NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-ß and further enhanced by hypoxia. The effect of TGF-ß was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Exploring Anti-Fibrotic Effects of Adipose-Derived Stem Cells: Transcriptome Analysis upon Fibrotic, Inflammatory, and Hypoxic Conditioning.

Autologous fat transfers show promise in treating fibrotic skin diseases, reversing scarring and stiffness, and improving quality of life. Adipose-derived stem cells (ADSCs) within these grafts are believed to be crucial for this effect, particularly their secreted factors, though the specific mechanisms remain unclear. This study investigates transcriptomic changes in ADSCs after in vitro fibrotic, inflammatory, and hypoxic conditioning. High-throughput gene expression assays were conducted on ADSCs exposed to IL1-ß, TGF-ß1, and hypoxia and in media with fetal bovine serum (FBS). Flow cytometry characterized the ADSCs. RNA-Seq analysis revealed distinct gene expression patterns between the conditions. FBS upregulated pathways were related to the cell cycle, replication, wound healing, and ossification. IL1-ß induced immunomodulatory pathways, including granulocyte chemotaxis and cytokine production. TGF-ß1 treatment upregulated wound healing and muscle tissue development pathways. Hypoxia led to the downregulation of mitochondria and cellular activity.

Role of toll-like receptor 4 in skeletal muscle damage in chronic limb-threatening ischemia.

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors in the innate immune system. In particular, the TLR4-mediated immune response has been implicated in ischemia-induced tissue injury. Mounting evidence supports a detrimental role of the innate immune system in the pathophysiology of skeletal muscle damage in patients with chronic limb-threatening ischemia (CLTI), in whom patient-oriented functional outcomes are poor. The overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction and damage in CLTI. Methods: The role of TLR4 in ischemic muscle was investigated by (1) studying TLR4 expression and distribution in human gastrocnemius muscle biopsies, (2) evaluating the functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) assessing the therapeutic potential of modulating TLR4 signaling in ischemic muscle in a mouse hindlimb ischemia model. Results: TLR4 was found to be expressed in human muscle biopsies, with significant upregulation in samples from patients with CLTI. In vitro studies using cultured human myotubes demonstrated upregulation of TLR4 in ischemia, with activation of the downstream signaling pathway. Inhibition of TLR4 before ischemia was associated with reduced ischemia-induced apoptosis. Upregulation of TLR4 also occurred in ischemia in vivo and TLR4 inhibition was associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb. Conclusions: TLR4 is upregulated and activated in ischemic skeletal muscle in patients with CLTI. Modulating TLR4 signaling in vitro and in vivo was associated with attenuation of ischemia-induced skeletal muscle damage. This strategy could be explored further for potential clinical application.

Emerging diagnostic and therapeutic challenges for skin fibrosis in systemic sclerosis.

Systemic sclerosis (also called scleroderma, SSc) is a chronic autoimmune disorder characterized by excessive collagen deposition leading to skin fibrosis and various internal organ manifestations. The emergent diagnostics and therapeutic strategies for scleroderma focus on early detection and targeted interventions to improve patient outcomes and quality of life. Diagnostics for SSc have evolved significantly in recent years, driven by advancements in serological markers and imaging techniques. Autoantibody profiling, especially antinuclear antibodies (ANA) and specific scleroderma-associated autoantibodies, aids in identifying subsets of scleroderma and predicting disease progression. Furthermore, novel imaging modalities, such as high-frequency ultrasonography and optical coherence tomography, enable early detection of skin fibrosis and internal organ involvement, enhancing the diagnostic precision and allowing for tailored management. Therapeutic strategies for SSc are multifaceted, targeting immune dysregulation, vascular abnormalities, and fibrotic processes. Emerging biologic agents have shown promise in clinical trials, including monoclonal antibodies directed against key cytokines involved in fibrosis, such as transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6). Additionally, small-molecule inhibitors that disrupt fibrotic pathways, like tyrosine kinase inhibitors, have exhibited potential in limiting collagen deposition and preventing disease progression. Stem cell therapy, cell ablation and gene editing techniques hold great potential in regenerating damaged tissue and halting fibrotic processes. Early intervention remains crucial in managing SSc, as irreversible tissue damage often occurs in advanced stages. Novel diagnostic methods, such as biomarkers and gene expression profiling, are being explored to identify individuals at high risk for developing progressive severe disease and intervene proactively. Furthermore, patient-tailored therapeutic approaches, employing a combination of immunosuppressive agents and targeted anti-fibrotic therapies, are being investigated to improve treatment efficacy while minimizing adverse effects. The emergent diagnostics and therapeutic strategies in scleroderma are transforming the management of this challenging disease. Nevertheless, ongoing research and clinical trials are needed to optimize the efficacy and safety of these novel approaches in the complex and diverse spectrum of SSc manifestations.

