Effects of SARS-COV-2 on molecules involved in vascularization and autophagy in placenta tissues.

SARS-CoV-2 infection is considered as a multi-organ disease, and several studies highlighted the relevance of the virus infection in the induction of vascular injury and tissue morphological alterations, including placenta. In this study, immunohistochemical analyses were carried out on placenta samples derived from women with COVID-19 infection at delivery (SARS-CoV-2 PCR+) or women healed from a COVID-19 infection (SARS-CoV-2 negative at delivery, SARS-CoV-2 PCR-) or women who gave birth before 2019 (Control). Angiotensin Converting Enzyme 2 (ACE2) receptor, Cluster of differentiation 147 (CD147), endothelial CD34 marker, Vascular Endothelial Growth Factor (VEGF) and total Microtubule-associated protein 1 Light Chain 3B marker (LC3B) were investigated in parallel with SPIKE protein by standard IHC. Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS) was used to examine antigen co-expression in the same specimen. SPIKE protein was detected in villi and decidua from women with ongoing infection, with no significant differences in SPIKE staining between both biopsy sites. VEGF was significantly increased in SARS-CoV-2 PCR + biopsies compared to control and SARS-CoV-2 PCR- samples, and MICSSS method showed the co-localization of SPIKE with VEGF and CD34. The induction of autophagy, as suggested by the LC3B increase in SARS-CoV-2 PCR + biopsies and the co-expression of LC3B with SPIKE protein, may explain one of the different mechanisms by which placenta may react to infection. These data could provide important information on the impact that SARS-CoV-2 may have on the placenta and mother-to-fetus transmission.

[Therapeutic Plasma Exchange in a Patient with Chronic Hemodialysis and a New Diagnosis of Myasthenia Gravis].

Case ReportC.S.T. (♂, 71 years old) is a patient with multiple and severe comorbidities, undergoing thrice-weekly chronic hemodialysis since 2008 due to the progression of post-lithiasic uropathy. Over the past 2 months, the patient had been experiencing progressive ptosis of the eyelids, muscle weakness, and ultimately dysphagia and dysarthria that emerged in the last few days. Urgently admitted to the Neurology department, electromyography (EMG) was performed, leading to a diagnosis of predominant cranial myasthenia gravis (with borderline anti-acetylcholine receptor antibody serology). Prompt treatment with pyridostigmine and steroids was initiated. Considering the high risk of acute myasthenic decompensation, therapeutic plasma exchange (TPE) with centrifugation technique was promptly undertaken after femoral CVC placement. TPE sessions were alternated with hemodialysis. The patient's condition complicated after the third TPE session, with septic shock caused by Methicillin-Sensitive Staphylococcus Aureus (MSSA). The patient was transferred to the Intensive Care Unit (ICU). Due to hemodynamic instability, continuous veno-venous hemodiafiltration (CVVHDF) with citrate anticoagulation was administered for 72 hours. After resolving the septic condition, intermittent treatment with Acetate-Free Biofiltration (AFB) technique was resumed. The patient completed the remaining three TPE sessions and, once the acute condition was resolved, was transferred back to Neurology. Here, the patient continued the treatment and underwent a rehabilitation program, showing significant motor and functional recovery until discharge. Conclusions. The multidisciplinary interaction among Nephrologists, Neurologists, Anesthesiologists, and experts from the Immunohematology and Transfusion Medicine Service enabled the management and treatment of a rare condition (MG) in a high-risk chronic hemodialysis patient.

Rituximab in people living with HIV affected by immune-mediated renal diseases: a case-series.

