Prompt Thrombo-Inflammatory Response to Ischemia-Reperfusion Injury and Kidney Transplant Outcomes.

Introduction: In kidney transplantation (KT), the role of the intravascular innate immune system (IIIS) in response to ischemia-reperfusion injury (IRI) is not well-understood. Here, we studied parallel changes in the generation of key activation products of the proteolytic cascade systems of the IIIS following living donor (LD) and deceased donor (DD) transplantation and evaluated potential associations with clinical outcomes. Methods: In a cohort study, 63 patients undergoing LD (n = 26) and DD (n = 37) transplantation were prospectively included. Fifteen DD kidneys were preserved with hypothermic machine perfusion (HMP), and the remaining were cold stored. Activation products of the kallikrein-kinin, coagulation, and complement systems were measured in blood samples obtained systemically at baseline and locally from the transplant renal vein at 1, 10, and 30 minutes after reperfusion. Results: DD kidneys exhibited a prompt and interlinked activation of all 3 cascade systems of IIIS postreperfusion, indicating a robust and local thrombo-inflammatory response to IRI. In this initial response, the complement activation product sC5b-9 exhibited a robust correlation with other IIIS activation markers and displayed a strong association with short-term and mid-term (24-month) graft dysfunction. In contrast, LD kidneys did not exhibit this thrombo-inflammatory response. The use of HMP was associated with reduced thromboinflammation and preserved mid-term kidney function. Conclusion: Kidneys from DD are vulnerable to a prompt thrombo-inflammatory response to IRI, which adversely affects both short-term and long-term allograft function. Strategies aimed at minimizing graft immunogenicity prior to reperfusion are crucial to mitigate the intricate inflammatory response to IRI.

Siplizumab combination therapy with belatacept or abatacept broadly inhibits human T cell alloreactivity in vitro.

Combined antigen-specific T cell receptor stimulation and costimulation are needed for complete T cell activation. Belatacept and abatacept are nondepleting fusion proteins blocking CD28/B7 costimulation, whereas siplizumab is a depleting antiCD2 immunoglobulin G1 monoclonal antibody targeting CD2/CD58 costimulation. Herein, the effect of siplizumab combination therapy with abatacept or belatacept on T cell alloreactivity in mixed lymphocyte reactions was investigated. In contrast to monotherapy, the combination of siplizumab with belatacept or abatacept induced near-complete suppression of T cell proliferation and increased the potency of siplizumab-mediated T cell inhibition. Furthermore, dual targeting of CD2 and CD28 costimulation enhanced the selective depletion of memory T cells compared with monotherapy. Although siplizumab monotherapy leads to significant regulatory T cell enrichment, high doses of cytotoxic T-lymphocyte-associated antigen 4 and a human IgG1 Fc fragment in the combination therapy reduced this effect. These results support the clinical evaluation of dual costimulation blockade, combining siplizumab with abatacept or belatacept, for the prophylaxis of organ transplant rejection and improvement of long-term outcomes following transplantation. Ongoing investigative research will elucidate when other forms of siplizumab-based dual costimulatory blockade may be able to induce similarly strong inhibition of T cell activation although still allowing for enrichment of regulatory T cells.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity.

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro.

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

Development and validation of the first consensus gene-expression signature of operational tolerance in kidney transplantation, incorporating adjustment for immunosuppressive drug therapy.

