Effects of substance P on human cerebral microvascular endothelial cell line hCMEC/D3 are mediated exclusively through a truncated NK-1 receptor and depend on cell confluence.

The neuropeptide substance P (SP) mediates pain transmission, immune modulation, vasodilation and neurogenic inflammation. Its role in the peripheral nervous system has been well characterised. However, its actions on the blood-brain barrier (BBB) are less clear and warrant further study. The aim of this study was to characterise the effect of SP on the brain microvascular endothelial cells using the immortalized human brain microvascular endothelial cell line hCMEC/D3. As part of our studies, we have evaluated changes in expression, at mRNA and protein levels, of genes involved in the function of the blood-brain barrier such as occludin, induced by exposure to SP. We show that the effect of SP is dependent on cell confluence status. Thus, at low confluence but not at full confluence, SP treatment reduced occludin expression. The expression of the SP receptor, neurokinin-1 receptor (NK-1R) (the truncated form of the receptor expressed exclusively in this cell line) was also modulated in a similar pattern. SP treatment stimulated extracellular signal-regulated kinase (Erk2) phosphorylation which was not associated to changes in Interleukin-6 (IL-6), Interleukin-8 (IL-8), or Intercellular Adhesion Molecule 1 (ICAM-1) protein expression. In addition, SP treatment effectively recovered nitric oxide production on cells exposed to tumour necrosis factor alpha (TNF-α). SP did not trigger intracellular calcium release in hCMEC/D3 cells. We conclude that hCMEC/D3 cells are partially responsive to SP, that the effects are mediated through the truncated form of the receptor and are dependent on the confluence status of these cells.

Gas-exchange deficit and systemic hypoperfusion in COVID-19 and non-COVID-19 young adult patients with pneumonia.

BACKGROUND: Lung damage leading to gas-exchange deficit and sepsis leading to systemic hypoperfusion are well-known features of severe pneumonia. Although frequently described in COVID-19, their prognostic impact in COVID-19-related pneumonia vs COVID-19-urelated pneumonia has never been compared. This study assesses fundamental gas-exchange and hemodynamic parameters and explores their prognostic impact in COVID-19 pneumonia and non-COVID-19 pneumonia. METHODS: We prospectively evaluated arterial pO2/FiO2, alveolar to arterial O2 gradient, shock index, and serum lactate in 126 COVID-19 pneumonia patients, aged 18- 65, presenting to the emergency department with acute, non-hypercapnic respiratory failure. As a control group we identified 1:1 age-, sex-, and pO2/FiO2-matched COVID-19-urelated pneumonia patients. Univariate and multivariable predictors of 30-day survival were identified in both groups. RESULTS: COVID-19 patients showed lower arterial serum lactate concentration (p<0.001) and shock index (p<0.001) values as compared to non-COVID-19 patients. While we did not observe differences in lactate concentration or in shock index values in deceased vs surviving COVID-19 patients (respectively, p=0.7 and p=0.6), non-COVID-19 deceased patients showed significantly higher lactate and shock index than non-COVID-19 survivors (p<0.001 and p=0.03). The pO2/FiO2 was the most powerful determinant of survival by Cox regression multivariate analysis in COVID-19 patients (p=0.006), while it was lactate in non-COVID-19 patients (p=0.001). CONCLUSIONS: As compared to COVID19-unrelated pneumonia, COVID-19 pneumonia outcome seems more strictly correlated to the extent of lung damage, rather than to the systemic circulatory and metabolic derangements typical of sepsis.

Optimizing Cell Synchronization Using Nocodazole or Double Thymidine Block.

Cell synchronization is crucial when studying events that take place at specific points of the cell cycle. Several chemical agents can be used to achieve the cell culture synchronization but not all type of cells respond equally to a given concentration of these drugs. Here we describe a simple optimization method to select concentrations and timings for nocodazole or thymidine treatments using fluorescence staining. In addition, we provide detailed protocols to arrest an asynchronous culture of either suspension or adherent cells in G1/S or in G2/M.

Early predictors of clinical outcomes of COVID-19 outbreak in Milan, Italy.

BACKGROUND: National health-system hospitals of Lombardy faced a heavy burden of admissions for acute respiratory distress syndromes associated with coronavirus disease (COVID-19). Data on patients of European origin affected by COVID-19 are limited. METHODS: All consecutive patients aged ≥18 years, coming from North-East of Milan's province and admitted at San Raffaele Hospital with COVID-19, between February 25th and March 24th, were reported, all patients were followed for at least one month. Clinical and radiological features at admission and predictors of clinical outcomes were evaluated. RESULTS: Of the 500 patients admitted to the Emergency Unit, 410 patients were hospitalized and analyzed: median age was 65 (IQR 56-75) years, and the majority of patients were males (72.9%). Median (IQR) days from COVID-19 symptoms onset was 8 (5-11) days. At hospital admission, fever (≥ 37.5 °C) was present in 67.5% of patients. Median oxygen saturation (SpO2) was 93% (range 60-99), with median PaO2/FiO2 ratio, 267 (IQR 184-314). Median Radiographic Assessment of Lung Edema (RALE) score was 9 (IQR 4-16). More than half of the patients (56.3%) had comorbidities, with hypertension, coronary heart disease, diabetes and chronic kidney failure being the most common. The probability of overall survival at day 28 was 66%. Multivariable analysis showed older age, coronary artery disease, cancer, low lymphocyte count and high RALE score as factors independently associated with an increased risk of mortality. CONCLUSION: In a large cohort of COVID-19 patients of European origin, main risk factors for mortality were older age, comorbidities, low lymphocyte count and high RALE.

