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OBJECTIVE: The aim of this work is to test the stability of exogenous GHB in whole blood and urine samples collected from living and deceased GHB free-users, spiked with known concentrations of GHB and stored at different temperatures (-20°C, 4°C and 20°C) up to 4 weeks. MATERIALS AND METHODS: GHB was added to GHB-free ante-mortem blood and urine samples at the concentration of 5 and 10 mg/L, respectively whereas in post-mortem blood and urine specimens at 50 and 10 mg/L respectively. All samples were stored at three different temperatures: -20°C, 4°C and 20°C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks in duplicate. No preservatives were added. GHB was quantified by GC-MS after LLE according to a previously published method. RESULTS: Post-mortem blood specimens showed a reduction of GHB levels higher than 10% only after a period of 4 weeks of storage for samples kept at +4°C and +20°C, whereas samples stored at -20°C showed a mean reduction of 8.7%. In post-mortem urine samples, there was a mean reduction of GHB levels higher than 20% at all storage temperatures, after 4 weeks of storage. Antemortem blood samples showed a reduction of GHB levels lower than 10% only after 3 days of storage at -20°C and at +4°C (samples stored at +20°C showed a mean reduction of 10.4%). After 4 weeks of storage, there was a mean reduction of GHB concentrations higher than 20% at all storage temperatures. Ante-mortem urine samples showed a reduction of GHB levels higher than 10% after just 3 days of storage for samples kept at all tested temperatures. After 4 weeks of storage, there was a mean reduction of GHB concentrations higher than 25% at all storage temperatures. CONCLUSIONS: According to our findings, it would be useful to perform GHB analysis both in blood and urine specimens within 3 days of sampling and the specimens should be stored at -20°C or 4°C in order to avoid instability issues. [ABSTRACT FROM AUTHOR]

Abstract: Dendritic cells (DCs) induce and regulate T-cell responses, and tolerogenic DCs can promote the development of regulatory T cells with suppressive activity. The possibility of manipulating DCs using different pharmacological or biological agents, enabling them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection. [Copyright &y& Elsevier]

A multi-centre, open, within-patient controlled study was performed on 23 adult burnt patients to investigate the effectiveness, safety and tolerability of Veloderm((R)) in comparison with Algisite Mtrade mark and Jaloskin((R)) in split-thickness skin graft donor site care. The areas dressed with Veloderm((R)) completely healed within 10-13 days in a significant higher proportion than the other two dressings (47.6% for Veloderm((R)) versus 26.3% for Algisite Mtrade mark and 10% for Jaloskin((R)), P<0.03), showing during the whole study less incidence of exudates and of peri-lesional erythema. The aesthetic outcome of the treated lesions after healing was significantly better for Veloderm((R)) (P=0.0016). Veloderm((R)) and Jaloskin((R)) required very few renewals of the medication during the first week of treatment, while Algisite Mtrade mark needed several multiple re-dressings. Veloderm((R)) was judged better than the other two treatments as far as the acceptability (P<0.001), ease of use (P<0.001) and efficacy (P<0.00001). Both pain during application or at removal of dressings and local infections were negligible with all treatments. No scars were formed in any skin donor site. In conclusion Veloderm((R)) is a safe and effective dressing for the re-epithelialization of the skin graft donor sites: it showed higher activity than the other two compared dressings. [ABSTRACT FROM AUTHOR]

Adult skeletal muscle is a dynamic tissue able to regenerate quite efficiently, thanks to the presence of stem cell machinery. Besides the quiescent satellite cells that are activated upon injury or paracrine factors, other stem cells are described to be directly or indirectly involved in adult myogenesis. Mesoangioblasts (MABs) are vessel-associated stem cells originally isolated from embryonic dorsal aorta and, at later stages, from the adult muscle interstitium expressing pericyte markers. Adult MABs entered clinical trials for the treatment of Duchenne muscular dystrophy and the transcriptome of human fetal MABs has been described. In addition, single cell RNA-seq analyses provide novel information on adult murine MABs and more in general in interstitial muscle stem cells. This chapter provides state-of-the-art techniques to isolate and characterize murine MABs, fetal and adult human MABs., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yedigaryan, Martínez-Sarrà, Giacomazzi, Giarratana, van der Veer, Rotini, Querceto, Grosemans, Cortés-Calabuig, Salucci, Battistelli, Falcieri, Gijsbers, Quattrocelli, Peng Koh, De Waele, Buyse, Derua and Sampaolesi.)