BACKGROUND: Kidney transplant recipients (KTRs) with "operational tolerance" (OT) maintain a functioning graft without immunosuppressive (IS) drugs, thus avoiding treatment complications. Nevertheless, IS drugs can influence gene-expression signatures aiming to identify OT among treated KTRs. METHODS: We compared five published signatures of OT in peripheral blood samples from 18 tolerant, 183 stable, and 34 chronic rejector KTRs, using gene-expression levels with and without adjustment for IS drugs and regularised logistic regression. FINDINGS: IS drugs explained up to 50% of the variability in gene-expression and 20-30% of the variability in the probability of OT predicted by signatures without drug adjustment. We present a parsimonious consensus gene-set to identify OT, derived from joint analysis of IS-drug-adjusted expression of five published signature gene-sets. This signature, including CD40, CTLA4, HSD11B1, IGKV4-1, MZB1, NR3C2, and RAB40C genes, showed an area under the curve 0⋅92 (95% confidence interval 0⋅88-0⋅94) in cross-validation and 0⋅97 (0⋅93-1⋅00) in six months follow-up samples. INTERPRETATION: We advocate including adjustment for IS drug therapy in the development stage of gene-expression signatures of OT to reduce the risk of capturing features of treatment, which could be lost following IS drug minimisation or withdrawal. Our signature, however, would require further validation in an independent dataset and a biomarker-led trial. FUNDING: FP7-HEALTH-2012-INNOVATION-1 [305147:BIO-DrIM] (SC,IR-M,PM,DSt); MRC [G0801537/ID:88245] (MPH-F); MRC [MR/J006742/1] (IR-M); Guy's&StThomas' Charity [R080530]&[R090782]; CONICYT-Bicentennial-Becas-Chile (EN-L); EU:FP7/2007-2013 [HEALTH-F5-2010-260687: The ONE Study] (MPH-F); Czech Ministry of Health [NV19-06-00031] (OV); NIHR-BRC Guy's&StThomas' NHS Foundation Trust and KCL (SC); UK Clinical Research Networks [portfolio:7521].

Phase I trial evaluating safety and efficacy of intratumorally administered inflammatory allogeneic dendritic cells (ilixadencel) in advanced gastrointestinal stromal tumors.

BACKGROUND: The majority of patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Ilixadencel (allogeneic inflammatory dendritic cells) is a cell-based immune primer injected intratumorally that previously has been clinically investigated in metastatic renal cell carcinoma and hepatocellular carcinoma. METHODS: The trial was a single arm phase I trial assessing safety and efficacy of ilixadencel in subjects with progressing advanced/metastatic GIST despite ongoing treatment with second or later lines of tyrosine kinase inhibitors (TKI). Three patients were progressing while on sunitinib (second line), one on regorafenib (third line), and two on pazopanib (fourth line). TKI treatment was maintained throughout, while two intratumoral injections of ilixadencel (10 × 106 viable and HLA-DR expressing cells per dose) were administered. RESULTS: No severe adverse events were found to be related to ilixadencel administration. Four patients showed continued tumor progression at 3 months per RECIST 1.1 and Choi criteria. One patient (on third line regorafenib) had stable disease for 9 months and another patient (on second line sunitinib) had stable disease at end of study (12 months) as per RECIST 1.1. These two patients developed a partial response as per Choi criteria with a duration of 3 and 6 months, respectively. The median progression-free survival (PFS) was 4.0 months. CONCLUSION: Ilixadencel treatment presented an acceptable safety profile among advanced GIST patients who developed resistance to TKI. Encouraging radiological tumor responses were detected in 33% of treated patients, supporting further investigation. Clinical trial registration www.clinicaltrials.gov ; NCT: 02432846; registration date: February 22, 2016.

CD2 Immunobiology.

The glycoprotein CD2 is a costimulatory receptor expressed mainly on T and NK cells that binds to LFA3, a cell surface protein expressed on e.g., antigen-presenting cells. CD2 has an important role in the formation and organization of the immunological synapse that is formed between T cells and antigen-presenting cells upon cell-cell conjugation and associated intracellular signaling. CD2 expression is upregulated on memory T cells as well as activated T cells and plays an important role in activation of memory T cells despite the coexistence of several other costimulatory pathways. Anti-CD2 monoclonal antibodies have been shown to induce immune modulatory effects in vitro and clinical studies have proven the safety and efficacy of CD2-targeting biologics. Investigators have highlighted that the lack of attention to the CD2/LFA3 costimulatory pathway is a missed opportunity. Overall, CD2 is an attractive target for monoclonal antibodies intended for treatment of pathologies characterized by undesired T cell activation and offers an avenue to more selectively target memory T cells while favoring immune regulation.

Polyvinyl Alcohol Carbazate as a Polymer-Based Antitumoral Agent.

