflammatory cytokines in skin blister fluids and serum from patients with CRPS and patients with upper-limb pain of other origin (non-CRPS) in the early stage (< 1 year) and after 6 months of pain treatment. Blister fluid was collected from the affected and contralateral nonaffected side. We used a multiplex-10 bead array cytokine assay and Luminex technology to measure protein concentrations of the cytokines interleukin-1 receptor antagonist (IL-1RA), IL-2, IL-6, IL-8, IL-10, IL-12p40, and tumor necrosis factor-alpha (TNF-α) and the chemokines eotaxin, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1ß (MIP-1ß). We found bilaterally increased proinflammatory TNF-α and MIP-1ß and decreased antiinflammatory IL-1RA protein levels in CRPS patients compared to non-CRPS patients. Neither group showed side differences. After 6 months under analgesic treatment, protein levels of all measured cytokines in CRPS patients, except for IL-6, significantly changed bilaterally to the level of non-CRPS patients. These changes were not related to treatment outcome. In serum, only IL-8, TNF-α, eotaxin, MCP-1, and MIP-1ß were detectable without intergroup differences. Blister fluid of CRPS patients showed a bilateral proinflammatory cytokine profile. This profile seems to be relevant only at the early stage of CRPS. Almost all measured cytokine levels were comparable to those of non-CRPS patients after 6 months of analgesic treatment and were not related to treatment outcome.

Evaluation of immunological escape mechanisms in a mouse model of colorectal liver metastases.

BACKGROUND: The local and systemic activation and regulation of the immune system by malignant cells during carcinogenesis is highly complex with involvement of the innate and acquired immune system. Despite the fact that malignant cells do have antigenic properties their immunogenic effects are minor suggesting tumor induced mechanisms to circumvent cancer immunosurveillance. The aim of this study is the analysis of tumor immune escape mechanisms in a colorectal liver metastases mouse model at different points in time during tumor growth. METHODS: CT26.WT murine colon carcinoma cells were injected intraportally in Balb/c mice after median laparotomy using a standardized injection technique. Metastatic tumor growth in the liver was examined by standard histological procedures at defined points in time during metastatic growth. Liver tissue with metastases was additionally analyzed for cytokines, T cell markers and Fas/Fas-L expression using immunohistochemistry, immunofluorescence and RT-PCR. Comparisons were performed by analysis of variance or paired and unpaired t test when appropriate. RESULTS: Intraportal injection of colon carcinoma cells resulted in a gradual and time dependent metastatic growth. T cells of regulatory phenotype (CD4+CD25+Foxp3+) which might play a role in protumoral immune response were found to infiltrate peritumoral tissue increasingly during carcinogenesis. Expression of cytokines IL-10, TGF-beta and TNF-alpha were increased during tumor growth whereas IFN-gamma showed a decrease of the expression from day 10 on following an initial increase. Moreover, liver metastases of murine colon carcinoma show an up-regulation of FAS-L on tumor cell surface with a decreased expression of FAS from day 10 on. CD8+ T cells express FAS and show an increased rate of apoptosis at perimetastatic location. CONCLUSIONS: This study describes cellular and macromolecular changes contributing to immunological escape mechanisms during metastatic growth in a colorectal liver metastases mouse model simulating the situation in human cancer.

The intraportal injection model: a practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer.

BACKGROUND: The development of new therapeutic strategies for treatment of metastasized colorectal carcinoma requires biologically relevant and adequate animal models that generate both reproducible metastasis and the dissemination of tumor cells in the form of so-called minimal residual disease (MRD), an expression of the systemic character of neoplastic disease. METHODS: We injected immunoincompetent nude mice intraportally with different numbers (1 x 10(5), 1 x 10(6) and 5 x 10(6) cells) of the human colon carcinoma cell lines HT-29 and SW-620 and investigated by histological studies and CK-20 RT-PCR the occurrence of hematogenous metastases and the dissemination of human tumor cells in bone marrow. RESULTS: Only the injection of 1 x 10(6) cells of each colon carcinoma cell line produced acceptable perioperative mortality with reproducible induction of hepatic metastases in up to 89% of all animals. The injection of 1 x 10(6) cells also generated tumor cell dissemination in the bone marrow in up to 63% of animals with hepatic metastases. CONCLUSION: The present intraportal injection model in immunoincompetent nude mice represents a biologically relevant and adequate animal model for the induction of both reproducible hepatic metastasis and tumor cell dissemination in the bone marrow as a sign of MRD.