Genetic ablation of Hipk2 induces cardiac dysfunction and, in combination with the loss of Hmga1, causes respiratory distress and thyroid dysfunction in mice

The Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine–threonine kinase able to interact with homeobox proteins and other transcription factors. It is a sensor for various extracellular stimuli thus regulating several signal pathways such as apoptosis, embryonic development, DNA-damage response, and cellular proliferation. As consequence, HIPK2 alterations are involved in diseases such as cancer and fibrosis. HIPK2 knock-out (KO) mice have been generated and display a reduction in body size compared to WT mice and severe psychomotor behavioral abnormalities (such as dystonia, impaired coordination, reduced motility, clasping of their posterior limbs when suspended by tails and reduced responses to novelty) that are consistent with cerebellar defects. In this thesis, we have further investigated the in vivo role of HIPK2 analyzing other phenotypic features of the HIPK2-KO mice. Firstly, we investigated the cardiac phenotype consequent to the kinase depletion. Echocardiographic analysis performed on HIPK2- KO mice at 4 and 12 months showed a significant reduction in systolic function in the adulthood, by a left ventricle fractional shortening (LVFS), and an increased in left ventricular (LV) mass/body weight (BW) ratio in HIPK2-KO mice in comparison with wild-type (WT) littermates. Consistently, increased expression levels of cardiac failure hallmarks, such atrial and brain natriuretic peptides, ANP , BNP , and myosin heavy chain, β-MHC were observed. In addition, the histological analysis of the myocardium was performed by WGA staining of cardiac sections at different age (4, 6, 12 and 18 months of age), revealing areas of fibrosis, mild inflammatory infiltrate and sarcoplasmic red deposits. At cellular level, a lot of p62/SQSTM1-positive autophagic vacuoles were observed in sections of hearts from KO mice at 18 months of age. In order to investigate the molecular mechanisms by which HIPK2 depletion causes cardiac failure, H9C2 rat cardiomyoblasts have been stably silenced for HIPK2 gene expression through a doxycycline-inducible system. HIPK2-depleted clones showed a strong upregulation of ANP, BNP and β-MHC markers with respect to control ones, thus representing a good model to study the phenotype observed in KO mice. As HIPK2 protein interacts with the architectural chromatin protein HMGA1 (Pierantoni et al. 2001), Hmga1/Hipk2 double knock-out mice (DKO) have been generated in our laboratory, crossing Hmga1-KO (A1-KO) mice and Hipk2-KO mice, in order to understand the functional role of Hmga1 and Hipk2 complex in vivo. High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasia with a causal role in cancer initiation and progression. HIPK2 and HMGA1 proteins physically interact, and HIPK2 phosphorylates HMGA1 modulating its DNA binding capacity. We observed that the 50% of DKO newborn mice dies within one day of life (P1) and autoptic examination showed that lung phenotype was characterized by collapsed immature sac-like alveoli and unexpanded alveolar spaces. Through molecular analysis, we have demonstrated that HMGA1 and HIPK2 positively regulate surfactant protein expression, since DKO mice show a strongly decreased expression of surfactant genes, both during embryogenesis and at birth. Surfactant proteins are components of lung surfactant which is essential for several lung functions. In addition, autoptic examination of DKO mice at P1 revealed also thyroid abnormalities represented by irregular structures of thyroid follicles devoid of colloid. Immunohistochemistry (IHC) and molecular analysis of thyroid late differentiation markers on DKO thyroid glands showed a reduction of expression levels of Thyroglobulin (Tg), Tireoperoxidase (Tpo) and Thyrotropin receptor (Tshr) DKO mice compared to control mice at P1. The expression of transcription factors required for the expression of the thyroid specific differentiation markers, FOXE1, PAX8 and TTF-1 were strongly reduced in thyroids from DKO at P1 (Gerlini et al. Manuscript in submission). Altogether these data suggest that the lack of both Hmga1 and Hipk2 genes impairs the expression of PAX8 and FOXE1 in thyroid gland and, consequently, of thyroid differentiation markers, indicating that HMGA1/HIPK2 interaction is crucial also for thyroid development

Contribution of Oligodendrocytes, Microglia, and Astrocytes to Myelin Debris Uptake in an Explant Model of Inflammatory Demyelination in Rats