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to date is incurable. The major cause of death is dilated cardiomyopathy however, its pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived induced pluripotent stem cells (DMD-COs) and isogenic-corrected controls (DMD-Iso-COs) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-COs lack initial proliferative capacity, displayed a progressive loss of sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, cardiomyocyte deterioration, fibrosis and aberrant adipogenesis were observed in DMD-COs over time. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-COs which was associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marini, Marino, Aliberti, Giarratana, Pozzo, Duelen, Cortés Calabuig, La Rovere, Vervliet, Torella, Bultynck, Sampaolesi and Chai.)

Sentinox (STX) is an acid-oxidizing solution containing hypochlorous acid in spray whose virucidal activity against SARS-CoV-2 has been demonstrated. In this paper, results of a randomized controlled trial (RCT) on the efficacy of STX in reducing viral load in mild COVID-19 patients (NCT04909996) and a complementary in vitro study on its activity against different respiratory viruses are reported. In the RCT, 57 patients were randomized (1:1:1) to receive STX three (STX-3) or five (STX-5) times/day plus standard therapy or standard therapy only (controls). Compared with controls, the log 10 load reduction in groups STX-3 and STX-5 was 1.02 ( p = 0.14) and 0.18 ( p = 0.80), respectively. These results were likely driven by outliers with extreme baseline viral loads. When considering subjects with baseline cycle threshold values of 20-30, STX-3 showed a significant ( p = 0.016) 2.01 log 10 reduction. The proportion of subjects that turned negative by the end of treatment (day 5) was significantly higher in the STX-3 group than in controls, suggesting a shorter virus clearance time. STX was safe and well-tolerated. In the in vitro study, ≥99.9% reduction in titers against common respiratory viruses was observed. STX is a safe device with large virucidal spectrum and may reduce viral loads in mild COVID-19 patients.

Phenylketonuria (PKU) is a rare autosomal recessive inborn error of metabolism where the mainstay of treatment is a Phe restricted diet consisting of a combination of limited amounts of natural protein with supplementation of Phe-free or low-Phe protein substitutes and special low protein foods. Suboptimal outcomes may be related to the different absorption kinetics of free AAs, which have lower biological efficacy than natural proteins. Physiomimic Technology TM is a technology engineered to prolong AA (AA-PT) release allowing physiological absorption and masking the odor and taste of free AAs. The aim of these studies was to assess the impact of AA-PT formulation on selected functional and metabolic parameters both in acute and long-term experimental studies. Adult rats in fasting conditions were randomized in different groups and treated by oral gavage. Acute AA-PT administration resulted in significantly lower BUN at 90 min versus baseline. Both BUN and glycemia were modulated in the same direction as intact casein protein. Long-term treatment with AA-PT significantly reduces the protein expression of the muscle degradation marker Bnip3L (-46%) while significantly increasing the proliferation of market myostatin (+58%). Animals dosed for 15 days with AA-PT had significantly stronger grip strength (+30%) versus baseline. In conclusion, the results suggest that the AA-PT formulation may have beneficial effects on both AA oxidation and catabolism with a direct impact on muscle as well as on other metabolic pathways.

Fusion-negative rhabdomyosarcoma (FN-RMS) is the most common soft tissue sarcoma of childhood arising from undifferentiated skeletal muscle cells from uncertain origin. Currently used therapies are poorly tumor-specific and fail to tackle the molecular machinery underlying the tumorigenicity and uncontrolled proliferation of FN-RMS. We and other groups recently found that microRNAs (miRNA) network contributes to myogenic epigenetic memory and can influence pluripotent stem cell commitments. Here, we used the previously identified promyogenic miRNAs and tailored it to the murine FN-RMS. Subsequently, we addressed the effects of miRNAs in vivo by performing syngeneic transplant of pre-treated FN-RMS cell line in C57Bl/6 mice. miRNA pre-treatment affects murine FN-RMS cell proliferation in vivo as showed by bioluminescence imaging analysis, resulting in better muscle performances as highlighted by treadmill exhaustion tests. In conclusion, in our study we identified a novel miRNA combination tackling the anti-myogenic features of FN-RMS by reducing proliferation and described novel antitumorigenic therapeutic targets that can be further explored for future pre-clinical applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pozzo, Giarratana, Sassi, Elmastas, Killian, Wang, Marini, Ronzoni, Yustein, Uyttebroeck and Sampaolesi.)

