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Effect of tacrolimus on soluble costimulatory molecules in patients with refractory myasthenia gravis

ObjectivesTo investigate the expression and possible role of soluble costimulatory molecules in the treatment of refractory myasthenia gravis. MethodsThirty-two patients with refractory myasthenia gravis were enrolled into this study and given tacrolimus 3 mg/day. At the beginning of treatment and 12 months follow-up period, clinical data were collected and recorded. The clinical classification of myasthenia gravis Foundation (MGFA) was performed. The MGFA-quantitative myasthenia gravis score (MGFA-QMGS), manual muscle test (MMT), MG activity of daily living (MG-ADL) and the activity of daily living (MG-ADL), the 15-item myasthenia gravis quality of life (MG QOL-15) and the dose change of prednisone were used to evaluate the efficacy. The expression levels of soluble costimulatory molecules and their ligands (sPD-1/sPD-L1, sICOS/sICOSL, sCD40/sCD40L), soluble CD25 and IL-2 in serum were determined by enzyme-linked immunosorbent assay (ELISA). ResultsWe observed that oral administration of 3 mg tacrolimus daily for 1 year can significantly improve the clinical symptoms of patients with refractory myasthenia gravis, which is characterized by a significant reduction in clinical scores, such as QMG, MMT, ADL, MGQOL-15, and a reduction daily oral prednisolone (PSL) dose (P < 0.0001).We also found that the levels of plasma sPD-1, sCD40, IL-2 in refractory MG patients increased significantly, and those decreased significantly 12 months after tacrolimus treatment (P < 0.05). The level of sCD25 was negatively correlated with clinical severity scores (P < 0.05). After tacrolimus treatment, the level of sPD-L1 increased although there was no significant difference. ConclusionTacrolimus could relieve the symptoms of refractory MG and significantly decrease the levels of plasma sPD-1, sICOSL, sCD40, sCD25 and IL-2. Soluble costimulatory molecules might be potential biomarkers for MG and tacrolimus treatment.

Evaluation of therapeutic efficacy of oxytetracycline against caprine respiratory mycoplasmosis using clinical score card method

Mycoplasmosis in goats is one of the challenging and continuous threats to small ruminant farming causing huge economic losses. This study was carried out to evaluate the therapeutic efficacy of oxytetracycline against caprine respiratory mycoplasmosis. Nasal swabs collected from fourteen goats showing clinical signs like cough, nasal discharge and abnormal breath sounds were screened for the presence of Mycoplasma spp. by polymerase chain reaction. The severity of the disease as well as the clinical improvement was recorded using a clinical score card. Oxytetracycline was administered intravenously at 15 mg/kg/day for 5 days along with supportive medications. Significant reduction in clinical score was observed after treatment and complete recovery was attained in 62.5 per cent animals.

