Post-Coronavirus Disease 2019 Pandemic Antimicrobial Resistance.

BACKGROUND AND AIM: Antimicrobial resistance (AMR) is a chronic issue of our Westernized society, mainly because of the uncontrolled and improper use of antimicrobials. The coronavirus disease 2019 (COVID-19) pandemic has triggered and expanded AMR diffusion all over the world, and its clinical and therapeutic features have changed. Thus, we aimed to review evidence from the literature on the definition and causative agents of AMR in the frame of the COVID-19 post-pandemic era. METHODS: We conducted a search on PubMed and Medline for original articles, reviews, meta-analyses, and case series using the following keywords, their acronyms, and their associations: antibiotics, antimicrobial resistance, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), COVID-19 pandemic, personal protective equipment. RESULTS: AMR had a significant rise in incidence both in in-hospital and outpatient populations (ranging from 5 up to 50%) worldwide, but with a variegated profile according to the germ and microorganism considered. Not only bacteria but also fungi have developed more frequent and diffuse AMR. These findings are explained by the increased use and misuse of antibiotics and preventive measures during the first waves of the SARS-CoV2 pandemic, especially in hospitalized patients. Subsequently, the reduction in and end of the lockdown and the use of personal protective equipment have allowed for the indiscriminate circulation of resistant microorganisms from low-income countries to the rest of the world with the emergence of new multi- and polyresistant organisms. However, there is not a clear association between COVID-19 and AMR changes in the post-pandemic period. CONCLUSIONS: AMR in some microorganisms has significantly increased and changed its characteristics during and after the end of the pandemic phase of COVID-19. An integrated supranational monitoring approach to this challenge is warranted in the years to come. In detail, a rational, personalized, and regulated use of antibiotics and antimicrobials is needed.

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing.

BACKGROUND: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. METHODS: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FINDINGS: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. INTERPRETATION: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FUNDING: Foundation for Liver Research, London.

Giant mitochondria in human liver disease.

This thematic review aims to provide an overview of the current state of knowledge about the occurrence of giant mitochondria or megamitochondria in liver parenchymal cells. Their presence and accumulation are considered to be a major pathological hallmark of the health and fate of liver parenchymal cells that leads to overall tissue deterioration and eventually results in organ failure. The first description on giant mitochondria dates back to the 1960s, coinciding with the availability of the first generation of electron microscopes in clinical diagnostic laboratories. Detailed accounts on their ultrastructure have mostly been described in patients suffering from alcoholic liver disease, chronic hepatitis, hepatocellular carcinoma and non-alcoholic fatty liver disease. Interestingly, from this extensive literature survey, it became apparent that giant mitochondria or megamitochondria present themselves with or without highly organised crystal-like intramitochondrial inclusions. The origin, formation and potential role of giant mitochondria remain to-date largely unanswered. Likewise, the biochemical composition of the well-organised crystal-like inclusions and their possible impact on mitochondrial function is unclear. Herein, concepts about the possible mechanism of their formation and three-dimensional architecture will be approached. We will furthermore discuss their importance in diagnostics, including future research outlooks and potential therapeutic interventions to cure liver disease where giant mitochondria are implemented.

Epidemiological and Clinical Characteristics of Patients Admitted to a Secondary Hospital with Suspected MPOX Virus Infection: Is HIV Playing a Role?

MPOX (monkeypox) is a zoonotic viral disease, endemic in some Central and West African countries. However, in May 2022, cases began to be reported in non-endemic countries, demonstrating community transmission. Since the beginning of the outbreak, different epidemiological and clinical behaviors have been observed. We conducted an observational study at a secondary hospital in Madrid to characterize suspected and confirmed cases of MPOX epidemiologically and clinically. Besides the general descriptive analysis, we compared data between HIV-positive and HIV-negative subjects; 133 patients were evaluated with suspected MPOX, of which 100 were confirmed. Regarding positive cases, 71.0% were HIV positive, and 99.0% were men with a mean age of 33. In the previous year, 97.6% reported having sex with men, 53.6% used apps for sexual encounters, 22.9% practiced chemsex, and 16.7% went to saunas. Inguinal adenopathies were significantly higher in MPOX cases (54.0% vs. 12.1%, p < 0.001), as the involvement of genital and perianal area (57.0% vs. 27.3% and 17.0% vs. 1.0%, p = 0.006 and p = 0.082 respectively). Pustules were the most common skin lesion (45.0%). In HIV-positive cases, only 6.9% had a detectable viral load, and the mean CD4 count was 607.0/mm3. No significant differences were observed in the disease course, except for a greater tendency towards the appearance of perianal lesions. In conclusion, the MPOX 2022 outbreak in our area has been related to sexual intercourse among MSM, with no severe clinical cases nor apparent differences in HIV and non-HIV patients.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

Human Immunocompetent Model of Neuroendocrine Liver Metastases Recapitulates Patient-Specific Tumour Microenvironment.

