Analysis of prognostic factors in COVID-19 hospitalized patients: an Italian single-center case-control study.

OBJECTIVE: COVID-19 clinical presentation ranges from asymptomatic infection to an inflammatory cytokine storm with multi-organ failure and fatal outcomes. The identification of high-risk patients for severe disease is crucial to plan an early treatment and intensive follow-up. We aimed to investigate negative prognostic factors in a group of patients hospitalized for COVID-19. PATIENTS AND METHODS: 181 patients (90 men and 91 women, mean age 66.56 ± 13.53 years) were enrolled. Each patient received a work-up including medical history, clinical examination, arterial blood gas analysis, laboratory blood tests, feasible ventilatory support required during hospital stay, intensive care setting required, duration of illness and length of hospital stay (>or<25 days). For the assessment of the severity of COVID-19, three main indicators were considered: 1) the intensive care unit (ICU) admission 2) the hospitalization length >25 days; 3) the need of non-invasive ventilation (NIV). RESULTS: The independent risk factor associated with the ICU admission were lactic dehydrogenase elevation (p=0.046), C reactive protein elevation (p=0.014) at hospital admission and direct oral anticoagulant home therapy (p=0.048); for hospital length >25 days: early corticosteroid therapy (p=0.035); for NIV treatment: ferritin elevation at hospital admission (p=0.006). CONCLUSIONS: The presence of the above factors may be useful to identify patients at high risk of developing a severe COVID-19 that need an early treatment and intensive follow-up.

Caveolin-1 and polymerase I and transcript release factor: new players in insulin-like growth factor-I receptor signaling.

Caveolae are plasma membrane regions enriched in Caveolin proteins which regulate vesicular transport, endocytosis, and cell signaling. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates Caveolin-1 (Cav-1), the most represented caveolar protein. Cav-1 participates to IGF-IR internalization and signaling directly interacting with IGF-IR and its substrates. Recently, polymerase I and transcript release factor (PTRF) or Cavin-1, has been identified in the caveolar backbone. PTRF does not play a Cav-1 ancillary role and emerging data support a direct role of PTRF in IGF-IR signaling. PTRF and Cav-1 can bind IGF-IR and regulate IGF-IR internalization and plasma membrane replacement, mechanisms frequently deregulated in cancer cells. Although the exact roles of Cav-1 and IGF-IR in human cancer continue to be a matter of some debate, there is a strong evidence for an association between Cav-1 and IGF-IR in cancer development. With the discovery of IGF-IR interaction with PTRF in caveolae, new insight emerged to understand the growing functions of these domains in IGF-I action.

Circulating Reg1α proteins and autoantibodies to Reg1α proteins as biomarkers of ß-cell regeneration and damage in type 1 diabetes.

Type 1 diabetes is an autoimmune disease where ß-cells are in a constant process of death and renewal. Reg genes play a role in ß-cells regeneration. Reg proteins may be target of an autoimmune response in type 1 diabetes with consequent production of autoantibodies and failure of regeneration. The objective of this work was to characterize the role of Reg1α proteins and anti-Reg1α antibodies as biomarkers of ß-cell regeneration and damage. Serum levels of Reg1α protein were investigated in 87 type 1 diabetic subjects (31 newly diagnosed and 56 long standing), 63 type 2 diabetic subjects, 39 subjects with systemic lupus erythematosus (SLE), a nonpancreatic autoimmune disorder, and 64 healthy subjects. The presence of anti-Reg1α antibodies and correlation with metabolic, immune, and genetic parameters were analyzed in diabetic subjects. Increased levels of Reg1α protein were observed in newly diagnosed (p=0.002), and long standing (p=0.001) type 1 diabetes patients and type 2 diabetic subjects (p<0.001). Anti-Reg1α antibodies were found in 47% of patients with type 1 diabetes. No correlation was found with metabolic, immune, and genetic parameters. Patients with SLE showed no increase in Reg1α protein. We report here for the first time raised serum Reg1α protein in type 1 and type 2 diabetes and anti-Reg1α antibodies in type 1 diabetes. Reg1α levels appear not to be influenced by genetic or metabolic control. These findings allow considering future studies on Reg1α protein and autoantibody as new tools in the evaluation and monitoring of ß-cells regeneration and autoimmunity.

Hypoadiponectinemia in lipodystrophic HIV individuals: a metabolic marker of subclinical cardiac damage.