Microrna-31 Promotes T-Cell Pathogenicity in Chronic Graft-Versus-Host Disease

MicroRNAs (miRs) are short, non-coding RNAs that repress gene expression at post-transcriptional level. Certain miRs play critical roles on T-cell responses in inflammation and autoimmunity. To understand the biology of miRs in T-cell allo-responses, we measured expression profiles of miRs in donor T cells in allogeneic vs. syngeneic recipients following bone marrow transplantation (BMT). We found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in vivo(Fig. A). MiR-31 was shown to regulate autoimmunity and cancer. However, its roles in regulating T-cell response remain largely undetermined. Recent study shows miR-31 represses peripheral Treg differentiation in EAE development. In contrast, miR-31 inhibits CD8 T-cell function in anti-virus response. We hypothesize that miR-31 is required for CD4 T-cell pathogenicity in chronic graft-versus-host disease (cGVHD), but is dispensable for CD8 T-cell cytolytic function in graft-versus-leukemia (GVL) activity.To study how miR-31 regulates T-cell allo-response, we used mice with miR-31 conditional knock out (KO) in T cells (miR-31fl/fl CD4Cre). No differences were observed in the baseline composition of naïve WT (miR-31fl/fl) and miR-31 KO splenocytes. After being transferred into allogeneic recipients, KO T cells had impaired ability to proliferate, reflected by fewer divided (% CFSElow) cells and reduced donor T-cell expansion in recipient spleens (Fig. B). To further evaluate how miR-31 contributes to T-cell pathogenicity in cGVHD, we used a murine model of BMT (B6 → BALB/c), in which low dose spleonocyes can induce scleroderma manifestation. Significant reduction of clinical scores (Fig. C), including improved skin scores and body weight loss, were found in the recipients of miR-31 KO grafts compared to those with WT grafts. This finding was validated in another haploidentical BMT model of scleroderma (B6 → B6D2F1), indicating miR-31 is required for T-cell to induce cGVHD. Consistently, the pathological damage was dramatically reduced in skin, gut and lung in the recipients of miR-31 KO grafts (Fig. D).Mechanistically, miR-31 deficiency in donor T cells resulted in significantly reduced numbers of CD4 T cells that produce IFNγ, IL-4/5 or IL-17 in recipient spleen and mesenteric lymph nodes. In absence of miR-31, fewer effector and central memory cells were generated from donor CD4 T cells. Further, T cells that are capable of localizing to B-cell follicles (CXCR5+PD-1hi) were significantlyreduced in spleen (Fig. E) and peripheral lymph nodes in recipients of miR-31 KO grafts. Interestingly, among these cells, more foxp3+ follicular T-regulatory (Tfr) but fewer foxp3- follicular T-helper (Tfh) cells were observed in the recipients of KO grafts (Fig. F), indicating miR-31 contributes to the unbalance of Tfr and Tfh cells during cGVHD development. Consistently with the role of miR-31 in regulating Tfr and Tfh cells, we found Blimp1 is the predicted targets of miR-31, which is critically involved in the homeostasis of Tfr and Tfh cells. On donor B-cell compartment, lower levels of CD86 and MHC-II expression were observed in the recipients of miR-31 KO grafts. In addition, germinal center B cells and plasma cells were also reduced in those recipients, indicating miR-31 indirectly regulates B-cell activation and differentiation through controlling T-cell pathogenicity.For translational purpose, we evaluated how miR-31 impacts T cell-mediated GVL effect. Using B6 → BDF1 BMT model, we consistently found attenuated skin manifestation in recipients of KO T cells. Importantly, in the ongoing experiment, miR-31 KO T cells were capable of controlling P815(mastocytoma) growth as efficiently as WT T cells (Fig. G), suggesting miR-31 may be dispensable for T cells to mediating GVL activity after allogeneic BMT.Taken together, the current work reveals that miR-31 plays pleotropic roles in enhancing CD4 T-cell pathogenicity in cGVHD. MiR-31 represents a promising therapeutic target for the control of cGVHD while sparing GVL effects after allogeneic hematopoietic stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Beneficial role of Pistacia lentiscus aqueous extract in experimental colitis: anti-inflammatory and potential therapeutic effects.

Pistacia lentiscus L. (PL) is a flowering plant traditionally used in the treatment of gastrointestinal disorders. The extracts of this plant are endowed with strong pharmacological activities. The aim of our current study was to investigate the anti-inflammatory and potential therapeutic effects of PL leaves aqueous extract (PLAE) against Dextran Sulfate Sodium (DSS)-induced acute colitis. The therapeutic effect of PLAE was evaluated after orally administration of 3% DSS alone or concomitantly with PLAE (50, 100 or 200mg/Kg). Mucosal lesions were assessed by macroscopic and histopathological examination. In this context, hemorrhage, diarrhea, weight loss, and disease activity index (DAI) were determined daily throughout the experiment. In the same way, hematoxylin-eosin and Alcian blue staining of colonic mucosal were used to evaluate, respectively, mucosal damages and mucus production. Furthermore, the levels of nitric oxide (NO), and pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] were measured in plasma, as well as in colonic explants and peritoneal macrophages cultures supernatants. Administration of DSS + PLAE indicated a significant reduction in clinical score of acute colitis DAI compared to DSS alone administration. Interestingly, histological analysis of the mucosa showed that DSS + PLAE-treated groups exhibited almost normal histology evidenced by an intact epithelium structure and less inflammatory cell infiltration in the mucosa. Alcian bleu staining revealed that DSS + PLAE-treated groups displayed almost normal mucus production. Importantly, a significant decrease in pro-inflammatory mediators (NO, IL-6 and TNF-α) levels in dose-dependent manner was reported in plasma, and culture supernatants of colonic explants and peritoneal macrophages from DSS + PLAE-treated mice compared to the DSS group. Our results showed that the systemic and local anti-inflammatory activities of aqueous leaves extract of PL improve the clinical signs of acute colitis. Our data suggest that PLAE has beneficial effects and could constitute a promising approach against acute ulcerative colitis by targeting the deregulated immune response.

