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Abstract Background Value-based healthcare (VBHC) is an approach that focuses on delivering the highest possible value for patients while driving cost efficiency in health services. It emphasizes improving patient outcomes and experiences while optimizing the use of resources, shifting the healthcare system’s focus from the volume of services to the value delivered. Our study assessed the effectiveness of implementing a VBHC-principled, tailored preoperative evaluation in enhancing patient care and outcomes, as well as reducing healthcare costs. Methods We employed a quality improvement, before-and-after approach to assessing the effects of implementing VBHC strategies on the restructuring of the preoperative evaluation clinics at Humanitas Research Hospital. The intervention introduced a VBHC-tailored risk matrix during the postintervention phase (year 2021), and the results were compared with those of the preintervention phase (2019). The primary study outcome was the difference in the number of preoperative tests and visits at baseline and after the VBHC approach. Secondary outcomes were patient outcomes and costs. Results A total of 9722 patients were included: 5242 during 2019 (baseline) and 4,480 during 2021 (VBHC approach). The median age of the population was 63 (IQR 51–72), 23% of patients were classified as ASA 3 and 4, and 26.8% (2,955 cases) were day surgery cases. We found a considerable decrease in the number of preoperative tests ordered for each patient [6.2 (2.5) vs 5.3 (2.6) tests, p

Abstract Background Blood pressure has become one of the most important vital signs to monitor in the perioperative setting. Recently, the Italian Society of Anesthesia Analgesia Resuscitation and Intensive Care (SIAARTI) recommended, with low level of evidence, continuous monitoring of blood pressure during the intraoperative period. Continuous monitoring allows for early detection of hypotension, which may potentially lead to a timely treatment. Whether the ability to detect more hypotension events by continuous noninvasive blood pressure (C-NiBP) monitoring can improve patient outcomes is still unclear. Here, we report the rationale, study design, and statistical analysis plan of the niMON trial, which aims to evaluate the effect of intraoperative C-NiBP compared with intermittent (I-NiBP) monitoring on postoperative myocardial and renal injury. Methods The niMon trial is an investigator-initiated, multicenter, international, open-label, parallel-group, randomized clinical trial. Eligible patients will be randomized in a 1:1 ratio to receive C-NiBP or I-NiBP as an intraoperative monitoring strategy. The proportion of patients who develop myocardial injury in the first postoperative week is the primary outcome; the secondary outcomes are the proportions of patients who develop postoperative AKI, in-hospital mortality rate, and 30 and 90 postoperative days events. A sample size of 1265 patients will provide a power of 80% to detect a 4% absolute reduction in the rate of the primary outcome. Conclusions The niMON data will provide evidence to guide the choice of the most appropriate intraoperative blood pressure monitoring strategy. Clinical trial registration Clinical Trial Registration: NCT05496322, registered on the 5th of August 2023.

Abstract Background The intraoperative period is often characterized by hemodynamic instability, and intraoperative hypotension is a common complication. The optimal mean arterial pressure (MAP) target in hypertensive patients is still not clear. We hereby describe the protocol and detailed statistical analysis plan for the high versus standard blood pressure target in hypertensive high-risk patients undergoing elective major abdominal surgery: the HISTAP randomized clinical trial. The HISTAP trial aims at addressing whether the use of a higher intraoperative MAP target in high-risk hypertensive surgical patients scheduled for elective abdominal surgery would improve postoperative outcomes, as compared to the standard and recommended perioperative MAP, by using a composite outcome including a 30-day mortality from surgical intervention and at least one major organ dysfunction or new onset of sepsis and septic shock occurring 7 days after surgery. Methods The HISTAP trial is an investigator-initiated, pragmatic, parallel-grouped, randomized, stratified, analyst-blinded trial with adequate allocation sequence generation, and allocation concealment. We will allocate 636 patients to a MAP target ≥ 80 mmHg (treatment group) or to a MAP target ≥65 mmHg (control group). The primary outcome is a composite outcome including a 30-day mortality from the operation and major organ complications. Secondary outcomes are mortality at 30 days, intensive care unit (ICU) length of stay, ICU readmission, Sequential Organ Failure Assessment (SOFA) scores recorded up to postoperative day 7, overall intraoperative fluid balance, vasopressors use, and the need for reoperation. An unadjusted χ 2 test will be used for the primary outcome analysis. A Cox proportional hazards model will be used to adjust the association between the primary outcome and baseline covariates. Conclusions The HISTAP trial results will provide important evidence to guide clinicians’ choice regarding the intraoperative MAP target in high-risk hypertensive patients scheduled for elective abdominal surgery.

