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Background Immunoglobulin G4-related disease (IgG4-RD) promptly responds to glucocorticoids but relapses in a considerable fraction of patients. Reliable biomarkers of flare are currently lacking because the pathophysiology of IgG4-RD remains largely elusive. In the present work, we aimed to identify perturbations of B-cell subpopulations that might predict IgG4-RD relapse. Methods Thirty patients were treated with glucocorticoids according to international guidelines. Circulating CD19+ and CD20+ cells, naive B cells, memory B cells, plasmablasts, and plasma cells were measured by flow cytometry at baseline and every 6 months for 2 years after the initiation of corticosteroid therapy. Results Patients with active untreated IgG4-RD showed significantly reduced CD19+ B cells, CD20+ B cells, and naive B cells compared with healthy subjects (p

Objectives IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory disorder characterized by a dysregulated resolution of inflammation and wound healing response that might develop after an apoptotic insult induced by cytotoxic T lymphocytes (CTLs). Mer receptor tyrosine kinase (MerTK) and its ligand, protein S (ProS1), have a pivotal role in the resolution of inflammation, being implicated in the clearance of apoptotic cells, quenching of the immune response and development of tissue fibrosis. In the present work we aimed to investigate a possible involvement of the MerTK signalling pathway in the pathogenesis of IgG4-RD and development of tissue fibrosis. Methods MerTK and ProS1 expression patterns in IgG4-RD lesions were evaluated by immunohistochemistry and immunofluorescence studies. Circulating MerTK+ monocytes, soluble Mer and MerTK ligands were measured in the peripheral blood of IgG4-RD patients and healthy controls by flow cytometry and ELISA, respectively. Results MerTK was highly expressed by macrophages infiltrating IgG4-RD lesions. MerTK+ macrophages were more abundant in IgG4-RD than in Sjögren’s syndrome and interacted with apoptotic cells and ProS1-expressing T and B lymphocytes. Moreover, they expressed the pro-fibrotic cytokine TGF-β and their numbers declined following rituximab-induced disease remission. Circulating MerTK+ monocytes, soluble Mer and MerTK ligands were not increased in the peripheral blood of patients with IgG4-RD. Conclusions The MerTK–ProS1 axis is activated in IgG4-RD lesions, possibly leading to persistent stimulation of processes involved in the resolution of inflammation and tissue fibrosis.

Immunoglobulin G4-related disease (IgG4-RD) is an uncommon disorder that demonstrates characteristic clinicopathologic features including sclerosing lesions with storiform fibrosis, increased IgG4+ plasma cells with an increased IgG4+/IgG+ plasma cell ratio, obliterative phlebitis, and often an increased serum IgG4 level. This review summarizes the characteristic histopathologic and clinical features of IgG4-RD with detailed discussion of the histopathologic characteristics of the most commonly involved anatomic sites. We also present recent advances in our understanding of the pathophysiologic mechanisms of IgG4-RD and discuss updates on the treatment, prognosis, and outcomes of this rare disease, including discussion of the possible association between IgG4-RD and malignancy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent stromal reaction that has been variably implicated in both tumor growth and tumor suppression. B-lymphocytes have been recently implicated in PDAC progression but their contribution to the characteristic stromal desmoplasia has never been assessed before. In the present work, we aimed to verify whether B-lymphocytes contribute to stromal cell activation in PDAC. CD19+ B-lymphocytes purified from peripheral blood of patients with PDAC were cultivated in the presence of human pancreatic fibroblasts and cancer-associated fibroblasts. Released pro-fibrotic soluble factors and collagen production were assessed by ELISA and Luminex assays. Quantitative RT-PCR was used to assess fibroblast activation in the presence of B cells. The expression of selected pro-fibrotic and inflammatory molecules was confirmed on PDAC tissue sections by multi-color immunofluorescence studies. We herein demonstrate that B-cells from PDAC patients (i) produce the pro-fibrotic molecule PDGF-B and stimulate collagen production by fibroblasts; (ii) express enzymes implicated in extracellular matrix remodeling including LOXL2; and (iii) produce the chemotactic factors CCL-4, CCL-5, and CCL-11. In addition we demonstrate that circulating plasmablasts are expanded in the peripheral blood of patients with PDAC, stimulate collagen production by fibroblasts, and infiltrate pancreatic lesions. Our results indicate that PDAC is characterized by perturbations of the B-cell compartment with expansion of B-lymphocyte subsets that directly contribute to the stromal reaction observed at disease site. These findings provide an additional rationale for modulating B-cell activity in patients with pancreatic cancer.