Development of treatment resistance is a major concern during treatment of cancer, and there is an unmet need for therapeutic strategies with novel modes of action. Polyvinyl alcohol carbazate (PVAC) is a polymer compound with unique biological properties. Herein, we describe the antitumoral effects of PVAC. Three well-established cell lines GIST-T1, B16.F10, and A375 were used to determine the in vitro antitumoral effects of PVAC. Assessments included light microscopy, cell viability, cell cycle, and apoptosis assays. In vivo treatment safety and efficacy were characterized in one immunocompetent (B16.F10) mouse model and one athymic nude (MDA-MB-231) mouse model. Excised tumors were measured, weighed, stained for Ki-67, CD3, and histopathologically evaluated. Intact PVAC expressed a non-linear dose-response antitumoral effect in vitro, whereas its separate components, PVA and carbazate, did not display antitumoral effects alone. In vivo, PVAC induced a significant intratumoral CD3+ T-cell recruitment in immunocompetent mice (B16.F10), which was associated with tumor growth inhibition. Although growth inhibition was not significant in athymic mice (MDA-MB-231), histopathological evaluation detected an increase in stromal tissue and leukocyte infiltration. In conclusion, we present evidence for PVAC antitumoral effects both in vitro and in vivo. The mode of action was not elucidated in vitro, but a potential mechanism of in vivo activity was observed, characterized by an increase of immune cells into both immunocompetent and athymic mice. This finding warrants further study to validate its possible role as an immunomodulatory polymeric agent.

Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro.

Siplizumab, a humanized anti-CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney-bone marrow transplantation. Siplizumab-based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed-lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA-mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4+ and CD8+ effector memory T cells, which express higher CD2 levels than naïve T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA- FoxP3HI cells in MLRs. FoxP3 expression was stable over time in siplizumab-containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high-throughput TCRß CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor-reactive Tregs along with depletion of donor-reactive CD4+ effector/memory T cells in siplizumab-containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance-inducing regimens. Our studies also confirm that naïve in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naïve T cells.

Safety and pharmacodynamics of anti-CD2 monoclonal antibody treatment in cynomolgus macaques - an experimental study.

Anti-CD2 treatment provides targeted immunomodulatory properties that have demonstrated clinical usefulness to condition the immune system and to treat transplant rejection. The treatment is species-specific due to structural CD2 antigen differences between nonhuman primates and humans. Herein, we report the safety profile and efficacy of two modifications of the same anti-CD2 monoclonal antibody in cynomolgus macaques. Twelve subjects received one i.v. anti-CD2 (of rat or rhesus type) dose each, range 1-4 mg/kg, and were followed for 1-7 days. Treatment effects were evaluated with flow cytometry on peripheral blood and histopathological evaluation of secondary lymphoid organs. In vitro inhibitory activity on primary MHC disparate mixed lymphocyte reactions (MLRs) was determined. Upon anti-CD2 treatment, CD4+ , CD8+ memory subsets were substantially depleted. Naïve T cells and Tregs were relatively spared and exhibited lower CD2 expression than memory T cells. Early immune reconstitution was noted for naïve cells, while memory counts had not recovered after one week. Both antibodies displayed a concentration-dependent MLR inhibition. Lymph node examination revealed no significant lymphocyte depletion. None of the animals experienced any significant study drug-related adverse events. This study outlines the safety and pharmacodynamic profile of primate-specific anti-CD2 treatment, relevant for translation of anti-CD2-based animal models into clinical trials.

Pharmacokinetic and pharmacodynamic study of a clinically effective anti-CD2 monoclonal antibody.