Over the last two decades, rituximab (RTX) has played an important role in the treatment of some lymphoproliferative malignancies and immune-mediated diseases. RTX administration is generally safe and well-tolerated, but side effects including late-onset neutropenia, hypogammaglobulinemia, hepatitis B reactivation and rare cases of progressive multifocal leukoencephalopathy have been observed after its administration. Although there are no absolute contraindications regarding its use in people living with HIV (PLWH), the prescription of this drug has been principally limited in patients with oncohematological diseases. In this report, we described the outcome of four PLWH who underwent RTX therapy after the diagnosis of immune-mediated renal disease. The main RTX-associated adverse effects were leukopenia, late-onset neutropenia and decline of CD4+ and CD8+ T-cell counts. In addition, two of the four patients experienced pneumonia requiring hospitalization within six months from the last RTX infusion. We suggest that RTX should be used with caution in PLWH until further evidence emerges on its safety profile in this vulnerable population.

Physician-patient communication and patient-reported outcomes in the actinic keratosis treatment adherence initiative (AK-TRAIN): a multicenter, prospective, real-life study of treatment satisfaction, quality of life and adherence to topical field-directed therapy for the treatment of actinic keratosis in Italy.

BACKGROUND: Patients with multiple actinic keratoses (AKs) should be treated with field-directed therapy. Such treatments challenge patients' adherence due to out-of-pocket costs, length of treatment and severity of local skin reactions (LSRs). Effective physician-patient communication (PPC) may buffer therapy-related distress, thus improving quality of life, treatment satisfaction and adherence. OBJECTIVES: We evaluated the interplay between PPC, LSR intensity (safety) and lesion clearance rates (effectiveness) on treatment satisfaction, quality of life and treatment adherence among patients with multiple AKs receiving topical field-directed therapies. METHODS: In this observational, multicentre, longitudinal, cohort study, we included 1136 adult patients with discrete, clinically detectable, visible, multiple (three or more lesions in a 25 cm2 area), Grade I/II AKs, for whom the attending dermatologist has prescribed treatment with a topical field-directed therapy. We matched self-reported data and medical information recorded by dermatologists in standard clinical forms. Patients were followed up at two time points (T1: 8 days; T2: 25-30 days) RESULTS: Most patients were elderly, married, men with poor socio-economic status and multiple lesions of the scalp or face. The majority (n = 961) had a prescription of ingenol mebutate (IMB) and 175 received either diclofenac 3% in hyaluronic acid (DHA) or imiquimod 5% (IMQ). Clearance rate at 1 month was 84%. Most patients felt very supported (n = 819, 73%) and rated dermatologist's explanations very clear (n = 608, 54%). Treatment satisfaction (effectiveness and convenience scales) increased along the follow-up, especially for those on IMB (Δpre-post  = -4.00; other: Δpre-post  = -0.25; interaction P < 0.001). Communication clarity was associated with higher treatment satisfaction scores (ß = 0.4-0.6, P < 0.01) and lower risk of non-adherence among IMB patients (risk difference: 16%, P < 0.01). CONCLUSION: Communication clarity was associated with patient-reported outcomes and adherence beyond AK-related clinical parameters. Our study questions the current episodic approach to AK management and provides the rationale to develop chronic care models fostering patients' engagement and treatment alliance.

Italian expert consensus for the management of actinic keratosis in immunocompetent patients.

Actinic keratosis (AK) is a common skin disease which can potentially progress to invasive squamous cell carcinoma (iSCC). Given that mortality rates and health-care cost associated with iSCC are substantial, the management of AK represents an important public health issue. Several effective lesion-directed and field-directed treatments are available. Ablative procedures (e.g. cryosurgery, excision, laser ablation, curettage alone or with electrodessication) are considered cost-effective options for solitary lesions. Field-directed therapies (e.g. Ingenol Mebutate, imiquimod, PDT, 5-Fluorouracile, diclofenac 3%, 5-FU + Salicylic acid) can be used over large epidermal surfaces and are directed to treat both individual visible lesions and cancerization fields. In order to provide guidance for management choice in clinical practice, several guidelines concerning the diagnosis and treatment of AK have been published in the past decade. However, the introduction of novel therapeutic options requires continuous updates of recommendations and adaptation to national contexts. The present review summarizes the existing evidence and reports the results of a consensus workshop on the management of AK.