BACKGROUND AND AIM: To evaluate cardiovascular abnormalities in highly active antiretroviral therapy (HAART) treated HIV patients with no signs or symptoms of cardiovascular impairment, and to assess the relative role of multiple concomitant risk factors. METHODS AND RESULTS: Forty-four consecutive HIV subjects (mean age 41+/-6 yrs) were enrolled. Inclusion criteria were HIV infection, CD4+cell count>150/ml, HAART treatment for at least 4 years. Metabolic serum levels, morphological and functional echocardiographic parameters were assessed in all subjects. Sixteen healthy age and sex matched subjects with no cardiovascular risk factors were recruited as controls. HIV patients showed increased left ventricular mass index with reduced mid-wall fractional shortening (mFS) when compared to controls (50.2+/-10.5 vs. 38.6+/-14.4, p=0.05 and 18.3+/-0.6 vs. 21.9+/-0.7, p<0.05, respectively). Twenty-nine patients were lipodystrophic (LD) and showed a longer HAART period (p=0.0004) and greater use of protease inhibitors (PI) (p=0.001). Coronary flow reserve (CFR) was significantly reduced in HIV patients as compared to controls (p<0.0001), as it was in LD subjects when compared to non-lipodystrophic ones (NLD) (p<0.001). Adiponectin concentrations were found to be significantly lower in LD subjects than in NLD ones (7.8+/-0.8 vs. 13.8+/-1.2 microg/ml, p=0.01), and showed a direct correlation with CFR. In multiple regression analysis, insulin, HDL and adiponectin accounted for 63% of CFR variations. CONCLUSIONS: Left ventricular hypertrophy, depressed mFS and reduced CFR represent the main signs of subclinical cardiac damage in HIV subjects treated with HAART. Hypoadiponectinemia in these subjects seems to be a metabolic risk factor of cardiovascular impairment.

IGF-I induced rapid recruitment of integrin beta1 to lipid rafts is Caveolin-1 dependent.

Caveolin-1 (Cav-1) regulates both insulin like growth factor receptor (IGF-IR) and integrin beta1 function. However, the role of Cav-1 in IGF-IR/integrin beta1 cross talk remains to be established. In this study, we observed that IGF-I did not induce integrin beta1 internalization but its plasma membrane reorganization. In particular, we found a rapid and transient association between integrin beta1 and Cav-1 followed by the enrichment of integrin beta1 in lipid rafts. To determine the role of Cav-1 in this process, we transfected Hacat cells with small interfering RNA specific for Cav-1 (siRNA-Cav-1) and with a scrambled siRNA as control (siRNA-Ctr). Cav-1 down regulated Hacat cells were then stimulated with IGF-I and analyzed by immunofluorescence and flow cytometry. We found that Cav-1 silencing abolished the recruitment of integrin beta1 to lipid rafts in the presence of IGF-I. These data demonstrate that IGF-IR/integrin beta1 cross talk is followed by integrin beta1 lipid raft compartmentalization and that Cav-1 is required for this process.

Caveolin-1 is essential for glimepiride-induced insulin secretion in the pancreatic betaTC-6 cell line.

The K(ATP) channels play a pivotal role in the complex mechanism of insulin secretion. K(ATP) channels represent the target of sulphonylureas, a class of drugs widely used in type 2 diabetes to stimulate insulin secretion. We previously showed that caveolin-1 depletion impairs action of the sulphonylurea glimepiride in human endothelial cells. The aim of this work was to investigate the possible role of caveolin-1 in glimepiride-induced insulin secretion. Caveolin-1 was depleted using siRNA method in the pancreatic betaTC-6 cell line. Then stimulation of insulin secretion was performed with different secretagogues (glucose, KCl, and glimepiride). Here, we show that betaTC-6 caveolin-1 depleted cells maintained high rate of insulin secretion after KCl, but not after glucose and glimepiride stimulation. Moreover, we find a direct interaction between caveolin-1 and Kir6.2, one of the K(ATP) channel subunit. These results demonstrate that Cav-1 plays a critical role for glucose and sulfonylurea-stimulated insulin secretion.

Simulation supported scenario analysis for water resources planning: a case study in northern Italy.

The work presents the results of a comprehensive modelling study of surface and groundwater resources in the Muzza-Bassa Lodigiana irrigation district, in Northern Italy. It assesses the impact of changes in land use and irrigation water availability on the distribution of crop water consumption in space and time, as well as on the groundwater resources. A distributed, integrated surface water-groundwater simulation system was implemented and applied to the study area. The system is based on the coupling of a conceptual vadose zone model with the groundwater model MODFLOW. To assess the impact of land use and irrigation water availability on water deficit for crops as well as on groundwater system in the area, a number of management scenarios were identified and compared with a base scenario, reflecting the present conditions. Changes in land use may alter significantly both total crop water requirement and aquifer recharge. Water supply is sufficient to meet demand under present conditions and, from the crop water use viewpoint, a reduction of water availability has a positive effect on the overall irrigation system efficiency; however, evapotranspiration deficit increases, concentrated in July and August, when it may be critical for maize crops.