: The internalization and degradation of myelin in glia contributes to the resolution of neuroinflammation and influences disease progression. The identification of a three-dimensional experimental model to study myelin processing under neuroinflammation will offer a novel approach for studying treatment strategies favoring inflammation resolution and neuroprotection. Here, by using a model of neuroinflammation in hippocampal explants, we show that myelin debris accumulated immediately after insult and declined at 3 days, a time point at which tentative repair processes were observed. Olig2+ oligodendrocytes upregulated the LRP1 receptor and progressively increased MBP immunoreactivity both at peri-membrane sites and within the cytosol. Oligodendrocyte NG2+ precursors increased in number and immunoreactivity one day after insult, and moderately internalized MBP particles. Three days after insult MBP was intensely coexpressed by microglia and, to a much lesser extent, by astrocytes. The engulfment of both MBP+ debris and whole MBP+ cells contributed to the greatest microglia response. In addition to improving our understanding of the spatial-temporal contribution of glial scarring to myelin uptake under neuroinflammation, our findings suggest that the exposure of hippocampal explants to LPS + IFN-γ-induced neuroinflammation may represent a valuable demyelination model for studying both the extrinsic and intrinsic myelin processing by glia under neuroinflammation

Microgravity Induces Transient EMT in Human Keratinocytes by Early Down-Regulation of E-Cadherin and Cell-Adhesion Remodeling

Abstract: Changes in cell–matrix and cell-to-cell adhesion patterns are dramatically fostered by the microgravity exposure of living cells. The modification of adhesion properties could promote the emergence of a migrating and invasive phenotype. We previously demonstrated that short exposure to the simulated microgravity of human keratinocytes (HaCaT) promotes an early epithelial– mesenchymal transition (EMT). Herein, we developed this investigation to verify if the cells maintain the acquired invasive phenotype after an extended period of weightlessness exposure. We also evaluated cells’ capability in recovering epithelial characteristics when seeded again into a normal gravitational field after short microgravity exposure. We evaluated the ultra-structural junctional features of HaCaT cells by Transmission Electron Microscopy and the distribution pattern of vinculin and E-cadherin by confocal microscopy, observing a rearrangement in cell–cell and cell–matrix interactions. These results are mirrored by data provided by migration and invasion biological assay. Overall, our studies demonstrate that after extended periods of microgravity, HaCaT cells recover an epithelial phenotype by re-establishing E-cadherin-based junctions and cytoskeleton remodeling, both being instrumental in promoting a mesenchymal–epithelial transition (MET). Those findings suggest that cytoskeletal changes noticed during the first weightlessness period have a transitory character, given that they are later reversed and followed by adaptive modifications through which cells miss the acquired mesenchymal phenotyp

Rebound effects of NCX3 pharmacological inhibition: A novel strategy to accelerate myelin formation in oligodendrocytes.

Abstract The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage. To date, no information is available on the effects resulting from prolonged exposure to NCX3 blockers and subsequent drug washout in oligodendroglia. Here, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5–amino‐N‐butyl‐2–(4–ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+]i and [Ca2+]i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+]i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+]i. This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, our findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects, and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases

Non small-cell lung cancer with metastasis to thigh muscle and mandible: two case reports

INTRODUCTION: Lung cancer is the leading cause of cancer-related death in Europe and the US. Isolated metastases to skeletal muscle and the mandible are very uncommon. CASE PRESENTATION: This report presents two cases. Case 1 concerns a 45-year-old Caucasian woman affected by muscle metastasis of the right thigh from non-small-cell lung cancer. Case 2 concerns a 61-year-old Caucasian man affected by mandible metastasis from non-small-cell lung cancer. Both metastases were detected by diagnostic imaging studies. Both patients were treated with radiation therapy with palliative and antalgic intent. CONCLUSION: Radiation therapy was effective and well tolerated in both cases. Both our patients are alive, with follow-up of 18 months and five months, respectively

Fecal microbiota transplantation to improve efficacy of immune checkpoint inhibitors in renal cell carcinoma (TACITO trial)