Introduction: To eliminate the COVID-19 pandemic, the transmission of the virus SARS-CoV-2 among the population needs to be blocked and/or at least reduced. Upper respiratory tract viral loads are highest in the early stages of the disease, and high loads are associated with higher mortality rates. This study aims to evaluate the virucidal efficacy of AOS2020, a novel sprayable Acid-Oxidizing solution containing pure and stable hypochlorous acid (HClO), on human coronavirus SARS-Cov-2 in vitro, and the tolerability profile on nasal and oral mucosa suggesting to be a potential solution for upper respiratory hygiene., Method: Virucidal assays and intranasal and oral irritation tests were undertaken in accordance with relevant national and international guidance and methods., Results: In pre-clinical tests, the AOS2020, showed > 99.8% virucidal efficacy in < 1 min against SARS-Cov-2. The safety profile testing on both the nasal and oral mucosa indicates that AOS2020 is non-irritant., Conclusion: These initial results indicate that this product has the potential treatment to reduce viral load in the upper respiratory tract.

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hair levels of the direct ethanol biomarker ethyl glucuronide (EtG) are used to evaluate history of alcohol intake. The proximal 3 cm of the hair sample is most often analyzed and this is assumed to represent the intake of ethanol over approximately the past three months. The aim of this study was to investigate change of EtG levels during hair growth in an ethanol-abstinent period. Twenty-seven patients were recruited from an alcohol treatment clinic. A hair sample was collected after hospitalization and EtG was analyzed in the 0-3 cm hair segment (T1). Another hair sample was collected after one month of abstinence and EtG was analyzed in the 1-4 cm hair segment (T2), discarding the proximal 1 cm (0-1 cm) of the segment. As a result of the segment choice and assuming a hair growth rate of 1 cm per month, T2 should represent roughly the same time of alcohol exposure as segment T1. The median concentration of EtG in T1 was 100 pg/mg (range 7.7-1320) and the median concentration in T2 was 53.4 pg/mg (range < LOQ-692). EtG concentrations decreased significantly from T1 to T2 (p = 0.003) and the median change in EtG from T1 to T2 was -46.0%. This study shows decreasing EtG concentrations in most subjects in a hair segment during growth when comparing two segments that is assumed to represent roughly the same period of alcohol intake. Further research is needed to confirm if this observed decline of EtG is a result of wash-out-effects of EtG or other factors., (© 2018 John Wiley & Sons, Ltd.)

Most recent advances in tissue engineering in the fields of oral surgery and dentistry have aimed to restore hard and soft tissues. Further improvement of these therapies may involve more biological approaches and the use of dental tissue stem cells in combination with inorganic/organic scaffolds. In this study, we analyzed the osteoconductivity of two different inorganic scaffolds based on poly (lactic-co-glycolic) acid alone (PLGA-Fisiograft) or in combination with hydroxyapatite (PLGA/HA-Alos) in comparison with an organic material based on equine collagen (PARASORB Sombrero) both in vitro and in vivo. We developed a simple in vitro model in which periosteum-derived stem cells were grown in contact with chips of these scaffolds to mimic bone mineralization. The viability of cells and material osteoconductivity were evaluated by osteogenic gene expression and histological analyses at different time points. In addition, the capacity of scaffolds to improve bone healing in sinus lift was examined. Our results demonstrated that the osteoconductivity of PLGA/HA-Alos and the efficacy of scaffolds in promoting bone healing in the sinus lift were increased. Thus, new clinical approaches in sinus lift follow-up should be considered to elucidate the clinical potential of these two PLGA-based materials in dentistry., Competing Interests: The authors declare no conflict of interest.

