OBJECTIVE To evaluate the efficacy of aluminum-formulated intralymphatic glutamic acid decarboxylase (GAD-alum) therapy combined with vitamin D supplementation in preserving endogenous insulin secretion in all patients with type 1 diabetes (T1D) or in a genetically prespecified subgroup. RESEARCH DESIGN AND METHODS In a multicenter, randomized, placebo-controlled, double-blind trial, 109 patients aged 12–24 years (mean ± SD 16.4 ± 4.1) with a diabetes duration of 7–193 days (88.8 ± 51.4), elevated serum GAD65 autoantibodies, and a fasting serum C-peptide >0.12 nmol/L were recruited. Participants were randomized to receive either three intralymphatic injections (1 month apart) with 4 μg GAD-alum and oral vitamin D (2,000 IE daily for 120 days) or placebo. The primary outcome was the change in stimulated serum C-peptide (mean area under the curve [AUC] after a mixed-meal tolerance test) between baseline and 15 months. RESULTS Primary end point was not met in the full analysis set (treatment effect ratio 1.091 [CI 0.845–1.408]; P = 0.5009). However, GAD-alum–treated patients carrying HLA DR3-DQ2 (n = 29; defined as DRB1*03, DQB1*02:01) showed greater preservation of C-peptide AUC (treatment effect ratio 1.557 [CI 1.126–2.153]; P = 0.0078) after 15 months compared with individuals receiving placebo with the same genotype (n = 17). Several secondary end points showed supporting trends, and a positive effect was seen in partial remission (insulin dose–adjusted HbA1c ≤9; P = 0.0310). Minor transient injection site reactions were reported. CONCLUSION Intralymphatic administration of GAD-alum is a simple, well-tolerated treatment that together with vitamin D supplementation seems to preserve C-peptide in patients with recent-onset T1D carrying HLA DR3-DQ2. This constitutes a disease-modifying treatment for T1D with a precision medicine approach.

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In spite of intensive treatment Type 1 diabetes leads to serious complications. Preservation of residual beta cell function makes the disease milder, facilitates treatment, prevents complications and increase survival. So far immune interventions have had limited effect, and some serious adverse events and risks. In an open pilot trial we aimed to improve efficacy of GAD-alum treatment using lymph-node administration in combination with oral vitamin D. Here we report the clinical effect and focus on biomarkers for response to treatment. Patients (n = 12) aged 12 to 24 years with recent onset of Type 1 diabetes received 4 μg GAD-alum into lymph-node at day 30, 60, and 90, and oral Vitamin D 2000 U/d, days 1 to 120. Beta cell function was estimated by Mixed Meal Tolerance Tests. GADA, GADA subclasses, GAD 65 -induced cytokines and proliferation, and T cells markers were analyzed. The treatment was tolerable with no adverse events. Fasting C-peptide and insulin requirement remained stable at 15 months, while HbA1c was lower than baseline. Stimulated C-peptide showed no change at 6 months but declined after 15 months (81% of baseline). Eleven patients remained in partial remission (IDAAC < 9). Patients (n = 9) with better clinical outcome had reduced proportion of IgG1 and increased IgG2, IgG3, and IgG4, increased IL-10 secretion, and reduction of proliferation and CD8 + T cells activation. Patients with poorer clinical response had higher baseline levels of GAD 65- induced cytokines and T-cell activation, and an increased ratio of effector/central memory T cells. Intra-lymphatic GAD treatment combined with Vitamin D might preserve beta cell function and improve clinical course in T1D. Patients with less benefit have a different quality of immune response both before and after treatment. Clinical Trial Registration , identifier NCT02352974.

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5′NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5′NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5′NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5′NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Uncaria tomentosa is widely used in folk medicine for the treatment of numerous diseases, such as urinary tract disease. Hemorrhagic cystitis (HE) is an inflammatory condition of the bladder associated with the use of anticancer drugs such as cyclophosphamide (CYP). Sodium 2-mercaptoethanesulfonate (Mesna) has been used to prevent the occurrence of HE, although this compound is not effective in established lesions. It has been demonstrated that the purinergic system is involved in several pathophysiological events. Among purinergic receptors, P2X7 deserves attention because it is involved in HE induced by CYP and, therefore, can be considered a therapeutic target. The objective of this study was to investigate the potential therapeutic effect of the quinovic acid glycosides purified fraction (QAPF) from U. tomentosa in the mouse model of CYP-induced HE. Pretreatment with QAPF not only had a protective effect on HE-induced urothelial damage (edema, hemorrhage and bladder wet weight) but was also able to control visceral pain, decrease IL-1β levels and down-regulates P2X7 receptors, most likely by inhibit the neutrophils migration to the bladder. This research clearly demonstrates the promising anti-inflammatory properties of QAPF, supporting its use as complementary therapy. QAPF represents a promising therapeutic option for this pathological condition.