Correction: Serodiagnosis of Tuberculosis in Asian Elephants (Elephas maximus) in Southern India: A Latent Class Analysis.

[This corrects the article DOI: 10.1371/journal.pone.0049548.].

Autoantibodies Which Bind to and Activate Keratinocytes in Systemic Sclerosis.

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterised by pathological processes involving autoimmunity, vasculopathy and resultant extensive skin and organ fibrosis. Recent studies have demonstrated activation and aberrant wound healing responses in the epithelial layer of the skin in this disease, implicating the epithelial keratinocytes as a source of pro-fibrotic and inflammatory mediators. In this paper, we investigated the role of Immunoglobulin G (IgG) autoantibodies directed against epithelial cells, as potential initiators and propagators of pathological keratocyte activation and the ensuing SSc fibrotic cascade. A keratinocyte cell-based ELISA is used to evaluate the binding of SSc IgG. SSc skin biopsies were stained by immunofluorescence for the presence of IgG in the keratinocyte layer. Moreover, IgG purified from SSc sera was evaluated for the potential to activate keratinocytes in tissue culture and to induce TLR2 and 3 signalling in reporter cell lines. We demonstrate enhanced binding of SSc IgG to keratinocytes and the activation of these cells leading to the release of IL-1α, representing a potential initiating pathway in this disease.

Adjuvanted Fusion Protein Vaccine Induces Durable Immunity to Onchocerca volvulus in Mice and Non-Human Primates.

Onchocerciasis remains a debilitating neglected tropical disease. Due to the many challenges of current control methods, an effective vaccine against the causative agent Onchocerca volvulus is urgently needed. Mice and cynomolgus macaque non-human primates (NHPs) were immunized with a vaccine consisting of a fusion of two O. volvulus protein antigens, Ov-103 and Ov-RAL-2 (Ov-FUS-1), and three different adjuvants: Advax-CpG, alum, and AlT4. All vaccine formulations induced high antigen-specific IgG titers in both mice and NHPs. Challenging mice with O. volvulus L3 contained within subcutaneous diffusion chambers demonstrated that Ov-FUS-1/Advax-CpG-immunized animals developed protective immunity, durable for at least 11 weeks. Passive transfer of sera, collected at several time points, from both mice and NHPs immunized with Ov-FUS-1/Advax-CpG transferred protection to naïve mice. These results demonstrate that Ov-FUS-1 with the adjuvant Advax-CpG induces durable protective immunity against O. volvulus in mice and NHPs that is mediated by vaccine-induced humoral factors.

Prostacyclin mimetics inhibit DRP1-mediated pro-proliferative mitochondrial fragmentation in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disorder, involving the remodelling of the small pulmonary arteries. Underlying this remodelling is the hyper-proliferation of pulmonary arterial smooth muscle cells within the medial layers of these arteries and their encroachment on the lumen. Previous studies have demonstrated an association between excessive mitochondrial fragmentation, a consequence of increased expression and post-translational activation of the mitochondrial fission protein dynamin-related protein 1 (DRP1), and pathological proliferation in PASMCs derived from PAH patients. However, the impact of prostacyclin mimetics, widely used in the treatment of PAH, on this pathological mitochondrial fragmentation remains unexplored. We hypothesise that these agents, which are known to attenuate the proliferative phenotype of PAH PASMCs, do so in part by inhibiting mitochondrial fragmentation. In this study, we confirmed the previously reported increase in DRP1-mediated mitochondrial hyper-fragmentation in PAH PASMCs. We then showed that the prostacyclin mimetic treprostinil signals via either the Gs-coupled IP or EP2 receptor to inhibit mitochondrial fragmentation and the associated hyper-proliferation in a manner analogous to the DRP1 inhibitor Mdivi-1. We also showed that treprostinil recruits either the IP or EP2 receptor to activate PKA and induce the phosphorylation of DRP1 at the inhibitory residue S637 and inhibit that at the stimulatory residue S616, both of which are suggestive of reduced DRP1 fission activity. Like treprostinil, MRE-269, an IP receptor agonist, and butaprost, an EP2 receptor agonist, attenuated DRP1-mediated mitochondrial fragmentation through PKA. We conclude that prostacyclin mimetics produce their anti-proliferative effects on PAH PASMCs in part by inhibiting DRP1-mediated mitochondrial fragmentation.