Aims: Measurement of ethyl glucuronide (EtG) in nail, as a biomarker for alcohol intake, has recently been suggested as alternative to measurement in hair. The aim of this study was to compare levels of EtG in nail and hair, and to investigate the elimination kinetics of EtG in fingernails during an alcohol abstinent period., Methods: Overall, 40 subjects (median estimated daily intake of ethanol (EDI) 92.5 g/day) were recruited from an alcohol rehabilitation clinic. Nail and hair samples were collected at inclusion and nail clippings were collected every 7-10th day for up to 12 weeks., Results: All patients showed higher nail EtG/EDI ratios compared to hair EtG/EDI ratios (P < 0.001). The median value of the ratios between EtG in nail and EtG in hair was 5.0 (range: 1.07-56.1). There was a significant correlation between nail EtG/EDI and hair EtG/EDI (Spearman's ρ = 0.638, P < 0.001). EtG disappeared from nails after ~2 months of abstinence and the median calculated EtG half-life in nail clippings was 13.3 days (range: 5.5-29.0). There was a significant correlation between the time elapsed to last positive sample for nail EtG and nail EtG levels at time of inclusion (Spearman's ρ = 0.449, P = 0.004)., Conclusion: The present data indicate that EtG cut-off levels in nails should be higher compared to the established 30 pg/mg EtG cut-off in hair representing heavy drinking. EtG may disappear faster from nail than expected from nail growth physiology., Short Summary: Nails are an alternative matrix to hair when measuring ethyl glucuronide (EtG). The present study indicate that EtG cut-off levels in nails should be higher compared to the established 30 pg/mg EtG cut-off in hair representing heavy drinking, and EtG may disappear faster from nail than expected., (© The Author 2017. Medical Council on Alcohol and Oxford University Press. All rights reserved)

Objective: Biofilms represent a key challenge in the treatment of chronic wounds, as they are among the main reasons for delays in chronic wound healing. This in vitro study was aimed at evaluating the activity of a new acid-oxidizing solution (AOS) on biofilm formation. Acid-oxidizing solution contains free chlorine species with stabilized hypochlorous acid in high concentration (> 95%) and is characterized by acidic (pH less than 3) and super-oxidizing (Redox greater than 1000mV) features., Materials and Methods: A 3-dimensional in vitro model of reconstructed human epidermis was used to compare the activity of AOS vs 2 reference products (RP) containing betaine and polyhexanide (RP1) and sodium hypochlorite and hypochlorous acid (RP2). Different approaches were used to assess the prevention and eradication of methicillin-resistant Staphyloccocus aureus biofilm by the study products. Xylitol and chlorhexidine were used as positive controls. The activity of the study products on the biofilm structure was evaluated analyzing the ultrastructural modification by scanning electron microscopy, while skin compatibility was assessed on noncolonized tissues measuring the metabolic activity of the cells., Results: In all experiments, AOS showed to be active on the biofilm matrix, modifying its structure and allowing bacterial release from the matrix. In all experiments, no cytotoxicity was observed in the tissues treated with the product suggesting a good compatibility of AOS with skin tissues. Reference product 1 affected the biofilm, suggesting a disruption effect; RP2 was slightly less active than AOS in modifying the biofilm structure., Conclusion: Treatment with AOS affects biofilm by modifying its structure and therefore facilitating local bacteria accessibility to bactericidal agents, with consequent potential clinical benefits in the treatment of chronic wounds.

The aim of the research was to assess the bioequivalence between Rapidfilm, a new patented delivery system, versus the traditional orodispersible tablet (ODT). A randomized, two-way, single dose, crossover, bioequivalence study was conducted in 24 fasting, healthy volunteers with two formulations of ondansetron (Ondansetron Rapidfilm vs. Zofran Zydis Lingual ODT by GlaxoSmithKline GmbH & Co. KG). Plasma samples were analysed by a validated LC-MS/MS method during a collection period of 24 h post-dosing. The analysis of variance (ANOVA) on the targeted pharmacokinetic parameters did not show any significant difference between the two formulations and 90% confidence intervals (CIs) fell within the common acceptance range of 80-125%, satisfying the bioequivalence criteria. These results allow Rapidfilm to claim the same panel of indications of the conventional immediate release oral solid dosage forms, but offering several advantages also over the ODT: it can result in higher patient convenience for several applications., (2010 Elsevier B.V. All rights reserved.)

Chronic nonbacterial prostatitis is a poorly defined syndrome of putative autoimmune origin. To further understand its pathogenesis, we have analyzed autoimmune prostatitis in the NOD mouse, a strain genetically prone to develop different organ-specific autoimmune diseases. Spontaneous development of autoimmune prostatitis in the NOD male, defined by lymphomonuclear cell infiltration in the prostate gland, is well-established by approximately 20 wk of age and is stably maintained afterward. Disease development is indistinguishable in NOD and NOR mice, but is markedly delayed in IFN-gamma-deficient NOD mice. A T cell response to the prostate-specific autoantigen prostatic steroid-binding protein (PSBP) can be detected in NOD males before development of prostate infiltration, indicating lack of tolerance to this self Ag. The intraprostatic inflammatory infiltrate is characterized by Th1-type CD4(+) T cells, which are able to transfer autoimmune prostatitis into NOD.SCID recipients. We characterize here experimental autoimmune prostatitis, detected by intraprostatic infiltrate and PSBP-specific T cell responses, induced in 6- to 8-wk-old NOD males by immunization with synthetic peptides corresponding to the C1 subunit of PSBP. Three PSBP peptides induce in NOD mice vigorous T and B cell responses, paralleled by a marked lymphomononuclear cell infiltration in the prostate. Two of these peptides, PSBP(21-40) and PSBP(61-80), correspond to immunodominant self epitopes naturally processed in NOD mice after immunization with PSBP, whereas peptide PSBP(91-111) represents a cryptic epitope. These model systems address pathogenetic mechanisms in autoimmune prostatitis and will facilitate testing and mechanistic analysis of therapeutic approaches in this condition.