The humanized IgG1κ monoclonal antibody siplizumab and its rat parent monoclonal IgG2b antibody BTI-322 are directed against the CD2 antigen. Siplizumab is species-specific, reacting with human and chimpanzee cells but not with cells from any other species, including other non-human primates. Because siplizumab treatment has recently shown great potential in clinical transplantation, we now present the results of our previous pharmacokinetic, pharmacodynamic and safety studies of both antibodies. Fourteen chimpanzees received 1-3 doses of 0.143 to 5.0 mg/kg iv The effects were followed with flow cytometry on peripheral lymphocytes and staining of lymph nodes. Side effects were recorded. Serum antibody concentrations were followed. Across the doses, a rapid, transient depletion of CD2, CD3, CD4 and CD8 lymphocytes and NK cells was observed for both antibodies. Immune reconstitution was more rapid for BTI-322 compared to siplizumab. Paracortical lymph node T cell depletion was moderate, estimated at 45% with doses of >0.6 mg/kg. Restoration of lymph node architecture was seen after two weeks to two months for all animals. All four subjects receiving BTI-322 experienced AEs on the first dosing day, while the eight subjects dosed with siplizumab experienced few mild, transient AEs. Infusion with siplizumab and BTI-322 resulted in rapid depletion of CD2+ cells in circulation and tissue. Siplizumab had a longer t1/2 and fewer AEs compared to BTI-322.

Polyvinylalcohol-carbazate (PVAC) reduces red blood cell hemolysis.

BACKGROUND AND OBJECTIVES: The objective of this study was to investigate whether a soluble polymer and aldehyde-scavenger, polyvinylalcohol-carbazate (PVAC), can inhibit hemolysis in the storage of red blood cells (RBC). STUDY DESIGN AND METHODS: The effect of PVAC was assessed over a wide range of concentrations, using absorption spectroscopy to evaluate the level of hemolysis. Moreover, osmotic stability and aldehyde-scavenging potential of RBC were assessed after storage in PVAC. RESULTS: After test tube storage for two weeks, red blood cell hemolysis was lower with PVAC compared to controls (mean difference 23%, 95% CI 16-29%, p < 0.001). A higher level of hemolysis led to a pronounced effect with PVAC. RBC stored in PVAC improved both the binding of free aldehydes (p <0.001) and the osmotic stability (p = 0.0036). CONCLUSION: Erythrocytes stored with PVAC showed less hemolysis, which might be explained by the ability of PVACs to stabilize the cell membrane and decrease oxidative injury.

Cause of death and significant disease found at autopsy.

The use of clinical autopsy has been in decline for many years throughout healthcare systems of developed countries despite studies showing substantial discrepancies between autopsy results and pre-mortal clinical diagnoses. We conducted a study to evaluate over time the use and results of clinical autopsies in Sweden. We reviewed the autopsy reports and autopsy referrals of 2410 adult (age > 17) deceased patients referred to two University hospitals in Sweden during two plus two years, a decade apart. There was a decline in the number of autopsies performed over time, however, mainly in one of the two hospitals. The proportion of autopsy referrals from the emergency department increased from 9 to 16%, while the proportion of referrals from regular hospital wards was almost halved. The autopsies revealed a high prevalence of cardiovascular disease, with myocardial infarction and cerebrovascular lesion found in 40% and 19% of all cases, respectively. In a large proportion of cases (> 30%), significant findings of disease were not anticipated before autopsy, as judged from the referral document and additional data obtained in some but not all cases. In accordance with previous research, our study confirms a declining rate of autopsy even at tertiary, academic hospitals and points out factors possibly involved in the decline.

Systemic Inflammatory Reaction in Patients With Head and Neck Cancer-An Explorative Study.