Comparative study of the use of miltefosine, miltefosine plus allopurinol, and allopurinol in dogs with visceral leishmaniasis

Visceral leishmaniasis (VL) is an infectious disease caused by the protozoan parasite Leishmania (Leishmania) infantum, an intracytoplasmic parasite that affects humans and other species of domestic and wild mammals. In Brazil, the treatment of canine visceral leishmaniasis (CVL) with miltefosine has been implemented since 2016, and the reports on the clinical and immunological conditions of treated dogs are scarce. Thus, this study aimed to assess and monitor the clinical, laboratory, and immunological condition of dogs with CVL before (D0) and after (D29) using three pharmacotherapeutic protocols: miltefosine monotherapy (Milteforan™, Virbac) (G1), miltefosine plus allopurinol (G2), and allopurinol monotherapy (G3). Forty-five dogs with CVL were assigned to one of three treatment groups. The dogs were evaluated for clinical signs, was well as haematological, biochemical, serological, and cytokine levels. Significant reduction in clinical scores was observed in all protocols, with no differences between groups. We did not observe a clinical cure in any of the dogs in the groups. Haematological and biochemical parameters showed slow recovery, with better results observed in G2. Anti-Leishmania antibody titre remained increased in all groups. The quantification of serum cytokines demonstrated a mixed Th1/Th2 profile in CVL. The IL-2 levels decreased in all groups after treatment. Evaluation of IFN-y and IL-10 did not show changes in the groups analysed, and it did not contribute to short term therapeutic monitoring. All therapeutic protocols promoted, to varying degrees, an improvement in the general condition (clinical signs, haematological, and biochemical levels) of the animals. Through clinical-pathological exams, we found that the combination of miltefosine plus allopurinol promoted better effects in the short-term, representing the best choice for the treatment of CVL, even when compared to the only therapeutic protocol allowed in Brazil, miltefosine monotherapy. Through the quantification of cytokines, IL-2 proved to be a potential therapeutic marker for the monitoring and follow-up of dogs with CVL.

Effects of Ibuprofen and Fusion Protein Inhibitor (FPI) on neutrophil extracellular traps (NETs) in the lung tissue of calves experimentally infected with Bovine Respiratory Syncytial Virus (BRSV)

Abstract Neutrophil extracellular traps is a mode of neutrophil death targeting extracellular pathogens. They are composed of a complex of processed chromatin bound to granular and cytoplasmic proteins expelled from the cell onto pathogens. Although NETs play a role in trapping and killing extracellular pathogens they may be injurious and are considered to play a role in a wide array of inflammatory diseases. The exact contribution of NETs to clearance of pathogens versus inflammatory injury is poorly understood. Bovine respiratory syncytial virus (BRSV), like RSV, causes severe respiratory disease. In this study, we quantified NETs in lung tissue of calves experimentally infected with BRSV after treating with the anti-inflammatory drug Ibuprofen and/or an antiviral drug (FPI). Twenty four calves were infected with BRSV, placed into 6 treatment groups and treated with ibuprofen, FPI or both. Calves were euthanized on day 10 post infection and the lungs evaluated for pathological changes that were correlated with the IHC data. Cryosections of lung tissue were stained with primary antibodies [Rabbit anti citrinated histones(citH3), Rabbit anti neutrophil elastase (NE) and counter stained with secondary antibodies conjugated to Alexa 594 or 488. Slides were examined using an EVOS fluorescence microscope with at least 10 fields from each lung section at ×400 magnification evaluated. NETs were analyzed and quantitated using Materials Image Processing and Automated Reconstruction (MIPAR™) with the total NETs score calculated as the average of NETs in the 10 fields. Results show that a combination of Ibuprofen and FPI treatment from day 3 following experimental infection had a significant reduction in clinical score and average NETs in lung tissue.