Reduction of cardiovascular morbidity and mortality in type 2 diabetes. A rational approach to hypoglycemic therapy.

Type 2 diabetes mellitus is the single most important risk factor for the development of coronary artery disease. Unfortunately, the traditional therapeutic strategies for the treatment of hyperglycemia have proven to be ineffective in preventing cardiovascular complications. In recent years the number of available hypoglycemic agents has increased and considerable progress has been made regarding the comprehension of the pathophysiology of diabetes and its vascular complications. In the present article we firstly present benefits and risks of intensive vs standard hypoglycemic intervention, and the pros and cons of therapy targeted to postprandial hyperglycemia. Secondly, we discuss the cardiovascular effects of sulfonylurea agents and insulin, focusing on the role of intensive insulin treatment in the context of acute coronary syndromes. Thirdly, we review the epidemiological, clinical and experimental evidence linking insulin resistance and cardiovascular disease. Finally, we present the rationale and the role of metformin and thiazolidinedionetherapy in the prevention of cardiovascular complications. We conclude that the optimal use of the full spectrum of hypoglycemic agents has the potential to play a key role in the prevention of diabetes-related macrovascular complications.

The extracellular portion of the insulin receptor beta-subunit regulates the cellular trafficking of the insulin-insulin receptor complex. Studies on Chinese hamster ovary cells carrying the Cys 860-->Ser insulin receptor mutation.

OBJECTIVE: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IR(C860S)) showed a defective insulin internalization without affecting insulin binding and IR autophosphorylation. Moreover, this mutation reduces insulin receptor substrate (IRS)-1 tyrosine phosphorylation and insulin-induced metabolic and mitogenic effects. Altogether, these observations support a role of the extracellular domain of IR beta-subunit in insulin and receptor intracellular targeting as well as in insulin signaling. DESIGN AND METHODS: This study assesses in more details the effect of IR(C860S) mutation on the trafficking of the insulin-IR complex. In particular, IR internalization, phosphorylation, dissociation and recycling, as well as insulin degradation and retroendocytosis have been investigated in CHO cells overexpressing either wild type (CHO-IR(WT)) or mutated IRs. RESULTS: the C860S mutation significantly decreases IR internalization both insulin stimulated and constitutive. In spite of a similar dissociation of internalized insulin-IR complex, recycling of internalized IR was significantly faster (half life (t(1/2)): 21 min vs 40 min, P<0.001) and more extensive (P<0.01) for IR(C860S) than for IR(WT). On the other hand, insulin degradation and retroendocytosis were superimposable in both cell lines. As expected, insulin-induced phosphorylation was similar in both IRs, however dephosphorylation was much more rapid and was greater (P<0.01) in CHO-IR(WT) as compared with CHO-IR(C860S) cells. CONCLUSIONS: Transmembrane and intracellular domain of IR seem to be determinants for IR internalization. Now we report that Cys 860 in the IR beta-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.

Cys 786 and Cys 776 in the posttranslational processing of the insulin and IGF-I receptors.

The extracellular regions of insulin and IGF-I receptors (IR and IGF-IR) contain fibronectin type III repeats with cysteine residues potentially involved in S==S bond. In this report we show that Cys 786 in the IR and the corresponding Cys 776 in the IGF-IR regulate proreceptor dimerization with high specificity. Both C786S insulin and C776S IGF-I proreceptors reach the monomeric 210-kDa step, but do not proceed further. Mature IR(C786S) and IGF-IR(C776S) expression on plasmamembrane is abolished. No retention of C786S IR precursor was detected in the endoplasmic reticulum, which is degraded by a nonlysosomal mechanism. The rearrangement of the remaining cysteines in the insulin receptor beta subunit ectodomain does not rescue dimerization of C786S insulin proreceptor. As observed in other transmembrane receptors, iuxtamembrane cysteines, specifically Cys 786 in the IR and Cys 776 in the IGF-IR, are critical for correct processing of proreceptors.

Isolation and characterization of endothelium-dependent vasorelaxing compounds from grape seeds.