Single-Cell Analysis of ADSC Interactions with Fibroblasts and Endothelial Cells in Scleroderma Skin.

Adipose-derived stem cells (ADSCs) as part of autologous fat grafting have anti-fibrotic and anti-inflammatory effects, but the exact mechanisms of action remain unknown. By simulating the interaction of ADSCs with fibroblasts and endothelial cells (EC) from scleroderma (SSc) skin in silico, we aim to unravel these mechanisms. Publicly available single-cell RNA sequencing data from the stromal vascular fraction of 3 lean patients and biopsies from the skin of 10 control and 12 patients with SSc were obtained from the GEO and analysed using R and Seurat. Differentially expressed genes were used to compare the fibroblast and EC transcriptome between controls and SSc. GO and KEGG functional enrichment was performed. Ligand-receptor interactions of ADSCs with fibroblasts and ECs were explored with LIANA. Pro-inflammatory and extracellular matrix (ECM) interacting fibroblasts were identified in SSc. Arterial, capillary, venous and lymphatic ECs showed a pro-fibrotic and pro-inflammatory transcriptome. Most interactions with both cell types were based on ECM proteins. Differential interactions identified included NTN1, VEGFD, MMP2, FGF2, and FNDC5. The ADSC secretome may disrupt vascular and perivascular inflammation hubs in scleroderma by promoting angiogenesis and especially lymphangiogenesis. Key phenomena observed after fat grafting remain unexplained, including modulation of fibroblast behaviour.

Unravelling morphoea aetiopathogenesis by next-generation sequencing of paired skin biopsies.

BACKGROUND: Morphoea can have a significant disease burden. Aetiopathogenesis remains poorly understood, with very limited existing genetic studies. Linear morphoea (LM) may follow Blascho's lines of epidermal development, providing potential pathogenic clues. OBJECTIVE: The first objective of this study was to identify the presence of primary somatic epidermal mosaicism in LM. The second objective was tTo explore differential gene expression in morphoea epidermis and dermis to identify potential pathogenic molecular pathways and tissue layer cross-talk. METHODOLOGY: Skin biopsies from paired affected and contralateral unaffected skin were taken from 16 patients with LM. Epidermis and dermis were isolated using a 2-step chemical-physical separation protocol. Whole Genome Sequencing (WGS; n = 4 epidermal) and RNA-seq (n = 5-epidermal, n = 5-dermal) with gene expression analysis via GSEA-MSigDBv6.3 and PANTHER-v14.1 pathway analyses, were performed. RTqPCR and immunohistochemistry were used to replicate key results. RESULTS: Sixteen participants (93.8% female, mean age 27.7 yrs disease-onset) were included. Epidermal WGS identified no single affected gene or SNV. However, many potential disease-relevant pathogenic variants were present, including ADAMTSL1 and ADAMTS16. A highly proliferative, inflammatory and profibrotic epidermis was seen, with significantly-overexpressed TNFα-via-NFkB, TGFß, IL6/JAKSTAT and IFN-signaling, apoptosis, p53 and KRAS-responses. Upregulated IFI27 and downregulated LAMA4 potentially represent initiating epidermal 'damage' signals and enhanced epidermal-dermal communication. Morphoea dermis exhibited significant profibrotic, B-cell and IFN-signatures, and upregulated morphogenic patterning pathways such as Wnt. CONCLUSION: This study supports the absence of somatic epidermal mosaicism in LM, and identifies potential disease-driving epidermal mechanisms, epidermal-dermal interactions and disease-specific dermal differential-gene-expression in morphoea. We propose a potential molecular narrative for morphoea aetiopathogenesis which could help guide future targeted studies and therapies.