The nonobese diabetic (NOD) mouse represents probably the best spontaneous model for a human autoimmune disease. It has provided not only essential information on type 1 diabetes (T1D) pathogenesis, but also valuable insights into mechanisms of immunoregulation and tolerance. Importantly, it allows testing of immunointervention strategies potentially applicable to man. The fact that T1D incidence in the NOD mouse is sensitive to environmental conditions, and responds, sometimes dramatically, to immunomanipulation, does not represent a limit of the model, but is likely to render it even more similar to its human counterpart. In both cases, macrophages, dendritic cells, CD4+, CD8+, and B cells are present in the diseased islets. T1D is a polygenic disease, but, both in human and in NOD mouse T1D, the primary susceptibility gene is located within the MHC. On the other hand, T1D incidence is significantly higher in NOD females, although insulitis is similar in both sexes, whereas in humans, T1D occurs with about equal frequency in males and females. In addition, NOD mice have a more widespread autoimmune disorder, which is not the case in the majority of human T1D cases. Despite these differences, the NOD mouse remains the most representative model of human T1D, with similarities also in the putative target autoantigens, including glutamic acid decarboxylase IA-2, and insulin.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)(2)D(3) on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)(2)D(3) up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)(2)D(3) also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)(2)D(3) treatment markedly increased CD4(+) suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)(2)D(3) did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-alpha was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4(+) suppressor T cells was unaffected by 1,25(OH)(2)D(3). Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)(2)D(3) similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)(2)D(3) inhibited NF-kappaB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)(2)D(3) to modulate tolerogenic properties in P-DCs.

Type 1 diabetes (T1D) is an autoimmune disease characterized by leukocyte infiltration into the pancreatic islets, and we have previously shown that treatment of adult NOD mice with a vitamin D analog arrests the progression of insulitis, blocks Th1 cell infiltration into the pancreas, and markedly reduces T1D development, suggesting inhibition of chemokine production by islet cells. In this study, we show that all TLRs are expressed by mouse and human islet cells, and their engagement by pathogen-derived ligands markedly enhances proinflammatory chemokine production. The vitamin D analog significantly down-regulates in vitro and in vivo proinflammatory chemokine production by islet cells, inhibiting T cell recruitment into the pancreatic islets and T1D development. The inhibition of islet chemokine production in vivo persists after restimulation with TLR ligands and is associated with up-regulation of IkappaBalpha transcription, an inhibitor of NF-kappaB and with arrest of NF-kappaBp65 nuclear translocation, highlighting a novel mechanism of action exerted by vitamin D receptor ligands potentially relevant for the treatment of T1D and other autoimmune diseases.

Vitamin D receptor (VDR) ligands, in addition to controlling calcium metabolism, exert important effects on the growth and differentiation of many cell types and possess pronounced pro-tolerogenic immunoregulatory activities. VDR ligands can act directly on T cells, but antigen-presenting cells (APCs), and in particular dendritic cells (DCs), appear to be primary targets for their tolerogenic properties. The capacity of VDR ligands to target APCs and T cells is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor NF-kB that is down-regulated in APCs and in T cells. VDR ligands can induce in vitro and in vivo tolerogenic DCs able to enhance CD4(+)CD25(+) suppressor T cells that, in turn, inhibit Th1 cell responses. These mechanisms of action can explain some of the immunoregulatory properties of VDR ligands, and are potentially relevant for the treatment of Th1-mediated autoimmune diseases and allograft rejection.

Immunosuppressive and anti-inflammatory agents are able to generate tolerogenic DCs, leading, in some cases, to induction or enhancement of regulatory T cells with suppressive activity. This novel mechanism of action, shared by several immunosuppressive and anti-inflammatory agents, is becoming firmly established and contributes to explain their functional properties. The possibility to manipulate DCs in vivo using more or less conventional low molecular weight drugs, enabling them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection.