Neuroendocrine liver metastases (LM-NEN) develop in a considerable proportion of patients with gastroenteropancreatic neuroendocrine neoplasms. There is a paucity of experimental models that accurately recapitulate this complex metastatic human liver microenvironment precluding scientific and clinical advancements. Here, we describe the development of a novel personalised immunocompetent precision cut tumour slice (PCTS) model for LM-NEN using resected human liver tissue. The histological assessment throughout the culture demonstrated that slices maintain viability for at least 7 days and retain the cellular heterogeneity of the original tumour. Essential clinical features, such as patient-specific histoarchitecture, tumour grade, neuroendocrine differentiation and metabolic capacity, are preserved in the slices. The PCTS also replicate the tumor-specific immunological profile as shown by the innate and adaptive immunity markers analysis. Furthermore, the study of soluble immune checkpoint receptors in the culture supernatants proves that these immunomodulators are actively produced by LM-NEN and suggests that this process is epithelium-dependent. This model can be employed to investigate these pathways and provides a powerful platform for mechanistic, immunological and pre-clinical studies.

Extracellular Vesicle-Mediated Mitochondrial Reprogramming in Cancer.

Altered metabolism is a defining hallmark of cancer. Metabolic adaptations are often linked to a reprogramming of the mitochondria due to the importance of these organelles in energy production and biosynthesis. Cancer cells present heterogeneous metabolic phenotypes that can be modulated by signals originating from the tumor microenvironment. Extracellular vesicles (EVs) are recognized as key players in intercellular communications and mediate many of the hallmarks of cancer via the delivery of their diverse biological cargo molecules. Firstly, this review introduces the most characteristic changes that the EV-biogenesis machinery and mitochondria undergo in the context of cancer. Then, it focuses on the EV-driven processes which alter mitochondrial structure, composition, and function to provide a survival advantage to cancer cells in the context of the hallmarks of cancers, such as altered metabolic strategies, migration and invasiveness, immune surveillance escape, and evasion of apoptosis. Finally, it explores the as yet untapped potential of targeting mitochondria using EVs as delivery vectors as a promising cancer therapeutic strategy.

Interplay between Cellular and Non-Cellular Components of the Tumour Microenvironment in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Currently, treatments available for advanced HCC provide dismal chances of survival, thus there is an urgent need to develop more effective therapeutic strategies. While much of the focus of recent decades has been on targeting malignant cells, promising results have emerged from targeting the tumour microenvironment (TME). The extracellular matrix (ECM) is the main non-cellular component of the TME and it profoundly changes during tumorigenesis to promote the growth and survival of malignant cells. Despite this, many in vitro models for drug testing fail to consider the TME leading to a high failure rate in clinical trials. Here, we present an overview of the function and properties of the ECM in the liver and how these change during malignant transformation. We also discuss the relationship between immune cells and ECM in the TME in HCC. Lastly, we present advanced, 3D culture techniques of cancer modelling and argue that the incorporation of TME components into these is essential to better recapitulate the complex interactions within the TME.

Soluble TIM3 and Its Ligands Galectin-9 and CEACAM1 Are in Disequilibrium During Alcohol-Related Liver Disease and Promote Impairment of Anti-bacterial Immunity.