Non-controlled, open-label clinical trial to assess the effectiveness of a dietetic food on pruritus and dermatologic scoring in atopic dogs

BackgroundCanine atopic dermatitis (AD) is a common skin disease. The goal of this study was to evaluate food designed to improve skin barrier function and lower inflammation to reduce pruritus and clinical severity in client-owned atopic dogs. The food contained an antioxidant blend to reduce oxidative stress, plant polyphenols to stabilize mast cells, and polyunsaturated fatty acids to improve skin health and reduce inflammation.ResultsSeventeen dogs were included in the analysis. Initially 48 adult atopic dogs were enrolled and exclusively fed a dermatologic food for 8 weeks in a non-controlled, open-label study. Thirty-one dogs were excluded for the following reasons: oral and topical medication changes (n = 17), missing data (n = 4), fatty acid supplementation (n = 3), food refusal (n = 3), dropped out (n = 3), and owner concerns (n = 1). Using a scale from 0 (normal) - 4 (severe), veterinarians evaluated the presence and severity of clinical signs of atopy at weeks 0, 4, and 8. Pet owners also rated their pet’s clinical signs of atopy on a scale from 0 (not present) - 10 (present continuously) at weeks 0, 4, and 8. Compared with initial baseline scores (median 19, range 3–69), the total veterinarian scores were significantly lower at weeks 4 (median 11, range 1–15) and 8 (median 7, range 3–46) (p < 0.05). Similarly, owner assessments showed significant improvements in the least squares mean (LSM) from baseline to 4 weeks (itching, redness, licking, and scratching) continuing to 8 weeks (itching, redness, and scratching) (p < 0.05).ConclusionsIn this open, non-controlled study evaluating a dermatologic diet in seventeen client-owned dogs, owner and veterinarian assessments showed statistically significant reductions in clinical scores designed to measure severity of atopic dermatitis. While these results show promise for the management of canine atopic dermatitis, controlled clinical trials are also needed to affirm our findings.

Acid sensing ion channel 2: A new potential player in the pathophysiology of multiple sclerosis.

Acid‐sensing ion channels (ASICs) are proton‐gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3′UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild‐type (WT), knockout Asic1−/− and Asic2−/− mice and observed a significant reduction of clinical score in Asic1−/− mice and a significant reduction in the clinical score in Asic2−/− mice in a limited time window (i.e., at days 20–23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1−/− mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2−/− mice under physiological conditions, before immunization, as compared to WT mice and Asic1−/−. A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele‐specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.

Cannabidiol Attenuates Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis Through Induction of Myeloid-Derived Suppressor Cells.

Multiple sclerosis (MS) is a chronic debilitating autoimmune disease without a cure. While the use of marijuana cannabinoids for MS has recently been approved in some countries, the precise mechanism of action leading to attenuate neuroinflammation is not clear. We used experimental autoimmune encephalomyelitis (EAE), a murine model of MS, to explore the anti-inflammatory properties of cannabidiol (CBD), a non-psychoactive cannabinoid. Treatment with CBD caused attenuation of EAE disease paradigms as indicated by a significant reduction in clinical scores of paralysis, decreased T cell infiltration in the central nervous system, and reduced levels of IL-17 and IFNγ. Interestingly, CBD treatment led to a profound increase in myeloid-derived suppressor cells (MDSCs) in EAE mice when compared to the vehicle-treated EAE controls. These MDSCs caused robust inhibition of MOG-induced proliferation of T cells in vitro. Moreover, adoptive transfer of CBD-induced MDSCs ameliorated EAE while MDSC depletion reversed the beneficial effects of CBD treatment, thereby conclusively demonstrating that MDSCs played a crucial role in CBD-mediated attenuation of EAE. Together, these studies demonstrate for the first time that CBD treatment may ameliorate EAE through induction of immunosuppressive MDSCs.

Procalcitonin Under the Course of Budesonide Inhalation Therapy in Recurrent Airway Obstruction

Procalcitonin (PCT), a precursor protein of the hormone calcitonin, is a sensitive inflammatory marker useful in diagnosis of exacerbation of asthma and chronic obstructive pulmonary disease. In this study, PCT was evaluated as a potential biomarker for the success of budesonide inhalation therapy in equine recurrent airway obstruction (RAO). Twelve horses suffering from RAO were included in a prospective clinical study. Clinical examinations, exercise test, blood gas analysis, endoscopy, bronchoalveolar lavage fluid cytology, and thoracic radiography were performed before and after therapy and results included in a scoring system. Inhalative therapy using budesonide at a dosage of 1,500 μg twice daily was performed over 10 days. Equine-specific enzyme-linked immunosorbent assays were used to evaluate concentrations of PCT as well as interleukins-1ß and 6 in bronchoalveolar lavage fluid. A significant reduction in clinical score, in particular in dyspnea, amount, and viscosity of tracheal secretion, was found after 10 days of inhalation (P = .005). For PCT, no difference was found before and after therapy. The median PCT concentration increased insignificantly from 13.85 (6.8–42.09) ng/mL to 16.47 (2.04–151.01) ng/mL after therapy (P = .158). In conclusion, PCT does not seem to be a useful marker to monitor treatment success of glucocorticoid inhalation in RAO.