Background IgG4-related disease (IgG4-RD) is a fibroinflammatory condition marked by rapid clinical improvement after selective depletion of B lymphocytes with rituximab. This feature suggests that B cells might participate in fibrogenesis and wound healing. Objective In the present work we aimed to demonstrate that B lymphocytes contribute directly to tissue fibrosis in patients with IgG4-RD. Methods Total circulating CD19+ B lymphocytes, naive B cells, memory B cells, or plasmablasts from patients with IgG4-RD were cultivated with human fibroblasts. Profibrotic soluble factors and collagen production in cocultures were assessed by using ELISAs and Luminex assays. RNA sequencing and quantitative RT-PCR were used to assess fibroblast activation in the presence of B cells, as well as induction of profibrotic pathways in B-cell subsets. Relevant profibrotic and inflammatory molecules were confirmed in vitro by using functional experiments and on IgG4-RD tissue sections by using multicolor immunofluorescence studies. Results B cells from patients with IgG4-RD (1) produced the profibrotic molecule platelet-derived growth factor B and stimulated collagen production by fibroblasts; (2) expressed enzymes implicated in extracellular matrix remodeling, such as lysyl oxidase homolog 2; (3) produced the chemotactic factors CCL4, CCL5, and CCL11; and (4) induced production of these same chemokines by activated fibroblasts. Plasmablasts expressed sets of genes implicated in fibroblast activation and proliferation and therefore represent cells with intrinsic profibrotic properties. Conclusion We have demonstrated that B cells contribute directly to tissue fibrosis in patients with IgG4-RD. These unanticipated profibrotic properties of B lymphocytes, particularly plasmablasts, might be relevant for fibrogenesis in patients with other fibroinflammatory disorders and for wound-healing processes in physiologic conditions.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

Eleonora Comi,1,2 Marco Lanza,1 Flora Ferrari,1 Valeria Mauri,1,3 Gianfranco Caselli,1 Lucio Claudio Rovati1 1Department of Pharmacology and Toxicology, Rottapharm Biotech, 2PhD Program in Neuroscience, University of Milan-Bicocca, 3Department of Surgery and Translational Medicine, University ofMilan-Bicocca, Monza,Italy Purpose: CR4056, (2-phenyl-6-(1H-imidazol-1yl) quinazoline), an imidazoline-2 (I2) receptor ligand, is a promising analgesic drug that has been reported to be effective in several animal models of pain. The aim of this study was to evaluate the effects of CR4056 in two well-established rat models of osteoarthritis (OA), mimicking the painful and structural components of human OA.Methods: Knee OA was induced either by single intra-articular injection of monoiodoacetate (MIA) or by medial meniscal tear (MMT) in the right knee of male rats. In the MIA model, allodynia and hyperalgesia were measured as paw withdrawal threshold to mechanical stimulation. In the MMT model, pain behavior was analyzed as weight-bearing asymmetry (i.e. difference in hind paw weight distribution, HPWD) between the injured and the contralateral limbs.Results: Acute oral administration of CR4056, 14 days after MIA injection, significantly and dose-dependently reduced allodynia and hyperalgesia 90 minutes after treatment, whereas acute naproxen administration significantly reduced allodynia but not hyperalgesia. After 7days of repeated treatment, both CR4056 and naproxen showed significant anti-allodynic and anti-hyperalgesic effects in the MIA model. Rats undergoing MMT surgery developed a significant and progressive asymmetry in HPWD compared with sham-operated animals. Repeated treatment with CR4056 significantly reduced the progression of the pain behavior, whereas naproxen had no effects.Conclusion: The data presented here show that the I2 ligand CR4056 could be a new effective treatment for OA pain. The compound is currently under Phase II clinical evaluation for this indication. Keywords: osteoarthritis, pain, CR4056, imidazoline-2 receptors, MIA, MMT