BACKGROUND AND AIMS: Immunoregulatory checkpoint receptors (CR) contribute to the profound immunoparesis observed in alcohol-related liver disease (ALD) and in vitro neutralization of inhibitory-CRs TIM3/PD1 on anti-bacterial T-cells can rescue innate and adaptive anti-bacterial immunity. Recently described soluble-CR forms can modulate immunity in inflammatory conditions, but the contributions of soluble-TIM3 and soluble-PD1 and other soluble-CRs to immune derangements in ALD remain unclear. METHODS: In Alcoholic Hepatitis (AH; n = 19), alcohol-related cirrhosis (ARC; n = 53) and healthy control (HC; n = 27) subjects, we measured by Luminex technology (i) plasma levels of 16 soluble-CRs, 12 pro/anti-inflammatory cytokines and markers of gut bacterial translocation; (ii) pre-hepatic, post-hepatic and non-hepatic soluble-CR plasma levels in ARC patients undergoing TIPS; (iii) soluble-CRs production from ethanol-treated immunocompetent precision cut human liver slices (PCLS); (iv) whole-blood soluble-CR expression upon bacterial challenge. By FACS, we assessed the relationship between soluble-TIM3 and membrane-TIM3 and rescue of immunity in bacterial-challenged PBMCs. RESULTS: Soluble-TIM3 was the dominant plasma soluble-CR in ALD vs. HC (p = 0.00002) and multivariate analysis identified it as the main driver of differences between groups. Soluble-CRs were strongly correlated with pro-inflammatory cytokines, gut bacterial translocation markers and clinical indices of disease severity. Ethanol exposure or bacterial challenge did not induce soluble-TIM3 production from PCLS nor from whole-blood. Bacterial challenge prompted membrane-TIM3 hyperexpression on PBMCs from ALD patient's vs. HC (p < 0.002) and was inversely correlated with plasma soluble-TIM3 levels in matched patients. TIM3 ligands soluble-Galectin-9 and soluble-CEACAM1 were elevated in ALD plasma (AH > ARC; p < 0.002). In vitro neutralization of Galectin-9 and soluble-CEACAM1 improved the defective anti-bacterial and anti-inflammatory cytokine production from E. coli-challenged PBMCs in ALD patients. CONCLUSIONS: Alcohol-related liver disease patients exhibit supra-physiological plasma levels of soluble-TIM3, particularly those with greater disease severity. This is also associated with increased levels of soluble TIM3-ligands and membrane-TIM3 expression on immune cells. Soluble-TIM3 can block the TIM3-ligand synapse and improve anti-bacterial immunity; however, the increased levels of soluble TIM3-binding ligands in patients with ALD negate any potential immunostimulatory effects. We believe that anti-TIM3 neutralizing antibodies currently in Phase I clinical trials or soluble-TIM3 should be investigated further for their ability to enhance anti-bacterial immunity. These agents could potentially represent an innovative immune-based supportive approach to rescue anti-bacterial defenses in ALD patients.

Perturbations in Mitochondrial Dynamics Are Closely Involved in the Progression of Alcoholic Liver Disease.

BACKGROUND: Mitochondria play a fundamental role in the pathogenesis of alcoholic liver disease (ALD). The preservation of functional mitochondria during toxic alcohol insults is essential for cell survival and is maintained by key processes known as mitochondrial dynamics, including fragmentation and fusion, which are regulated by mitochondria-shaping proteins (MSP). We have shown mitochondrial dynamics to be distorted by alcohol in cellular and animal models, but the effect in humans remains unknown. METHODS: Hepatic gene expression of the main MSP involved in the mitochondrial fusion and fragmentation pathways was evaluated in patients with alcoholic hepatitis (AH) by DNA microarray (n = 15) and Reverse Transcription Polymerase Chain Reaction (n = 32). The activation of dynamin-1-like protein (Drp1) was also investigated in mitochondria isolated from liver biopsies of ALD patients (n = 8). The effects of alcohol on mitochondrial dynamics and on MSP protein expression were studied in human precision-cut liver slices (PCLS) exposed for 24 hours to increasing doses of ethanol (EtOH; 50 to 250 mM). RESULTS: A profound hyperactivation of the fragmentation pathway was observed in AH patients, with a significant increase in the expression of Drp1 and its adapters/receptors. The translocation of Drp1 to the mitochondria was also induced in patients with severe ALD and was affected in the PCLS with short-term exposure to EtOH but only mildly. The fusion pathway was not altered in ALD, and this was confirmed in the PCLS model. CONCLUSIONS: The present study reveals the role of mitochondrial dynamics in human ALD, confirming our previous observations in animal and cell culture models of ALD. Taken together, we show that alcohol has a significant impact on the fragmentation pathway, and we confirm Drp1 as a potential therapeutic target in severe ALD.

Deficient IL-6/Stat3 Signaling, High TLR7, and Type I Interferons in Early Human Alcoholic Liver Disease: A Triad for Liver Damage and Fibrosis.