Resveratrol augments therapeutic efficiency of mouse bone marrow mesenchymal stem cell-based therapy in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalitis (EAE) is an inflammatory demyelinating disease, which served as a useful model providing considerable insights into the pathogenesis of multiple sclerosis (MS). Mouse bone marrow mesenchymal stem cells (mBM-MSC) were shown to have neuroprotection capabilities in EAE. Resveratrol is a small polyphenolic compound and possess therapeutic activity in various immune-mediated diseases. The sensitivity of mBM-MSCs to resveratrol was determined by an established cell-viability assay. Resveratrol-treated mBM-MSCs were also characterized with flow cytometry using MSC-specific surface markers and analyzed for their multiple differentiation capacities. EAE was induced in C57BL/6 mice by immunization with MOG35-55. Interferon gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)/interleukin-10 (IL-10), the hallmark cytokines that direct T helper type 1 (Th1) and Th2 development, were detected with enzyme-linked immunosorbent assay (ELISA). In vivo efficacy experiments showed that mBM-MSCs or resveratrol alone led to a significant reduction in clinical scores, and combined treatment resulted in even more prominent reduction. The combined treatment with mBM-MSCs and resveratrol enhanced the immunomodulatory effects, showing suppressed proinflammatory cytokines (IFN-γ, TNF-α) and increased anti-inflammatory cytokines (IL-4, IL-10). The combination of mBM-MSCs and resveratrol provides a novel potential experimental protocol for alleviating EAE symptoms.

Transplantation of Neural Stem Cells Cotreated with Thyroid Hormone and GDNF Gene Induces Neuroprotection in Rats of Chronic Experimental Allergic Encephalomyelitis.

The present study investigates whether transplantation of NSCs treated with T3 alone (T3/NSCs), or in conjunction with GDNF gene (GDNF-T3/NSCs), provides a better therapeutic effect than NSCs for chronic EAE. EAE rats were, respectively, injected with NSCs, T3/NSCs, GDNF-T3/NSCs, and saline at 10 days and sacrificed at 60 days after EAE immunization. The three cell grafted groups showed a significant reduction in clinical scores, inflammatory infiltration, and demyelination compared with the saline-injected group, and among the cell grafted groups, the reduction in GDNF-T3/NSCs group was the most notable, followed by T3/NSCs group. Grafted T3/NSCs and GDNF-T3/NSCs acquired more MAP2, GalC, and less GFAP in brain compared with grafted NSCs, and grafted GDNF-T3/NSCs acquired most MAP2 and least GalC among the cell grafted groups. Furthermore, T3/NSCs and GDNF-T3/NSCs grafting increased the expression of mRNA for PDGFαR, GalC, and MBP in lesion areas of brain compared with NSCs grafting, and the expression of mRNA for GalC and MBP in GDNF-T3/NSCs group was higher than that in T3/NSCs group. In conclusion, T3/NSCs grafting, especially GDNF-T3/NSCs grafting, provides a better neuroprotective effect for EAE than NSCs transplantation.

A prospective, comparative, randomized, controlled study on the efficacy of the treatment with magnesium (Mg-) alginate in infants with gastroesophageal reflux