In the nonobese diabetic (NOD) mouse, pathogenic and suppressor CD4(+) T cells can be distinguished by the constitutive expression of CD25. In this study, we demonstrate that the progression of autoimmune diabetes in NOD mice reflects modifications in both T cell subsets. CD4(+)CD25(+) suppressor T cells from 8-, but not 16-wk-old NOD mice delayed the onset of diabetes transferred by 16-wk-old CD25-depleted spleen cells. These results were paralleled by the inhibition of alloantigen-induced proliferation of CD4(+)CD25(-) cells, indicating an age-dependent decrease in suppressive activity. In addition, CD4(+)CD25(-) pathogenic T cells became progressively less sensitive to immunoregulation by CD4(+)CD25(+) T cells during diabetes development. CD4(+)CD25(-) T cells showed a higher proliferation and produced more IFN-gamma, but less IL-4 and IL-10, whereas CD4(+)CD25(+) T suppressor cells produced significantly lower levels of IL-10 in 16- compared with 8-wk-old NOD mice. Consistent with these findings, a higher frequency of Th1 cells was observed in the pancreas of 16-wk-old compared with 8-wk-old NOD mice. An increased percentage of CD4(+)CD25(-) T cells expressing CD54 was present in 16-wk-old and in diabetic NOD, but not in BALB/c mice. Costimulation via CD54 increased the proliferation of CD4(+)CD25(-) T cells from 16-, but not 8-wk-old NOD mice, and blocking CD54 prevented their proliferation, consistent with the role of CD54 in diabetes development. Thus, the pathogenesis of autoimmune diabetes in NOD mice is correlated with both an enhanced pathogenicity of CD4(+)CD25(-) T cells and a decreased suppressive activity of CD4(+)CD25(+) T cells.

IL-12 administration to nonobese diabetic (NOD) mice induces IFN-gamma-secreting type 1 T cells and high circulating IFN-gamma levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-gamma production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-gamma-deficient as well as in IFN-gamma-sufficient NOD mice. Both in IFN-gamma(+/-) and IFN-gamma(-/-) NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c(+) dendritic cells, and increases the number of pancreatic CD4(+) cells secreting IL-2 and TNF-alpha. Surprisingly, IL-12-induced IFN-gamma hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4(+) T cells from IL-12-treated IFN-gamma(-/-) mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-gamma(+/-) mice because IL-12 administration in the absence of IFN-gamma leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in beta cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-gamma. Conversely, in IL-12-treated IFN-gamma-deficient mice, other pathways of beta cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-gamma. These data demonstrate that IFN-gamma has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL-12 and enhanced IL-10 secretion. We have found that a short treatment with 1,25(OH)(2)D(3) induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. This effect is enhanced by co-administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)(2)D(3) on DCs. Graft acceptance is associated with an increased percentage of CD4(+)CD25(+) regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4(+)CD25(+) cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non-hypercalcemic doses. The enhancement of CD4(+)CD25(+) regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach., (Copyright 2002 Wiley-Liss, Inc.)

Type 1 diabetes is a chronic progressive autoimmune disease characterized by mononuclear cell infiltration, dominated by interleukin-12 (IL-12)-dependent Th1 cells, of the pancreatic islets, with subsequent destruction of insulin-producing beta-cells. Here, we demonstrate that treatment of adult nonobese diabetic (NOD) mice with an analog of 1alpha,25-dihydroxyvitamin D(3), an immunomodulatory agent preventing dendritic cell maturation, decreases lipopolysaccharide-induced IL-12 and gamma-interferon production, arrests Th1 cell infiltration and progression of insulitis, and inhibits diabetes development at nonhypercalcemic doses. Arrest of disease progression is accompanied by an enhanced frequency in the pancreatic lymph nodes of CD4(+)CD25(+) regulatory T-cells that are able to inhibit the T-cell response to the pancreatic autoantigen insulinoma-associated protein 2 and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells. Thus, a short treatment of adult NOD mice with an analog of 1,25-dihydroxyvitamin D(3) inhibits IL-12 production, blocks pancreatic infiltration of Th1 cells, enhances CD4(+)CD25(+) regulatory cells, and arrests the progression of type 1 diabetes, suggesting its possible application in the treatment of human autoimmune diabetes.