Mechanisms underlying alcohol-induced liver injury and its progression still remain incompletely understood. Animal models can only address some aspects of the pathophysiology that requires studies directly in humans, which are scarce. We assessed liver inflammatory and immune responses at early stages of alcoholic liver disease in a unique cohort of alcohol-dependent patients undergoing a highly standardized alcohol withdrawal program. In active drinkers, quantitative real-time polymerase chain reaction revealed alcohol-induced activation of tumor necrosis factor alpha, interleukin (IL)-1ß, and nuclear factor kappa B in liver tissue already at early disease stages. Double immunofluorescence staining indicated that this proinflammatory response was restricted to activated, CD68-positive macrophages. In parallel, down-regulation of IL-6, inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway, as well as blunted cyclin D expression in hepatocytes, reduced proliferation and favored hepatocyte apoptosis. In addition, immunofluorescence and quantitative real-time polymerase chain reaction of liver tissue showed that alcohol also activated the toll-like receptor (TLR) 7-interferon (IFN) axis in hepatocytes, which was confirmed in alcohol-stimulated primary human hepatocytes and precision-cut liver slices in vitro. Activation of the TLR7-IFN axis strongly correlated with liver fibrosis markers and disease progression. Two weeks of abstinence attenuated the inflammatory response but did not allow recovery of the defective Stat3 pathway or effect on fibrosis-associated factors. Conclusion: In humans, inflammation, activation of the TLR7-IFN axis, and inhibition of Stat3-dependent repair mechanisms in early alcoholic liver disease pave the way for fibrosis development and ultimately disease progression.

41 Cases of Treatment of Cranial Cruciate Ligament Rupture with Porous TTA: Three Years of Follow Up.

Tibial Tuberosity Advancement (TTA) is a surgical technique based on a linear osteotomy that determines a cranial advancement of the tibial tuberosity in patients suffering from cranial cruciate ligament rupture (CCL). The aim is to neutralize the cranial tibial thrust (CTT) and to reach a 90° angle between the patellar tendon and the tibial plateau with a physiological knee extension of 135°. In our study, a Ti6AI4V ELI (Titanium Aluminium Vanadium) titanium scaffold for the Porous TTA, with excellent properties of osteointegration and osteoconduction when subjected to cyclic loading has been adopted. Based on the previous scientific work on an ovine model, the use of this type of porous scaffolds has subverted the previous models. Scaffold production technology is based on direct mechanical manufacturing called Electron Beam Melting (EBM). For this study, 41 dogs, different breeds, medium-large size, weighing between 10 and 80 kg, aged between 1 and 13 years, were enrolled. The inclusion criteria were based on clinical evaluations (different gaits), drawer test and tibial compression, LOAD score (Liverpool Osteoarthritis in Dogs questionnaire), radiographic diagnosis in sedation with a 135° positioning of the joint and baropodometric investigations (Stance Analyzer). The results show that Porous TTA is an excellent method for functional recovery of the knee joint following the partial and total rupture of the CCL.

Dynamin-1-Like Protein Inhibition Drives Megamitochondria Formation as an Adaptive Response in Alcohol-Induced Hepatotoxicity.

Despite the growing global burden of alcoholic liver diseases, therapeutic options are limited, and novel targets are urgently needed. Accumulating evidence suggests that mitochondria adapt in response to ethanol and formation of megamitochondria in the livers of patients is recognized as a hallmark of alcoholic liver diseases. The processes involved in ethanol-induced hepatic mitochondrial changes, the impact on mitochondria-shaping proteins, and the significance of megamitochondria formation remain unknown. In this study, we investigated the mitochondrial and cellular response to alcohol in hepatoma cell line VL-17A. The mitochondrial architecture rapidly changed after 3 or 14 days of ethanol exposure with double-pronged presentation of hyperfragmentation and megamitochondria, and cell growth was inhibited. Dynamin-1-like protein (Drp1) was identified as the main mediator driving these mitochondrial alterations, and its genetic inactivation was determined to foster megamitochondria development, preserving the capacity of the cells to grow despite alcohol toxicity. The role of Drp1 in mediating megamitochondria formation in mice with liver-specific inactivation of Drp1 was further confirmed. Finally, when these mice were fed with ethanol, the presentation of hepatic megamitochondria was exacerbated compared with wild type fed with the same diet. Ethanol-induced toxicity was also reduced. Our study demonstrates that megamitochondria formation is mediated by Drp1, and this phenomenon is a beneficial adaptive response during alcohol-induced hepatotoxicity.

Precision-cut liver slices: a versatile tool to advance liver research.