Purpose: Joint pain is the cardinal symptom of OA although it poorly correlates with OA joint structure changes. Interestingly, OA pain is driven by both nociceptive and neuropathic mechanisms. The analgesic efficacy of CR4056, an I2 receptor ligand currently in phase II clinical trials, was previously reported in the OA pain model obtained by the intra-articular injection of monosodium iodoacetate (MIA) in the rat knee. Moreover, CR4056 evidenced a remarkable analgesic activity in several animal models of inflammatory, neuropathic and postoperative pain. The aim of this study was to evaluate the effect of CR4056 in a well-established model of surgically-induced OA able to mimic the structural and painful components of human OA. Methods: OA was induced in male Sprague Dawley rats (335-370 g) by transection of the medial collateral ligament and medial meniscus of the femoro-tibial right joint (MMT model). Hind paw weight bearing distribution was assessed as indirect measure of spontaneous pain by using an incapacitance tester (2Biological Instruments Snc, Besozzo, Italy), prior to surgery and 14, 28, 35 and 42 days post-surgery. Control rats were subjected to sham surgery, while treatments after MMT consisted of 6 mg/kg CR4056, or 10 mg/kg naproxen as an active control, or vehicle (9 animals/group) and they were administered orally as subacute treatment from 28 to 42 days after surgery. Statistical analysis was performed by Two-way RM ANOVA, followed by Tukey's multiple comparisons test. Results: MMT surgery resulted in a significant development of right-left hind paw weight bearing imbalance at each experimental time point, compared with the sham group (data expressed as difference of weight bearing between contralateral and ipsilateral paw). The difference in weight bearing distribution between sham and MMT rats gradually increased throughout the study, reaching its peak 42 days post-surgery in animals receiving vehicle (mean ± sem, 0.83 ± 1.87 vs. 65.92 ± 2.26, respectively). Subacute oral administration of 6 mg/kg CR4056 for 2-weeks from 28 to 42 days after surgery induced a significant reduction of right-left hind paw weight bearing imbalance, compared with the MMT control group treated with vehicle (42 days post-surgery mean ± sem, 39.84 ± 3.47 vs. 65.92 ± 2.26, respectively), as illustrated in the Figure. Conversely, 10 mg/kg naproxen was devoid of noticeable effect on weight bearing imbalance after 2-weeks subacute treatment. Conclusions: The data presented here further evidence that the imidazoline I2 receptor ligand CR4056 could represent a new highly effective analgesic treatment option for OA pain.

OBJECTIVES: We evaluated the possibility that a pattern of abnormal microRNA (miRNA) expression could be fuelling the mechanisms causing HIV-associated lipodystrophy (HAL).METHODS: In this case-control study, samples of subcutaneous adipose tissue from eight consecutive HIV-infected patients on combination antiretroviral therapy with HAL (cases) were compared with those of eight HIV-negative subjects (controls). Human miRNA microarrays were used to probe the transcriptomes of the samples. Analysis of differentially expressed miRNAs was performed using DataAssist v2.0 software, applying a paired Student's t-test.RESULTS: Data showed that 21 miRNAs out of 754 were overexpressed in the patient group. Ten of these (i.e. miR-186, miR-199a-3p, miR-214, miR-374a, miR-487b, miR-532-5p, miR-628-5p, miR-874, miR-125-b-1* and miR-374b*) were up-regulated to a significant degree (fold change >2.5; P