Abstract Background Medulloblastoma (MB) is the most common cerebellar malignancy during childhood. Among MB, MYC-amplified Group 3 tumors display the worst prognosis. MYC is an oncogenic transcription factor currently thought to be undruggable. Nevertheless, targeting MYC-dependent processes (i.e. transcription and RNA processing regulation) represents a promising approach. Methods We have tested the sensitivity of MYC-driven Group 3 MB cells to a pool of transcription and splicing inhibitors that display a wide spectrum of targets. Among them, we focus on THZ531, an inhibitor of the transcriptional cyclin-dependent kinases (CDK) 12 and 13. High-throughput RNA-sequencing analyses followed by bioinformatics and functional analyses were carried out to elucidate the molecular mechanism(s) underlying the susceptibility of Group 3 MB to CDK12/13 chemical inhibition. Data from International Cancer Genome Consortium (ICGC) and other public databases were mined to evaluate the functional relevance of the cellular pathway/s affected by the treatment with THZ531 in Group 3 MB patients. Results We found that pharmacological inhibition of CDK12/13 is highly selective for MYC-high Group 3 MB cells with respect to MYC-low MB cells. We identified a subset of genes enriched in functional terms related to the DNA damage response (DDR) that are up-regulated in Group 3 MB and repressed by CDK12/13 inhibition. Accordingly, MYC- and CDK12/13-dependent higher expression of DDR genes in Group 3 MB cells limits the toxic effects of endogenous DNA lesions in these cells. More importantly, chemical inhibition of CDK12/13 impaired the DDR and induced irreparable DNA damage exclusively in MYC-high Group 3 MB cells. The augmented sensitivity of MYC-high MB cells to CDK12/13 inhibition relies on the higher elongation rate of the RNA polymerase II in DDR genes. Lastly, combined treatments with THZ531 and DNA damage-inducing agents synergically suppressed viability of MYC-high Group 3 MB cells. Conclusions Our study demonstrates that CDK12/13 activity represents an exploitable vulnerability in MYC-high Group 3 MB and may pave the ground for new therapeutic approaches for this high-risk brain tumor.

Abstract Background AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. Results We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1 +/− mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. Conclusions By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Abstract Although guidelines provide excellent expert guidance for managing patients with septic shock, they leave room for personalization according to patients’ condition. Hemodynamic monitoring depends on the evolution phase: salvage, optimization, stabilization, and de-escalation. Initially during the salvage phase, monitoring to identify shock etiology and severity should include arterial pressure and lactate measurements together with clinical examination, particularly skin mottling and capillary refill time. Low diastolic blood pressure may trigger vasopressor initiation. At this stage, echocardiography may be useful to identify significant cardiac dysfunction. During the optimization phase, echocardiographic monitoring should be pursued and completed by the assessment of tissue perfusion through central or mixed-venous oxygen saturation, lactate, and carbon dioxide veno-arterial gradient. Transpulmonary thermodilution and the pulmonary artery catheter should be considered in the most severe patients. Fluid therapy also depends on shock phases. While administered liberally during the resuscitation phase, fluid responsiveness should be assessed during the optimization phase. During stabilization, fluid infusion should be minimized. In the de-escalation phase, safe fluid withdrawal could be achieved by ensuring tissue perfusion is preserved. Norepinephrine is recommended as first-line vasopressor therapy, while vasopressin may be preferred in some patients. Essential questions remain regarding optimal vasopressor selection, combination therapy, and the most effective and safest escalation. Serum renin and the angiotensin I/II ratio may identify patients who benefit most from angiotensin II. The optimal therapeutic strategy for shock requiring high-dose vasopressors is scant. In all cases, vasopressor therapy should be individualized, based on clinical evaluation and blood flow measurements to avoid excessive vasoconstriction. Inotropes should be considered in patients with decreased cardiac contractility associated with impaired tissue perfusion. Based on pharmacologic properties, we suggest as the first test a limited dose of dobutamine, to add enoximone or milrinone in the second line and substitute or add levosimendan if inefficient. Regarding adjunctive therapies, while hydrocortisone is nowadays advised in patients receiving high doses of vasopressors, patients responding to corticosteroids may be identified in the future by the analysis of selected cytokines or specific transcriptomic endotypes. To conclude, although some general rules apply for shock management, a personalized approach should be considered for hemodynamic monitoring and support.