Human precision-cut liver slices represent a robust and versatile ex vivo model which retains the complex and multi-cellular histoarchitecture of the hepatic environment. As such, they represent an ideal model to investigate the mechanisms of liver injury and for the identification of novel therapeutic targets. Schematic overview to highlight the utility of precision-cut liver slices as a relevant and versatile ex-vivo model of liver disease. Top panel; Precision cut liver slices (PCLS) exposed to ethanol develop mega-mitochondria, a classical hallmark of Alcoholic Liver Disease (ALD). Right panel; PCLS from liver tumours can be used as a model for liver cancer and can be used to investigate cancer-immune cell interactions by co-culturing with matched immune cells. Bottom panel; Exposure to a mixture of oleic and linoleic acids can simulate Non-Alcoholic Fatty Liver Disease (NAFLD). Left panel; PCLS can be infected with Hepatitis B and C virus and used as a model to study viral infection and replication.

The development and testing of a module on child functioning for identifying children with disabilities on surveys. I: Background.

This is the first of three papers that will document the development of a survey module on child functioning developed by UNICEF in collaboration with the Washington Group on Disability Statistics (WG), and demonstrate - both conceptually and through test results - the strengths of that module compared with alternative tools for identifying children with disabilities in household surveys. This first paper in the series sets the background and reviews the literature leading to the development of the UNICEF/WG Child Functioning Module (CFM) and presents the WG Short Set of questions (WG-SS) and the Ten Question Screening Instrument (TQSI) as precursors, outlining some of their shortcomings and how the UNICEF/WG CFM was designed to meet those challenges. Subsequent articles will summarize results from the cognitive and field testing of the CFM including comparisons with results derived from the TQSI and the WG-SS.

Effects of the functional orthopaedic therapy on masticatory muscles activity.

BACKGROUND: The purpose of this study was to examine surface electromyographic (sEMG) activity of masticatory muscles before and after functional orthopaedic therapy with Sander appliance. MATERIAL AND METHODS: Ten adolescents (5 girls, 5 boys) with an Angle Class II, division I malocclusion, 9-13 years old, were submitted to sEMG before and after functional orthopaedic therapy. To verify the neuromuscular equilibrium, the standardized EMG activities of right and left masseter and anterior temporal muscles were recorded during maximum voluntary clench, and analysed calculating: POC (index of the symmetric distribution of the muscular activity determined by the occlusion); TC (index of presence of mandibular torque) and Ac (index suggesting the position of occlusal barycentre). The total muscular activity was also calculated. Pre- and post- functional therapy data were compared with Wilcoxon Signed-Rank Test. RESULTS: Before treatment, all subjects had a good neuromuscular equilibrium, which was not altered by treatment. CONCLUSIONS: sEMG evaluations allow to quantify the impact of occlusion on masticatory muscle activity and to control that the functional orthopaedic therapy maintain a good muscular coordination. Key words:Functional appliance, Sander appliance, electromyography, masticatory muscles.

Longitudinal effects of rapid maxillary expansion on masticatory muscles activity.

BACKGROUND: To investigate the modifications induced by rapid maxillary expansion (RME) on the electromyographic (EMG) activities of the anterior temporal and superficial masseter muscles, in patients without pre-treatment EMG alterations. MATERIAL AND METHODS: Twenty-one patients with unilateral posterior cross-bite selected from the orthodontic department of the University of L'Aquila (Italy), were enrolled. There was no control group in this study since each subject acted as a control of her/himself. Two surface EMG recordings were taken: T0 (before RME) and at T1 (3 month after the end of expansion). To verify the neuromuscular equilibrium, the EMG activities of both right and left masseter and anterior temporal muscles were recorded during a test of maximum clench. EMG indexes were compared by paired Student's t-test. RESULTS: In both occasions, all indices showed a good symmetry between the right and left side masticatory muscles. No statistically significant differences were found between the two recordings. CONCLUSIONS: In children without pre-treatment EMG alterations, no variations in standardized muscular activity after RME were found. The treatment did not alter the equilibrium of the masseter and temporal muscles. Key words:Rapid maxillary expansion, electromyography, masticatory muscles.

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis.

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Orthodontic management of bilateral maxillary canine-first premolar transposition and bilateral agenesis of maxillary lateral incisors: a case report.

INTRODUCTION: Maxillary canine-first premolar transposition (Mx.C.P1) is an uncommon dental positional anomaly that may create many orthodontic problems from both esthetic and functional points of view. OBJECTIVE: In this report we show the orthodontic management of a case of Mx.C.P1 associated with bilateral maxillary lateral incisor agenesis and unilateral mandibular second premolar agenesis METHODS: The patient was treated with a multibracket appliance and the extraction of the lower premolar. RESULTS: treatment was completed without the need for any prosthetic replacement.