A rodent model for Dirofilaria immitis, canine heartworm: parasite growth, development, and drug sensitivity in NSG mice.

Heartworm disease, caused by Dirofilaria immitis, remains a significant threat to canines and felines. The development of parasites resistant to macrocyclic lactones (ML) has created a significant challenge to the control of the infection. The goal of this study was to determine if mice lacking a functional immune response would be susceptible to D. immitis. Immunodeficient NSG mice were susceptible to the infection, sustaining parasites for at least 15 weeks, with infective third-stage larvae molting and developing into the late fourth-stage larvae. Proteomic analysis of host responses to the infection revealed a complex pattern of changes after infection, with at least some of the responses directed at reducing immune control mechanisms that remain in NSG mice. NSG mice were infected with isolates of D. immitis that were either susceptible or resistant to MLs, as a population. The susceptible isolate was killed by ivermectin whereas the resistant isolate had improved survivability, while both isolates were affected by moxidectin. It was concluded that D. immitis survives in NSG mice for at least 15 weeks. NSG mice provide an ideal model for monitoring host responses to the infection and for testing parasites in vivo for susceptibility to direct chemotherapeutic activity of new agents.

Relative Recovery of Non-Alcoholic Fatty Liver Disease (NAFLD) in Diet-Induced Obese Rats.

Consumption of a high-carbohydrate diet has a critical role in the induction of weight gain and obesity-related pathologies. This study tested the hypothesis that a carbohydrate-rich diet induces weight gain, ectopic fat deposition, associated metabolic risks and development of non-alcoholic fatty liver disease (NAFLD), which are partially reversible following carbohydrate reduction. Sprague Dawley (SD) rats were fed a carbohydrate-enriched cafeteria diet (CAF) or normal chow (NC) ad libitum for 16-18 weeks. In the reversible group (REV), the CAF was replaced with NC for a further 3 weeks (18-21 weeks). Animals fed the CAF diet showed significantly increased body weight compared to those fed NC, accompanied by abnormal changes in their systemic insulin and triglycerides, elevation of hepatic triglyceride and hepatic steatosis. In the REV group, when the CAF diet was stopped, a modest, non-significant weight loss was associated with improvement in systemic insulin and appearance of the liver, with lower gross fatty deposits and hepatic triglyceride. In conclusion, a carbohydrate-enriched diet led to many features of metabolic syndrome, including hyperinsulinemia, while a dietary reduction in this macronutrient, even for a short period, was able to restore normoinsulinemia, and reversed some of the obesity-related hepatic abnormalities, without significant weight loss.

Exploring metabolism in scleroderma reveals opportunities for pharmacological intervention for therapy in fibrosis.