Aim: To assess the longitudinal pattern of pro-inflammatory cytokines and growth factors in serum up to 1 year following treatment for head and neck cancer. Materials and Methods: Patients with newly diagnosed, curable head and neck cancer were included (n = 30). The most common subsite was oropharynx (n = 13) followed by oral cavity (n = 9). Blood was drawn from all patients at regular intervals (before treatment, 7 weeks after the start of the treatment, and at 3 months and 1 year after termination of treatment) and analyzed for cytokines (Il-1ß, Il-2, Il-4, Il-5, Il-6, Il-8, Il-10, GM-CSF, TNF-α, and IFN-γ) and growth factors (G-CSF, FGF-2, EGF, and VEGF). Results: The time point of the peak level of pro-inflammatory cytokines was 7 weeks after start of treatment which corresponded for the majority of patients with termination of radiotherapy or chemoradiotherapy. Patients undergoing chemoradiotherapy exhibited a significant increase of IL-1ß, IL-6, and IL-10 at 7 weeks as compared to pre-treatment levels. At 1 year after termination of treatment four patients experienced recurrence of disease while 26 patients were considered disease-free. The patients with recurrence had significantly higher levels of IL-1ß, IL-6, IL-8, and IL-10 at 7 weeks after the start of treatment than patients without recurrence. Correlated with T stadium patients with T3-T4 had higher levels of IL-1ß and IL-8 than patients with T1-T2 7 weeks after the start of treatment. Conclusions: The observed immune response in this explorative study demonstrates that chemoradiotherapy may induce not only a local treatment effect on the immune system but also effects far outside the irradiated field. The result of the study indicates that analysis of a pro-inflammatory panel of cytokines in serum at 7 weeks after the start of treatment could be of prognostic value in patients with head and neck cancer. Further study of a larger cohort could help identify patients at larger risk for recurrent disease with measurements of pro-inflammatory cytokines under and after treatment.

Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

Clinical Significance of Alloantibodies in Hand Transplantation: A Multicenter Study.

BACKGROUND: Donor-specific antibodies (DSAs) have a strong negative correlation with long-term survival in solid organ transplantation. Although the clinical significance of DSA and antibody-mediated rejection (AMR) in upper extremity transplantation (UET) remains to be established, a growing number of single-center reports indicate their presence and potential clinical impact. METHODS: We present a multicenter study assessing the occurrence and significance of alloantibodies in UET in reference to immunological parameters and functional outcome. RESULTS: Our study revealed a high prevalence and early development of de novo DSA and non-DSA (43%, the majority detected within the first 3 postoperative y). HLA class II mismatch correlated with antibody development, which in turn significantly correlated with the incidence of acute cellular rejection. Cellular rejections preceded antibody development in almost all cases. A strong correlation between DSA and graft survival or function cannot be statistically established at this early stage but a correlation with a lesser outcome seems to emerge. CONCLUSIONS: While the phenotype and true clinical effect of AMR remain to be better defined, the high prevalence of DSA and the correlation with acute rejection highlight the need for optimizing immunosuppression, close monitoring, and the relevance of an HLA class II match in UET recipients.

Intratumoral expression of FoxP3-positive regulatory T-cells in T-cell lymphoma: no correlation with survival.

Background. In cancer, regulatory T-cells (Tregs) were previously believed to inhibit tumor immunity, leading to reduced survival. However, in hematologic malignancies, including T-cell lymphoma (TCL), a correlation between increased numbers of tumor-infiltrating Tregs and a favorable prognosis has been reported. We aimed to investigate the expression of the Treg biomarker forkhead box protein 3 (FoxP3) in TCL in immunocompetent individuals and explore a possible correlation to overall survival. Methods. In total, 35 diagnostic biopsies of TCL were stained using a FoxP3-specific monoclonal antibody (clone 236A/E7). Visual scoring was performed by counting positive cells in 15 high-power fields. Clinical data were collected retrospectively from medical records. Results. All the TCLs contained FoxP3+ cells, median 342 FoxP3+ cells/mm2 (range 1-3047). The degree of intratumoral expression of FoxP3 varied between the different subtypes of TCL, with the highest frequency found in angioimmunoblastic TCL. The frequency of intratumoral FoxP3+ cells had no impact on overall survival; neither when using a cutoff value of 200 FoxP3+ cells/mm2 (P = 0.84) nor with FoxP3 as a continuous variable (P = 0.63). Conclusions. Intratumoral Tregs are frequently found in TCL in immunocompetent individuals. In this heterogeneous group of TCL, there was no correlation between the density of intratumoral FoxP3+ cells and overall survival.

WAKMAR2, a Long Noncoding RNA Downregulated in Human Chronic Wounds, Modulates Keratinocyte Motility and Production of Inflammatory Chemokines.