Objective: ADAMTS5 (aggrecanase-2) has been demonstrated to be crucial in the development of osteoarthritis (OA), by use of several mouse mutants carrying either truncated, catalytically inactive enzymes or aggrecanase-resistant mutant aggrecan. We have selected recombinant monoclonal antibodies directed against ADAMTS5, by using Intracellular Antibody Capture Technology (IACT). CRB0017 revealed very high affinity for the enzyme in Biacore analyses and very good specificity in a panel of binding assays. Therefore, we tested CRB0017 in a relevant spontaneous OA model, the STR/ort mouse. Design: STR/ort male mice were recruited at 5 months of age, and treated intra-articularly in each knee with CRB0017 1.2μg, CRB0017 12μg, or vehicle. After 6 weeks, the intra-articular administration of CRB0017 was repeated with the same doses. After 3 months from recruitment, the animals were sacrificed and the femorotibial joints processed for histology and scored in a blind fashion according to both Mankin's and the OARSI methods. Results and conclusions: All histological scores were significantly decreased in the CRB0017 12μg/knee group compared to vehicle, while administration of CRB0017 1.2μg was associated with a trend to a decrease in the same parameters. Therefore, CRB0017 administered twice in 3 months could modify the course of OA in the STR/ort mouse, by delaying cartilage breakdown as assessed histologically. The procedure of blind scoring of the histological samples clearly showed that knee intra-articular administration of CRB0017, an anti-ADAMTS5 antibody, dose-dependently improved disease progression in a relevant animal model of OA. © 2013 Osteoarthritis Research Society International.

Objective To evaluate the efficacy of tunneled lotus petal flap in terms of anatomical and cosmetic results in patients who underwent vulvoperineal reconstruction for vulvar malignancy. Methods Between March 2010 and July 2011, 22 women underwent vulvoperineal reconstruction using tunneled lotus petal flap for primary or recurrent disease at San Gerardo Hospital, Monza. In 16 cases, lotus flaps were bilateral, whereas in 6 cases, they were monolateral. Results The median age was 72 years (range, 53–87 years). The mean operating time was 85 minutes. The mean length of follow-up was 10 months (range, 2–16 months). Postoperative complications occurred in 2 patients, including one case of partial flap necrosis and one case of donor site breakdown. Conclusions Tunneled lotus petal flap is safe, easy and fast to perform, has a low rate of complications, and good functional and cosmetic results. This technique represents an optimal solution for plastic reconstruction in case of primary or recurrent vulvar disease, or in case of introital stenosis after primary inadequate closure.

Berberine (BERB) and a combination (COMB) of berberine (CAS 2086-83-1) with policosanol (CAS 557-61-9), red yeast extract (containing monacolin, CAS 557-61-9), folic acid and astaxanthin were orally administered daily for 4 weeks to 40 subjects with moderate dyslipidemias divided in two parallel groups each of 20 subjects. Total cholesterol (TC), LDL, HDL, Non HDL, ApoB, ApoA, Lp(a) and triglycerides (TG) were measured before and at the end of treatments. BERB and COMB significantly reduced TC (respectively by 16% and 20%), LDL (by 20% and 25%), ApoB (by 15% and 29%) and TG (by 22% and 26%), and increased HDL (by 6.6% and 5.1%). Adverse events or impairments of liver transaminases or of CPK were not observed. In conclusion, food supplements containing natural products such as berberine, policosanol, red yeast extracts, folic acid and astaxanthin could be a useful support to diet and life style changes to correct dyslipidemias and to reduce cardiovascular risk in subjects with moderate mixed dyslipidemias.

Proglumide, a cholecystokinin antagonist, did not improve Parkinson's disease in a preliminary drug treatment trial.