Background: Recent evidence has indicated that alterations in energy metabolism play a critical role in the pathogenesis of fibrotic diseases. Studies have suggested that 'metabolic reprogramming' involving the glycolysis and oxidative phosphorylation (OXPHOS) in cells lead to an enhanced generation of energy and biosynthesis. The aim of this study was to assess the molecular basis of changes in fibrotic metabolism in systemic sclerosis (Scleroderma; SSc) and highlight the most appropriate targets for anti-fibrotic therapies. Materials and methods: Dermal fibroblasts were isolated from five SSc patients and five healthy donors. Cells were cultured in medium with/without TGF-ß1 and with/without ALK5, pan-PIM or ATM kinase inhibitors. Extracellular flux analyses were performed to evaluate glycolytic and mitochondrial respiratory function. The mitochondrial network in TMRM-stained cells was visualized by confocal laser-scanning microscopy, followed by semi-automatic analysis on the ImageJ platform. Protein expression of ECM and fibroblast components, glycolytic enzymes, subunits of the five OXPHOS complexes, and dynamin-related GTPases and receptors involved in mitochondrial fission/fusion were assessed by western blotting. Results: Enhanced mitochondrial respiration coupled to ATP production was observed in SSc fibroblasts at the expense of spare respiratory capacity. Although no difference was found in glycolysis when comparing SSc with healthy control fibroblasts, levels of phophofructokinase-1 isoform PFKM were significantly lower in SSc fibroblasts (P<0.05). Our results suggest that the number of respirasomes is decreased in the SSc mitochondria; however, the organelles formed a hyperfused network, which is thought to increase mitochondrial ATP production through complementation. The increased mitochondrial fusion correlated with a change in expression levels of regulators of mitochondrial morphology, including decreased levels of DRP1, increased levels of MIEF2 and changes in OPA1 isoform ratios. TGF-ß1 treatment strongly stimulated glycolysis and mitochondrial respiration and induced the expression of fibrotic markers. The pan-PIM kinase inhibitor had no effect, whereas both ALK5 and ATM kinase inhibition abrogated TGF-ß1-mediated fibroblast activation, and upregulation of glycolysis and respiration. Conclusions: Our data provide evidence for a novel mechanism(s) by which SSc fibroblasts exhibit altered metabolic programs and highlight changes in respiration and dysregulated mitochondrial morphology and function, which can be selectively targeted by small molecule kinase inhibitors.

Downregulation of CYP17A1 by 20-hydroxyecdysone: plasma progesterone and its vasodilatory properties.

Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC-MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation.

Is Strongyloides stercoralis hyperinfection induced by glucocorticoids a result of both suppressed host immunity and altered parasite genetics?

The gastrointestinal (GI) nematode Strongyloides stercoralis (S.s.) causes human strongyloidiasis, a potentially life-threatening disease that currently affects over 600 million people globally. The uniquely pernicious aspect of S.s. infection, as compared to all other GI nematodes, is its autoinfective larval stage (L3a) that maintains a low-grade chronic infection, allowing undetectable persistence for decades. Infected individuals who are administered glucocorticoid therapy can develop a rapid and often lethal hyperinfection syndrome within days. Hyperinfection patients often present with dramatic increases in first- and second-stage larvae and L3a in their GI tract, with L3a widely disseminating throughout host organs leading to sepsis. How glucocorticoid administration drives hyperinfection remains a critical unanswered question; specifically, it is unknown whether these steroids promote hyperinfection through eliminating essential host protective mechanisms and/or through dysregulating parasite development. This current deficiency in understanding is largely due to the previous absence of a genetically defined mouse model that would support all S.s. life-cycle stages and the lack of successful approaches for S.s. genetic manipulation. However, there are currently new possibilities through the recent demonstration that immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice support sub-clinical infections that can be transformed to lethal hyperinfection syndrome following glucocorticoid administration. This is coupled with advances in transcriptomics, transgenesis, and gene inactivation strategies that now allow rigorous scientific inquiry into S.s. biology. We propose that combining in vivo manipulation of host immunity and deep immunoprofiling strategies with the latest advances in S.s. transcriptomics, piggyBac transposon-mediated transgene insertion, and CRISPR/Cas-9-mediated gene inactivation will facilitate new insights into the mechanisms that could be targeted to block lethality in humans with S.s. hyperinfection.

Co-Administration of Adjuvanted Recombinant Ov-103 and Ov-RAL-2 Vaccines Confer Protection against Natural Challenge in A Bovine Onchocerca ochengi Infection Model of Human Onchocerciasis.

Onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus, is a neglected tropical disease mainly of sub-Saharan Africa. Worldwide, an estimated 20.9 million individuals live with infection and a further 205 million are at risk of disease. Current control methods rely on mass drug administration of ivermectin to kill microfilariae and inhibit female worm fecundity. The identification and development of efficacious vaccines as complementary preventive tools to support ongoing elimination efforts are therefore an important objective of onchocerciasis research. We evaluated the protective effects of co-administering leading O. volvulus-derived recombinant vaccine candidates (Ov-103 and Ov-RAL-2) with subsequent natural exposure to the closely related cattle parasite Onchocerca ochengi. Over a 24-month exposure period, vaccinated calves (n = 11) were shown to acquire infection and microfilaridermia at a significantly lower rate compared to unvaccinated control animals (n = 10). Furthermore, adult female worm burdens were negatively correlated with anti-Ov-103 and Ov-RAL-2 IgG1 and IgG2 responses. Peptide arrays identified several Ov-103 and Ov-RAL-2-specific epitopes homologous to those identified as human B-cell and helper T-cell epitope candidates and by naturally-infected human subjects in previous studies. Overall, this study demonstrates co-administration of Ov-103 and Ov-RAL-2 with Montanide™ ISA 206 VG is highly immunogenic in cattle, conferring partial protection against natural challenge with O. ochengi. The strong, antigen-specific IgG1 and IgG2 responses associated with vaccine-induced protection are highly suggestive of a mixed Th1/Th2 associated antibody responses. Collectively, this evidence suggests vaccine formulations for human onchocerciasis should aim to elicit similarly balanced Th1/Th2 immune responses.

The Yin and Yang of IL-17 in Systemic Sclerosis.

IL-17 (IL-17A) is a pro-inflammatory cytokine produced by a sub-set of T helper cells termed Th17 cells primarily in response to cytokines like TGF-ß and IL-23 and play an important role in host defense. IL-17 signals via the IL-17RA/RC heterodimer and the adaptor protein Act1 to activate both canonical and non-canonical pathways inducing transcriptional activation and stabilization of mRNAs. IL-17 appears to act not directly on immune cells but stimulates stromal cells such as endothelial and epithelial cells and fibroblasts to secrete other immunomodulatory factors. Fibroblast activated by IL-17 can support the growth and differentiation of immune cells. Studies have begun to uncover a dual role for IL-17; on one hand enhancing immune reactions and promoting inflammatory diseases and on the other decreasing responses and immune activity in established disease settings. The balance of double-edged sword effect of IL-17 and autoimmunity is illustrated in a variety of human diseases and experimental models of diseases. Specifically, the emerging interest in autoimmunity in systemic sclerosis (Scleroderma, SSc) has led to potential role of IL-17A as a target therapy in this disease.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Randomised single centre double-blind placebo controlled phase II trial of Tocovid SupraBio in combination with pentoxifylline in patients suffering long-term gastrointestinal adverse effects of radiotherapy for pelvic cancer: The PPALM study.

BACKGROUND: Preclinical data suggest that combined gamma-tocotrienol with pentoxifylline ameliorates radiotherapy-induced gastrointestinal damage. AIM: To test whether gastrointestinal symptoms arising after radiotherapy, and persisting after maximal medical therapy, can be improved using Tocovid SupraBio 200 mg and pentoxifylline 400 mg orally twice daily for one year. Patients stratified by severity of symptoms, and randomised to active treatment or matched placebo were assessed after 12 months. The primary end point was improvement in gastrointestinal symptoms measured using the Inflammatory Bowel Disease Questionnaire, bowel subset score. Changes in bio-markers of fibrosis were assessed. RESULTS: 62 patients, median age 66, 34(55%) treated for prostate, 21(34%) gynaecological, 6(10%) anal and one(1%) rectal cancer were recruited; 40(65%) randomised to treatment, 22(35%) to placebo, 39 months (median) after radiotherapy completion. Gamma tocotrienol was not detected in serum in 41% of treated patients, despite good compliance with study medication. Treatment was completed in 28(70%) and 17(77%) patients in the treatment and placebo groups respectively. No improvement in symptom scores nor in quality of life was identified. Thirteen serious adverse events occurred. A transient ischaemic attack, was possibly related to pentoxifylline, others were assessed as unlikely to be related to treatment. Levels of EGF, PDGF and FGF were significantly reduced and consistent trends in reduced inflammation were seen during treatment but were not sustained once treatment ended. SUMMARY: This single centre study closed prematurely and therefore data interpretation is of necessity limited. No clinical benefit was demonstrated. However, biochemical data suggest that this intervention does have anti-inflammatory and anti-fibrotic effects.

Dysregulated B cell function and disease pathogenesis in systemic sclerosis.

Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-ß, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.