Aim: Gastroesophageal reflux (GER) is a frequent condition during the first year of life, that can be treated with lifestyle changes and reassurance. However, some children show distressing symptoms, which may require treatments that can improve the quality of life. The aim of our study was to evaluate the efficacy of Mg-alginate in the treatment of GER in infant compared with rice starch thickened formula or with reassurance. Methods: This prospective, comparative, randomized, controlled trial was conducted in patients younger than 1 year, affected by suggestive symptoms of gastroesophageal reflux. All patients underwent a validate questionnaire for assessment of clinical score (I-GERQ). The patients were randomized for 8 weeks into 3 groups according to the treatment (Group A: Mg-alginate; Group B: tickened formula feeding; Group C: reassurance, that consist in lifestyle changes and reassurance on the goodness of the condition). A clinical evaluation and score of symptoms was practiced after one month (T1) and after two months (T2) of treatment. Results: We enrolled 35 patients (mean age 5.56 ± 2.34). Fourteen children were treated with Mg-alginate (Group A), 10 with thickened formula (Group B) and 11 were only reassured (Group C). The mean values of the results of the questionnaire I-GERQ at T0 were similar in the three groups. At the end of the study (T2) in Group A 12 patients (85.7%) out of 14 showed a resolution of the symptoms, while the patients healed in Group B and in Group C were 6/10 (60%) and 6/11 (54.5%), respectively (ORG-TF: 4; ORG-R: 5; p < 0.05). Moreover, patients treated with alginate showed a lower clinical score in T2 compared with the other groups (Group A: 2.36; Group B: 5.67; Group C: 7.18). Conclusions: The results of our study showed a significant reduction in clinical score in patients treated with alginate, compared with those treated with rice starch thickened formula or only reassurance.

Effective combination of human bone marrow mesenchymal stem cells and minocycline in experimental autoimmune encephalomyelitis mice

IntroductionMultiple sclerosis (MS) is the most common inflammatory demyelinating disorder of the central nervous system (CNS). Minocycline ameliorates the clinical severity of MS and exhibits antiinflammatory, neuroprotective activities, and good tolerance for long-term use, whereas it is toxic to the CNS. Recently, the immunomodulation and neuroprotection capabilities of human bone marrow mesenchymal stem cells (hBM-MSCs) were shown in experimental autoimmune encephalomyelitis (EAE). In this study, we evaluated whether the combination of hBM-MSCs and a low-dose minocycline could produce beneficial effects in EAE mice.MethodsThe sensitivity of hBM-MSCs to minocycline was determined by an established cell-viability assay. Minocycline-treated hBM-MSCs were also characterized with flow cytometry by using MSC surface markers and analyzed for their multiple differentiation capacities. EAE was induced in C57BL/6 mice by using immunization with MOG35-55. Immunopathology assays were used to detect the inflammatory cells, demyelination, and neuroprotection. Interferon gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)/interleukin-10 (IL-10), the hallmark cytokines that direct Th1 and Th2 development, were detected with enzyme-linked immunosorbent assay (ELISA). terminal dUTP nick-end labeling (TUNEL) staining was performed to elucidate the cell apoptosis in the spinal cords of EAE mice.ResultsMinocycline did not affect the viability, surface phenotypes, or differentiation capacity of hBM-MSCs, while minocycline affected the viability of astrocytes at a high dose. In vivo efficacy experiments showed that combined treatment, compared to the use of minocycline or hBM-MSCs alone, resulted in a significant reduction in clinical scores, along with attenuation of inflammation, demyelination, and neurodegeneration. Moreover, the combined treatment with hBM-MSCs and minocycline enhanced the immunomodulatory effects, which suppressed proinflammatory cytokines (IFN-γ, TNF-α) and conversely increased anti-inflammatory cytokines (IL-4, IL-10). In addition, TUNEL staining also demonstrated a significant decrease of the number of apoptotic cells in the combined treatment compared with either treatment alone.ConclusionsThe combination of hBM-MSCs and minocycline provides a novel experimental protocol to enhance the therapeutic effects in MS.

OP0197 CGEN-15001, A Novel Negative Co-Stimulator Fusion Protein is Effective in the Collagen-Induced Arthritis Mouse Model of Rheumatoid Arthritis

BackgroundCGEN-15001 is a fusion protein composed of the extracellular domain of CGEN-15001T fused to IgG Fc. CGEN-15001T was recently identified as a novel member of the B7/CD28 costimulatory family based...

Clinical evaluation of the efficiency of low-level laser therapy for oral lichen planus: a prospective case series

Oral lichen planus (OLP) is an inflammatory disease that can be painful, mainly in the atrophic and erosive forms. Numerous drugs have been used with dissimilar results, but most treatments are empirical. However, to date, the most commonly employed and useful agents for the treatment of OLP are topical corticosteroids. The study objective was to detail the clinical effectiveness of low-level laser therapy (LLLT) for the management of OLP unresponsive to standard topical therapy. The authors studied a prospective cohort of 30 patients affected by OLP, who received biostimulation with a 980-nm gallium-aluminum-arsenide (GaAIAs) diode laser (DM980, distributed by DMT S.r.l., Via Nobel 33, 20035, Lissone, Italy). Outcome variables, statistically evaluated, were: the size of lesions; visual analogue score of pain and stability of the therapeutic results in the follow-up period. Eighty-two lesions were treated. We reported significant reduction in clinical scores of the treated lesions and in reported pain. No detailed complications or therapy side effects were observed during the study. As previously reported by our group with a preliminary report, this study suggests that LLLT could be a possible treatment choice for patients with unresponsive symptomatic OLP, also reducing the possible invasiveness correlated with other therapies.