Previous work has shown that red wines, grape juices, and other grape products cause endothelium-dependent relaxation (EDR) of blood vessels in vitro by increasing nitric oxide production. In this paper we describe the isolation and characterization of some of the compounds responsible for EDR activity. Concord grape seeds were extracted with methanol and the compounds were separated by Toyopearl TSK HW-40S chromatography. Resulting fractions (primarily phenolic acids, catechins, and proanthocyanidins) were further separated semipreparatively by reversed-phase HPLC, and peaks were collected and bioassayed for EDR activity using the rat aorta preparation. EDR-active compounds were subsequently characterized by HPLC retention times and electrospray-ion-trap mass spectrometry. The compounds exhibiting the most EDR activity were proanthocyanidin trimers, tetramers, pentamers, and polymers and their gallates, as well as a dimer gallate (EC50 values in the range of 0.6-2.5 microg catechin equivalents/mL). These compounds should be useful for in vitro and in vivo studies, particularly as they relate to improvement of cardiovascular function.

Role of proline 193 in the insulin receptor post-translational processing.

AIMS/HYPOTHESIS: A point mutation, P193L, in the insulin receptor alpha subunit, has been previously identified in a patient affected by an extreme form of insulin resistance due to reduced insulin binding. In our study we investigated the cellular mechanisms by which P193L substitution causes a reduction of insulin receptor numbers on the cell surface. METHODS: Mutated insulin receptors have been generated and expressed in COS1 cells. Transcription as well as translation of P193L insulin receptor have been measured and compared with wild type insulin receptor. RESULTS: P193L insulin receptor is normally transcribed and progresses to the step of insulin proreceptor, which does not proceed to dimerization, resulting in the accumulation of the 210 kDa form. These findings suggest that the P193L insulin proreceptor is retained in the endoplasmic reticulum, where several molecular chaperones drive the folding of protein precursors. Therefore the interaction between mutated insulin receptor precursor and two endoplasmic reticulum resident chaperones (GRP78 and calnexin) were investigated. P193L insulin proreceptor co-immunoprecipitates with greater amounts of GRP78 and its interaction with calnexin is greatly delayed compared with wild type insulin receptor precursor. Co-transfection of wild type and mutated insulin receptors causes a considerable reduction of cell surface wild type insulin receptors. CONCLUSION/INTERPRETATION: P193 is critical for insulin proreceptor folding. The monomeric form of P193L insulin proreceptor is retained in the endoplasmic reticulum by a calnexin and GRP78 mediated mechanism that reduces mature insulin receptor expression on the cell surface.

Cys860 in the extracellular domain of insulin receptor beta-subunit is critical for internalization and signal transduction.

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.

A ser for Cys mutation in the extracellular portion of insulin receptor beta subunit impairs the insulin-insulin receptor complex internalization in CHO cells.

We investigated the effects of a ser for cys 860 mutation, located in the extracellular portion of insulin receptor beta subunit, on several receptor functions. CHO cells, stably transfected with the mutated cDNA, were used for this study. In the present paper, we show that the ser 860 mutation does not affect the 125I-insulin binding, but severely impairs the insulin-insulin receptor complex internalization. The kinetic analysis of internalization indicates that this process is inhibited at steps preceding the coated pit endocytosis. The beta subunit autophosphorylation of the mutated receptor is higher both in the basal and insulin stimulated states, compared with autophosphorylation measured in wild type insulin receptors. The ser 860 mutation impairs also the insulin receptor down regulation, thus suggesting an effect on the intracellular sorting of insulin-insulin receptor complex. On the basis of these results we suggest that the cys 860 plays an important role in insulin receptor lateral moving on cell surface, after insulin binding, and on the intracellular sorting to degradation pathways.

The overexpression of insulin receptor makes CHO cells resistant to the action of IGF-1: role of IRS-1.

We investigated the influence of the relative abundance of insulin and IGF-1 receptors on cellular growth, by measuring the stimulation of c-fos expression and of H3-thymidine incorporation into DNA by insulin and IGF-1 in CHO cells overexpressing insulin Receptor (CHO-IR). We found that CHO-IR cells were resistant to the action of IGF-1, but were more responsive to insulin, compared to parental clone. This result suggest the presence of a limiting step, distal to the IGF-1 receptor, in the transduction pathway. To address this question we measured the IGF-1 effect on c-fos expression in CHO-IR cells, transiently transfected with the cDNA for IRS-1, the common intracellular substrate for both insulin and IGF-1 receptors (CHO-IR/IRS-1 cells). In these cells IGF-1 stimulated a 10 fold higher c-fos expression compared to CHO-IR cells. These results suggest that the abundance of IRS-1, relative to the number of insulin and IGF-1 receptors, represents a limiting step for the intracellular transduction of insulin and IGF-1 biological messages.