Background: Renal cell carcinoma (RCC) is the 6° most common cancer in men and the 8° in women in the USA. In Italy RCC incidence was 11,500 new cases in 2017, while mortality was 3,371 cases in 2015. Increasing evidence suggests that response to immune checkpoint inhibitors (ICIs), a novel treatment for advanced RCC (aRCC) and other epithelial tumors, can be influenced by the patient gut microbiota. Fecal microbiota transplantation (FMT) is a novel treatment option aimed to restore healthy gut microbiota, and is the most effective therapy for recurrent C. difficile infection. Preliminary nonrandomized findings show that FMT is able to improve efficacy of ICIs in patients with advanced melanoma. The aim of this study is to evaluate, through a double-blinded placebo-controlled randomized clinical trial, the efficacy of targeted FMT (from donors who are responding to ICIs) in improving response rates to ICIs in subjects with aRCC. Methods: 50 patients who are about to receive, or have started by <8 weeks, pembrolizumab + axitinib as first-line therapy for aRCC will be enrolled. Exclusion criteria include major comorbidities, concomitant GI or autoimmune disorders, or HIV, HBV, HCV infection, continuative corticosteroid therapy, previous treatment with systemic immune-suppressants or immune-modulatory drugs, antibiotic therapy within 4 weeks prior to enrollment. Stool samples and clinical data will be collected at baseline. Then, patients will be randomized to donor FMT or placebo FMT. They will receive the first infusion by colonoscopy and then oral frozen fecal or placebo capsules (8 capsules t.i.d.) 90 and 180 days after the first FMT. Stool donors will be searched among long-term (>12 months) responders to ICIs, and will be selected by following protocols recommended by international guidelines. Patients in the FMT group will always receive feces from the same donor throughout the three fecal transplants. Frozen fecal batches and frozen fecal capsules will be manufactured according to international guidelines. Patients will be followed-up 7, 15, 30, 90, 180, 270, and 360 days after randomization for clinical evaluation and collection of stool samples. Patients will also undergo radiological assessment at 90, 180, 270 and 360 days after randomization. Microbiome analysis will be performed with shotgun metagenomics. The primary endpoint is the progression-free survival (PFS) at 12 months. Secondary endpoints are: objective response rate at 12 months; overall survival at 12 months; adverse events after FMT; microbiome changes after FMT. Sample size calculation was based on the hypothesis that FMT can improve the 1-year PFS rate from 60% (reported 1-year PFS for SOC) to 80% wen associated to SOC. Clinical trial information: NCT04758507

Outcomes of COVID-19 patients treated with continuous positive airway pressure outside ICU

Aim We aim at characterizing a large population of Coronavirus 19 (COVID-19) patients with moderate-to-severe hypoxemic acute respiratory failure (ARF) receiving CPAP outside intensive care unit (ICU), and ascertaining whether the duration of CPAP application increased the risk of mortality for patients requiring intubation. Methods In this retrospective, multicentre cohort study, we included COVID-19 adult patients, treated with CPAP outside ICU for hypoxemic ARF from March 1 st to April 15th, 2020. We collected demographic and clinical data, including CPAP therapeutic goal, hospital length of stay (LOS), and 60- day in-hospital mortality. Results The study includes 537 patients with a median age of 69 (IQR, 60-76) years. Males were 391 (73%). According to predefined CPAP therapeutic goal, 397 (74%) patients were included in full treatment subgroup, and 140 (26%) in the do-not intubate (DNI) subgroup. Median CPAP duration was 4 (IQR, 1-8) days, while hospital LOS 16 (IQR, 9-27) days. Sixty-day in-hospital mortality was overall 34% (95%CI, 0.304-0.384), and 21% (95%CI, 0.169-0.249) and 73% (95%CI, 0.648-0.787) for full treatment and DNI subgroups, respectively. In the full treatment subgroup, in-hospital mortality was 42% (95%CI, 0.345-0.488) for 180 (45%) CPAP failures requiring intubation, while 2% (95%CI, 0.008- 0.035) for the remaining 217 (55%) patients who succeeded. Delaying intubation was associated with increased mortality [HR, 1.093 (95%CI, 1.010-1.184)]. Conclusions We described a large population of COVID-19 patients treated with CPAP outside ICU. Intubation delay represents a risk factor for mortality. Further investigation is needed for early identification of CPAP failures