Efficacy of Gemifloxacin for the Treatment of Experimental Staphylococcus aureus Keratitis

The objective of the present study was to evaluate the effectiveness of topically applied gemifloxacin for the treatment of experimental Staphylococcus aureus keratitis in a rabbit model. Rabbit corneas were intrastromally injected with ~100 colony-forming units (CFU) of S. aureus ATCC25923. Eight hours (early treatment) or 16 h (late treatment) after the injection, 1 topical drop of balanced salt solution (BSS), gemifloxacin ophthalmic solution (0.5%), levofloxacin ophthalmic solution (0.5%), or gatifloxacin eye gel (0.3%) was applied to each eye every 15 min for 5 doses and then, every 30 min for 14 doses. The eyes were examined both before and after treatment. The corneas were harvested from treated and untreated rabbits for the quantitation of bacteria and histological observation. In the early-treatment groups, all 3 fluoroquinolones significantly lowered the clinical severity of infection and the median erosion area of the cornea compared with the BSS control (P=0.000). In the late-treatment groups, gemifloxacin and levofloxacin did not cause a significant reduction in clinical scores compared with the BSS control (P=0.107 and 0.531, respectively), but the gatifloxacin caused a significant reduction in clinical scores compared with the BSS control (P=0.011). The median erosion area significantly decreased with treatment with gemifloxacin, gatifloxacin, and levofloxacin in both early- and late-treatment groups, when compared with the control group (P≤0.022). In the early-treatment groups, the gemifloxacin, gatifloxacin, and levofloxacin groups had significantly lower CFU recovered from the corneas compared with the control group (P<0.01), while in the late-treatment groups, levofloxacin failed to reduce the CFU recovered from the corneas compared with the control group (P=0.695). The minimal inhibitory concentrations for gemifloxacin, gatifloxacin, and levofloxacin against S. aureus ATCC25923 were 0.0625, 0.0625, and 0.125 mg/L, respectively. Gemifloxacin, similar to gatifloxacin and levofloxacin, can significantly lower the clinical severity and CFU per cornea observed in S. aureus keratitis when early treatment is implemented. Significantly, gemifloxacin showed a significant efficacy improvement in reducing the bacterial load recovered from the corneas in the late-treatment experiment.

Blockade of NKG2D ameliorates disease in mice with collagen‐induced arthritis: A potential pathogenic role in chronic inflammatory arthritis

To assess the role of the activating receptor NKG2D in arthritis. Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-gamma and TNF-alpha, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-gamma and TNF-alpha by TGF-beta. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-alpha and IFN-gamma production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-alpha levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.

Suppression of EAE by oral tolerance is independent of endogenous IFN‐β whereas treatment with recombinant IFN‐β ameliorates EAE

IFN-beta is anticipated to have an important function in mucosal tolerance, as it is one of the major cytokines produced by plasmacytoid dendritic cells, and has recently been suggested as central to the maintenance of mucosal homeostasis. Here, we have investigated whether oral tolerance is dependent on endogenous IFN-beta by feeding low-dose self-antigen myelin basic protein to IFN-beta(-/-) mice with subsequent induction of experimental autoimmune encephalomyelitis (EAE). Our study shows that oral tolerance was readily induced in IFN-beta(-/-) mice compared with their wild-type littermates (IFN-beta(+/+)). The non-self-antigen ovalbumin induced oral tolerance in both groups. These data indicate that endogenous IFN-beta is not required for induction of oral tolerance, whereas delivery of recombinant IFN-beta results in significant reduction in clinical score of EAE. Oral tolerance induction was associated with lower production of antigen-specific IFN-gamma, no shift toward antigen-specific Th2, Th17 or TGF-beta response was observed. Oral tolerance in IFN-beta(-/-) mice was also associated with the induction of regulatory and memory T cells in the mucosal-associated immune organs, however this was not a prerequisite for establishment of oral tolerance.