Conventional vs. daylight methyl aminolevulinate photodynamic therapy for actinic keratosis of the face and scalp: an intra-patient, prospective, comparison study in Italy.

INTRODUCTION: Daylight photodynamic therapy (DL-PDT) with methyl aminolevulinate (MAL) is a simplified PDT procedure that was recently shown in a few trials to be effective for grade I actinic keratosis (AK), with improved tolerability and reduced time of clinical attendance as compared to conventional PDT (c-PDT). OBJECTIVE: To evaluate the efficacy and tolerability of DL-PDT vs. c-PDT with MAL in the treatment of grade I AK on the face and scalp in Italy. METHODS: Thirty-five patients with AKs on the face (n = 17) or scalp (n = 18) were prospectively enrolled in an intra-patient, left-right, prospective, comparison study between DL-PDT and c-PDT at a single centre between September and October 2013. Weather conditions and outdoor temperature during daylight exposure were recorded for each DL-PDT session. Pain was assessed after the PDT session and local adverse events 2 days after treatment. Lesion response rate was evaluated on both sides at 3 months. AKs with complete regression were followed until 6 months. Patient's preference for either treatment was recorded. RESULTS: There was no difference in complete response (CR) rate of AK I at 3 months between DL-PDT and c-PDT (87% vs. 91%; RR = 0.96; P = 0.16). A lower CR rate was observed with DL-PDT than with c-PDT for AK II (36% vs. 61%; RR = 0.58, P = 0.06) and III (25% vs. 46%; RR = 0.50, P = 0.20). Recurrence rate at 6 months was slightly higher for cleared AK I after DL-PDT than after c-PDT (17% vs. 12%, RR = 1.50, P < 0.05). DL-PDT was associated with lower pain (ΔVAS = -2.2, P < 0.01) and reduced severity of local adverse events (ΔLSR = -1.4, P < 0.01) than c-PDT. Increasing outdoor temperature was associated with the efficacy of DL-PDT and the severity of adverse events. DL-PDT was preferred by 88% of the patients. CONCLUSION: MAL DL-PDT showed similar efficacy to c-PDT in the treatment of AK I of the face/scalp but was less effective than c-PDT for AKs II and III. DL-PDT was better tolerated being associated with lower pain and occurrence of fewer adverse events. Clinical response to DL-PDT was significantly moderated by outdoor temperature, increasing at higher temperatures.

Physicians' opinions and clinical practice patterns for actinic keratosis management in Italy.

AIM: We report dermatologists' opinions and clinical practice patterns about clinical factors driving decision making in the management of actinic keratosis (AK) in Italy. METHODS: We carried out a cross-sectional survey among 33 Italian dermatologists. Physicians were asked to report their management choices in consecutive patients with AK seen at their practice within 2 weeks since study initiation. We collected patients' clinical and socio-demographic characteristics with a standardized data collection form and assessed physicians' opinions on AK management with a self-reported questionnaire. RESULTS: Six hundred fifty-seven patients with new, single AK lesions without evidence of photo-damaged skin in the surrounding areas, were predominantly treated with lesion-directed therapies (primarily cryotherapy). In contrast, physicians preferentially prescribed field-directed therapies to patients with multiple lesions and evidence of photo-damaged skin in AK surrounding areas. However we observed a wide variation in treatment choices and physicians' opinions on AK management. Dermatologists underlined the importance of fostering patients' adherence and minimize therapy side effects. CONCLUSION: Overall, our results show that current guidelines regarding management of AK are only partially integrated in dermatology practice. The active dissemination of up-to-date national guidelines might help harmonize clinical decision making in this complex and fast growing therapeutic area.

Physicians' concerns towards prescription adherence and treatment effectiveness in the clinical management of actinic keratosis.