Background: The immune system reflects the microbiome (microbiota). Modulation of the immune system during early tendon remodeling by dexamethasone treatment can improve rat Achilles tendon healing. The authors tested whether changes in the microbiota could influence the effect of dexamethasone treatment. Hypothesis: A change in microbiome would influence the response to dexamethasone on regenerate remodeling, specifically tendon material properties (peak stress). Study Design: Controlled laboratory study. Methods: Specific opportunist and pathogen-free female rats were housed separately (n = 41) or together with specific pathogen-free rats carrying opportunistic microbes such as Staphylococcus aureus (n = 41). After 6 weeks, all co-housed rats appeared healthy but now carried S aureus. Changes in the gut bacterial flora were tested by API and RapID biochemical tests. All rats (clean and contaminated) underwent Achilles tendon transection under aseptic conditions. Flow cytometry was performed 8 days postoperatively on tendon tissue. Sixty rats received subcutaneous dexamethasone or saline injections on days 5 through 9 after transection. The tendons were tested mechanically on day 12. The predetermined primary outcome was the interaction between contamination and dexamethasone regarding peak stress, tested by 2-way analysis of variance. Results: Dexamethasone increased peak stress in all groups but more in contaminated rats (105%) than in clean rats (53%) (interaction, P = .018). A similar interaction was found for an estimate of elastic modulus ( P = .021). Furthermore, dexamethasone treatment reduced transverse area but had small effects on peak force and stiffness. In rats treated with saline only, contamination reduced peak stress by 16% ( P = .04) and elastic modulus by 35% ( P = .004). Contamination led to changes in the gut bacterial flora and higher levels of T cells (CD3+CD4+) in the healing tendon ( P < .05). Conclusion: Changes in the microbiome influence tendon healing and enhance the positive effects of dexamethasone treatment during the early remodeling phase of tendon healing. Clinical Relevance: The positive effect of dexamethasone on early tendon remodeling in rats is strikingly strong. If similar effects could be shown in humans, immune modulation by a few days of systemic corticosteroids, or more specific compounds, could open new approaches to rehabilitation after tendon injury.

According to the World Health Organization, bladder cancer is the seventh most common cancer among men in the world. The current treatments for this malignancy are not efficient to prevent the recurrence and progression of tumors. Then, researches continue looking for better therapeutic targets which can end up in new and more efficient treatments. One of the recent findings was the identification that the purinergic system was involved in bladder tumorigenesis. The ectonucleotidases, mainly ecto-5′-nucleotidase/CD73 have been revealed as new players in cancer progression and malignity. In this work, we investigated the NTPDase3 and ecto-5′-nucleotidase/CD73 expression in cancer progression in vivo. Bladder tumor was induced in mice by the addition of 0.05 % of N-butyl-N-(hydroxybutyl)-nitrosamine (BBN) in the drinking water for 4, 8, 12, 18, and 24 weeks. After this period, mice bladders were removed for histopathology analysis and immunofluorescence assays. The bladder of animals which has received BBN had alterations, mainly inflammation, in initial times of tumor induction. After 18 weeks, mice's bladder has developed histological alterations similar to human transitional cell carcinoma. The cancerous urothelium, from mice that received BBN for 18 and 24 weeks, presented a weak immunostaining to NTPDase3, in contrast to an increased expression of ecto-5′-nucleotidase/CD73. The altered expression of NTPDase3 and ecto-5′-nucleotidase/ CD73 presented herein adds further evidence to support the idea that alterations in ectonucleotidases are involved in bladder tumorigenesis and reinforce the ecto-5′-nucleotidase/ CD73 as a future biomarker and/or a target for pharmacological therapy of bladder cancer.

Background and purposeIn 3 papers in Acta Orthopaedica 10 years ago, we described that platelet-rich plasma (PRP) improves tendon healing in a rat Achilles transection model. Later, we found that microtrauma has similar effects, probably acting via inflammation. This raised the suspicion that the effect ascribed to growth factors within PRP could instead be due to unspecific influences on inflammation. While testing this hypothesis, we noted that the effect seemed to be related to the microbiota.Material and methodsWe tried to reproduce our old findings with local injection of PRP 6 h after tendon transection, followed by mechanical testing after 11 days. This failed. After fruitless variations in PRP production protocols, leukocyte concentration, and physical activity, we finally tried rats carrying potentially pathogenic bacteria. In all, 242 rats were used.ResultsIn 4 consecutive experiments on pathogen-free rats, no effect of PRP on healing was found. In contrast, apparently healthy rats carrying Staphylococcus aureus showed increased strength of the healing tendon after PRP treatment. These rats had higher levels of cytotoxic T-cells in their spleens.InterpretationThe failure to reproduce older experiments in clean rats was striking, and the difference in response between these and Staphylococcus-carrying rats suggests that the PRP effect is dependent on the immune status. PRP functions may be more complex than just the release of growth factors. Extrapolation from our previous findings with PRP to the situation in humans therefore becomes even more uncertain.