Chronic wounds represent a major and growing health and economic burden worldwide. A better understanding of molecular mechanisms of normal as well as impaired wound healing is needed to develop effective treatment. Herein we studied the potential role of long noncoding RNA LOC100130476 in skin wound repair. LOC100130476 is an RNA polymerase II-encoded polyadenylated transcript present in both cytoplasm and nucleus. We found that its expression was lower in wound-edge keratinocytes of human chronic wounds compared to normal wounds of healthy donors and intact skin. In cultured keratinocytes, LOC100130476 expression was induced by TGF-ß signaling. By reducing LOC100130476 expression with antisense oligos or activating its transcription with CRISPR/Cas9 Synergistic Activation Mediator system, we showed that LOC100130476 restricted the production of inflammatory chemokines by keratinocytes, while enhancing cell migration. In line with this, knockdown of LOC100130476 impaired re-epithelization of human ex vivo wounds. Based on these results, we named LOC100130476 wound and keratinocyte migration-associated long noncoding RNA 2 (WAKMAR2). Moreover, we identified a molecular network that may mediate the biological function of WAKMAR2 in keratinocytes using microarray. In summary, our data suggest that WAKMAR2 is an important regulator of skin wound healing and its deficiency may contribute to the pathogenesis of chronic wounds.

Steroid regulation: An overlooked aspect of tolerance and chronic rejection in kidney transplantation.

Steroid conversion (HSD11B1, HSD11B2, H6PD) and receptor genes (NR3C1, NR3C2) were examined in kidney-transplant recipients with "operational tolerance" and chronic rejection (CR), independently and within the context of 88 tolerance-associated genes. Associations with cellular types were explored. Peripheral whole-blood gene-expression levels (RT-qPCR-based) and cell counts were adjusted for immunosuppressant drug intake. Tolerant (n = 17), stable (n = 190) and CR patients (n = 37) were compared. Healthy controls (n = 14) were used as reference. The anti-inflammatory glucocorticoid receptor (NR3C1) and the cortisol-activating HSD11B1 and H6PD genes were up-regulated in CR and were lowest in tolerant patients. The pro-inflammatory mineralocorticoid gene (NR3C2) was downregulated in stable and CR patients. NR3C1 was associated with neutrophils and NR3C2 with T-cells. Steroid conversion and receptor genes, alone, enabled classification of tolerant patients and were major contributors to gene-expression signatures of both, tolerance and CR, alongside known tolerance-associated genes, revealing a key role of steroid regulation and response in kidney transplantation.

Standardizing the freeze-thaw preparation of growth factors from platelet lysate.

BACKGROUND: Over the past decades, the focus on the regenerative properties of platelets (PLTs) has intensified and many PLT-derived growth factors are readily used in medical settings. A general lack of standardization in the preparation of these growth factors remains, however, and this study therefore examines the dynamics of growth factors throughout the freeze-thaw procedure. STUDY DESIGN AND METHODS: Plateletpheresis (PA) and PLT-poor plasma (PPP) samples were collected from 10 healthy donors. PA was lysed to produce PLT lysate (PL) for 1, 3, 5, 10, and 30 freeze-thaw cycles. The resulting growth factor and cytokine concentrations from PPP, PA, and PL of different cycles were analyzed and compared using enzyme-linked immunosorbent assay and multiplex bead assays. RESULTS: PL produced by the freeze-thaw procedure resulted in approximately four- to 10-fold enrichment of transforming growth factor-ß1, epidermal growth factor, PLT-derived growth factor (PDGF)-AB/BB, PLT factor-4, and fibroblast growth factor-2. The increase in concentrations plateaued at Cycles 3 and 5 and in some cases declined with further cycles. The concentrations of insulin-like growth factor-1, hepatocyte growth factor, vascular endothelial growth factor, and bone morphogenetic protein-2 in PL were essentially comparable to those in PPP. CONCLUSION: Using the freeze-thaw method, optimal preparation of PL with regard to the concentration of growth factors was achieved at Cycles 3 to 5. Based on our findings, the clinical significance of using a greater number of cycles is likely limited.