AIM: We report concerns toward prescription adherence and treatment effectiveness in the clinical management of actinic keratosis (AK) in Italy. METHODS: We carried out a cross-sectional web-based survey among Italian dermatologists across Italy. Physicians were asked to answer a self-administered questionnaire about their concerns around AK therapy and barriers to patients' adherence. Each physician also profiled his last patient and answered items concerning his experience with topical treatments and the suitability of current and future treatment options for the profiled patient. RESULTS: Fifty practitioners answered the survey. Most dermatologists agreed that field-therapy is a key element for the management of AK in most patients, and 76% (N.=38) agreed that topical treatments were the best option in such cases given their ability to target subclinical lesions. However most interviewee underlined the importance of fostering patients' adherence and minimizing side effects in order to maximize benefits from therapy. CONCLUSION: We showed that features of current therapeutic options for field-directed therapy (namely long duration of treatment, intensity and duration of local skin reaction) raise practitioners' concerns toward patients' prescription adherence and real-world effectiveness.

Targeting the PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its therapeutic potential.

B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/ß and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

The influence of intraoperative central venous pressure on delayed graft function in renal transplantation: a single-center experience.

INTRODUCTION: Delayed graft function (DGF) is a common complication in kidney transplantation. We sought to evaluate possible correlates for DGF including intraoperative parameters, focusing on fluid replacement and central venous pressure (CVP) values among patients undergoing kidney transplantation at our center. METHODS: One hundred fifty-five cadaveric donor transplantations performed at our center between 2001 and 2005 were selected for the study. We compared intraoperative parameters together with 15 other clinical and socio-demographic recipient and donor variables among patients experiencing DGF (n = 58) versus those with immediate graft function (IGF; n = 97). All significant variables at P < .05 upon univariate analysis were entered into a multivariate logistic regression model to identify risk factors for DGF. RESULTS: CVP at awakening of ≤8 mm Hg (odds ratio [OR] = 3.53; 95% confidence interval [CI], 1.63-7.63), fluid input during surgery ≤2.250 mL (OR = 2.12; 95% CI, 1.00-4.51), and recipient age ≥50 years (OR = 2.72; 95% CI, 1.11-6.68) were the strongest correlates of DGF. CONCLUSIONS: Our data suggested that reduced intraoperative perfusion as measured using CVP monitoring might increase DGF risk. This study provides the rationale to further investigate the optimal CVP target during this surgery.

Protein kinase C isoforms and lipid second messengers: a critical nuclear partnership?

A growing body of evidence, accumulated over the past 15 years, has highlighted that the protein kinase C family of isozymes is capable of translocating to the nucleus or is resident within the nucleus. The comprehension of protein kinase C isoform regulation within this organelle is under development. At present, it is emerging that lipid second messengers may play at least two roles in the control of nuclear protein kinase C: on one side they serve as chemical attractants, on the other they directly modulate the activity of specific isoforms. One of the best characterized lipid second messenger that could be involved in the regulation of nuclear PKC activity is DAG. The existence of two separate pools of nuclear DAG suggests that this lipid second messenger might be involved in distinct pathways that lead to different cell responses. Nuclear phosphatidylglycerol, D-3 phosphorylated inositol lipids and nuclear fatty acids are involved in a striking variety of critical biological functions which may act by specific PKC activation. The fine tuning of PKC regulation in cells subjected to proliferating or differentiating stimuli, might prove to be of great interest also for cancer therapy, given the fact that PKC-dependent signaling pathways are increasingly being seen as possible pharmacological target in some forms of neoplastic diseases. In this article, we review the current knowledge about lipid second messengers that are involved in regulating the translocation and/or the activity of different protein kinase C isoforms identified at the nuclear level.

Alloxan decreases intracellular potassium content of the isolated frog skin epithelium.