Refractory anti-NMDAR encephalitis successfully treated with bortezomib and associated movements disorders controlled with tramadol: a case report with literature review.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a potentially fatal autoimmune disease, characterized by autoantibody-mediated neurotransmission impairment in multiple brain locations. The course of this condition often comprises altered mental status, autonomic dysfunctions, refractory seizures and hyperkinetic movement disorders. Available disease-modifying therapies include corticosteroids, i.v. immunoglobulins, plasma exchange, rituximab and cyclophosphamide. In a subgroup of patients not responding to B-cell depletion, bortezomib, a proteasome inhibitor, has shown promising evidence of efficacy. The time course of recovery from acute phase may be very slow (weeks/months), and only few data are available in literature about the concurrent management of encephalitis-associated movement disorders. We report a case of severe anti-NMDAR encephalitis in a 29-year-old woman, not responsive to first- and second-line treatments, with persistent involuntary motor manifestations. Starting three months after symptom onset, four cycles of bortezomib have been administered; subsequently we observed a progressive improvement of neurological status. Meanwhile, motor manifestations were controlled after the administration of tramadol, a non-competitive NMDA receptor antagonist.

Fast reshaping of intensive care unit facilities in a large metropolitan hospital in Milan, Italy: facing the COVID-19 pandemic emergency

At the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak spread from China all around the world, causing thousands of deaths. In Italy, the hardest hit region was Lombardy, with the first reported case on 20 February 2020. San Raffaele Scientific Institute ­ a large tertiary hospital and research centre in Milan, Italy ­ was immediately involved in the management of the public health emergency. Since the beginning of the outbreak, the elective surgical activity of the hospital was rapidly reduced and large areas of the hospital were simultaneously reorganised to admit and assist patients with coronavirus disease 2019 (COVID-19). In addition, the hospital became the regional referral hub for cardiovascular emergencies in order to keep ensuring a high level of health care to non-COVID-19 patients in northern Italy. In a few days, a COVID-19 emergency department was created, improving the general ward capacity to a total number of 279 beds dedicated to patients with COVID-19. Moreover, the number of intensive care unit (ICU) beds was increased from 28 to 72 (54 of them dedicated to patients with COVID-19, and 18 to cardiology and cardiac surgery hub emergencies), both converting pre-existing areas and creating new high technology spaces. All the involved health care personnel were rapidly trained to use personal protection equipment and to manage this particular category of patients both in general wards and ICUs. Furthermore, besides clinical activities, continuously important research projects were carried out in order to find new strategies and more effective therapies to better face an unprecedented health emergency in Italy.

Long-Term Survival Rate in Patients With Acute Respiratory Failure Treated With Noninvasive Ventilation in Ordinary Wards.

OBJECTIVE: Noninvasive ventilation is a life-saving technique increasingly used to treat acute respiratory failure. Noninvasive ventilation has been applied mostly in ICUs, but several reasons brought to an increasing application of noninvasive ventilation in ordinary wards. Few articles evaluated the outcomes of patients receiving noninvasive ventilation including long-term follow-up. The aim of the present study was to assess 1-year survival rate of patients treated with noninvasive ventilation outside the ICU for acute respiratory failure of heterogeneous causes and to identify the predictors of long-term mortality. DESIGN: Prospective, observational, pragmatic study. SETTING: Ordinary wards of a teaching hospital. PATIENTS: Consecutive patients treated with noninvasive ventilation for acute respiratory failure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Two-hundred and twenty-patients were enrolled. Mortality rates at 30-day, 90-days, and 1-year follow-up were 20%, 26%, and 34%. When excluding patients with "do-not-resuscitate" status, mortality rates were 13%, 19%, and 28%. The multivariate analyses identified solid cancer, pneumonia in hematologic patients, and do-not-resuscitate status as independent predictors of mortality with postoperative acute respiratory failure associated with improved survival. The same predictors were confirmed when excluding do-not-resuscitate patients from the analyses. CONCLUSIONS: Noninvasive ventilation applied in ordinary wards was effective, with long-term outcomes not different from those reported for ICU settings. Solid cancer, pneumonia in hematologic malignancies, and do-not-resuscitate status predicted mortality, whereas patients with postoperative acute respiratory failure had the best survival rate. Additional studies are required to evaluate noninvasive ventilation efficacy in the wards compared with ICU.

Fulfilling the metabolic requirements for cell proliferation.

The activity of key metabolic enzymes is regulated by the ubiquitin ligases that control the function of the cyclins; therefore the activity of these ubiquitin ligases explains the coordination of cell-cycle progression with the supply of substrates necessary for cell duplication. APC/C (anaphase-promoting complex/cyclosome)-Cdh1, the ubiquitin ligase that controls G(1)- to S-phase transition by targeting specific degradation motifs in cell-cycle proteins, also regulates the glycolysis-promoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) and GLS1 (glutaminase 1), a critical enzyme in glutaminolysis. A decrease in the activity of APC/C-Cdh1 in mid-to-late G(1) releases both proteins, thus explaining the simultaneous increase in the utilization of glucose and glutamine during cell proliferation. This occurs at a time consistent with the point in G(1) that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G(1) to S. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-ß-TrCP (ß-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G(1), whereas the activity of GLS1 remains high throughout S-phase. The differential regulation of the activity of these proteins indicates that a finely-tuned set of mechanisms is activated to fulfil specific metabolic demands at different stages of the cell cycle. These findings have implications for the understanding of cell proliferation in general and, in particular, of cancer, its prevention and treatment.