Alloxan has been widely used to provoke diabetes mellitus. This compound induces necrosis of the beta-pancreatic cells and the renal tubules. However, the mechanism of this action has not been fully established. There is some evidence that this drug may act by an alteration of several ionic transport mechanisms. Nevertheless, there is scant information on the effect of alloxan on these ionic transport mechanisms of the membrane in epithelial cells. We reported that this drug induces a decrease in sodium transport in the frog skin. In order to obtain information about the mechanism involved in the sodium transport diminution provoked by alloxan, in this study the function of Na+-K+ ATPase enzyme on transepithelial sodium transport altered by alloxan is explored. We measured changes in the short circuit current and in the intracellular content of sodium and potassium under conditions of maximally stimulated enzyme activity. Short circuit current was not modified by the treatment with alloxan during the period of highest activity of the enzyme, suggesting a site of action independent of this ATPase. Cell potassium was reduced in alloxan-treated epithelia, without significant changes in Na+ content. This finding points out the existence of an alteration induced by alloxan of some modulator mechanisms of the intracellular K+ concentration. The treatment of the frog skin with cesium chloride, a K+ channel blocker, prevented the decrease of Na+ transport produced by alloxan. This result suggests an action of this diabetogenic drug on the K+ channels of the frog skin epithelium.

Improved function of rat islets upon co-microencapsulation with Sertoli's cells in alginate/poly-L-ornithine.

The purpose of this study was to assess whether Sertoli's cells would improve functional performance of homologous pancreatic islets within microcapsules. Purified rat Sertoli's cells were co-enveloped with islets in microcapsules that had been fabricated with alginic acid and poly-L-ornithine. Confocal laser microscopy was used to determine any mitogenic effects of Sertoli's cells on islets beta-cells. Insulin secretion from islets, with or without Sertoli's cells, was examined, and grafts of Sertoli's cells with islets in microcapsules into diabetic mice were carried out. Co-incubation of Sertoli's cells with islets resulted in a significant increase in the islet beta-cell mitotic rate, which was coupled with significantly higher insulin release under glucose stimulation, as compared to controls. Grafts of co-microencapsulated Sertoli's cells with islets resulted in prolongation of the achieved normoglycemia in the animals receiving Sertoli's cells with islets as compared to controls that received islets only. Sertoli's cells do promote mitogenic activities upon in vitro co-incubation with islets, whose in vitro functional and in vivo post-transplant consequences were evident. Sertoli's cells could, therefore, be co-microencapsulated with islets for transplantation in diabetic recipients.

Phosphatidylinositol 3-kinase translocates to the nucleus of osteoblast-like MC3T3-E1 cells in response to insulin-like growth factor I and platelet-derived growth factor but not to the proapoptotic cytokine tumor necrosis factor alpha.

Changes in the metabolism of nuclear inositides phosphorylated in the D3 position of the inositol ring, which may act as second messengers, mainly have been linked to cell differentiation. To clarify a possible role of this peculiar class of inositides also during cell proliferation and/or apoptosis, we have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 it may be observed an insulin-like growth factor-I (IGF-I)- and platelet-derived growth factor (PDGF)-dependent nuclear translocation of an active phosphatidylinositol 3-kinase (PI 3-K). We found that both the growth factors increased rapidly and transiently both the amount and the activity of immunoprecipitable nuclear PI 3-K. Intranuclear PI 3-K exhibited a massive tyrosine phosphorylation on the p85 regulatory subunit. Moreover, by means of coimmunoprecipitation experiments, we showed the presence, in isolated nuclei, of the p110beta catalytic subunit of PI 3-K. Enzyme translocation was blocked by the specific PI 3-K inhibitor LY294002. In contrast, intranuclear translocation of PI 3-K did not occur in response to the proapoptotic cytokine tumor necrosis factor alpha (TNF-alpha). IGF-I was able to counteract the apoptotic stimulus of TNF-alpha and this was accompanied by the intranuclear translocation of PI 3-K. LY294002 inhibited both intranuclear translocation of PI 3-K and the rescuing effect of IGF-I. These findings strongly suggest that an important step in the signaling pathways that mediate both cell proliferation and survival is represented by the intranuclear translocation of PI 3-K.

Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors.

An active phosphatidylinositol 3-kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P(3) activated PKC, the zeta isozyme. We have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 there may be observed an insulin-like growth factor-I- (IGF-I) and platelet-derived growth factor- (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF-I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S-phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre-treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation.

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors.

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.