Abstract Since the advent of critical care in the twentieth century, the core elements that are the foundation for critical care systems, namely to care for critically ill and injured patients and to save lives, have evolved enormously. The past half-century has seen dramatic advancements in diagnostic, organ support, and treatment modalities in critical care, with further improvements now needed to achieve personalized critical care of the highest quality. For critical care to be even higher quality in the future, advancements in the following areas are key: the physical ICU space; the people that care for critically ill patients; the equipment and technologies; the information systems and data; and the research systems that impact critically ill patients and families. With acutely and critically ill patients and their families as the absolute focal point, advancements across these areas will hopefully transform care and outcomes over the coming years.

Abstract Hemodynamic monitoring is the centerpiece of patient monitoring in acute care settings. Its effectiveness in terms of improved patient outcomes is difficult to quantify. This review focused on effectiveness of monitoring-linked resuscitation strategies from: (1) process-specific monitoring that allows for non-specific prevention of new onset cardiovascular insufficiency (CVI) in perioperative care. Such goal-directed therapy is associated with decreased perioperative complications and length of stay in high-risk surgery patients. (2) Patient-specific personalized resuscitation approaches for CVI. These approaches including dynamic measures to define volume responsiveness and vasomotor tone, limiting less fluid administration and vasopressor duration, reduced length of care. (3) Hemodynamic monitoring to predict future CVI using machine learning approaches. These approaches presently focus on predicting hypotension. Future clinical trials assessing hemodynamic monitoring need to focus on process-specific monitoring based on modifying therapeutic interventions known to improve patient-centered outcomes.

Abstract Background The COVID-19 pandemic presented major challenges for critical care facilities worldwide. Infections which develop alongside or subsequent to viral pneumonitis are a challenge under sporadic and pandemic conditions; however, data have suggested that patterns of these differ between COVID-19 and other viral pneumonitides. This secondary analysis aimed to explore patterns of co-infection and intensive care unit-acquired infections (ICU-AI) and the relationship to use of corticosteroids in a large, international cohort of critically ill COVID-19 patients. Methods This is a multicenter, international, observational study, including adult patients with PCR-confirmed COVID-19 diagnosis admitted to ICUs at the peak of wave one of COVID-19 (February 15th to May 15th, 2020). Data collected included investigator-assessed co-infection at ICU admission, infection acquired in ICU, infection with multi-drug resistant organisms (MDRO) and antibiotic use. Frequencies were compared by Pearson’s Chi-squared and continuous variables by Mann–Whitney U test. Propensity score matching for variables associated with ICU-acquired infection was undertaken using R library MatchIT using the “full” matching method. Results Data were available from 4994 patients. Bacterial co-infection at admission was detected in 716 patients (14%), whilst 85% of patients received antibiotics at that stage. ICU-AI developed in 2715 (54%). The most common ICU-AI was bacterial pneumonia (44% of infections), whilst 9% of patients developed fungal pneumonia; 25% of infections involved MDRO. Patients developing infections in ICU had greater antimicrobial exposure than those without such infections. Incident density (ICU-AI per 1000 ICU days) was in considerable excess of reports from pre-pandemic surveillance. Corticosteroid use was heterogenous between ICUs. In univariate analysis, 58% of patients receiving corticosteroids and 43% of those not receiving steroids developed ICU-AI. Adjusting for potential confounders in the propensity-matched cohort, 71% of patients receiving corticosteroids developed ICU-AI vs 52% of those not receiving corticosteroids. Duration of corticosteroid therapy was also associated with development of ICU-AI and infection with an MDRO. Conclusions In patients with severe COVID-19 in the first wave, co-infection at admission to ICU was relatively rare but antibiotic use was in substantial excess to that indication. ICU-AI were common and were significantly associated with use of corticosteroids. Trial registration ClinicalTrials.gov: NCT04836065 (retrospectively registered April 8th 2021). Graphical abstract

Abstract Background Noninvasive ventilation (NIV) is a promising alternative to invasive mechanical ventilation (IMV) with a particular importance amidst the shortage of intensive care unit (ICU) beds during the COVID-19 pandemic. We aimed to evaluate the use of NIV in Europe and factors associated with outcomes of patients treated with NIV. Methods This is a substudy of COVIP study—an international prospective observational study enrolling patients aged ≥ 70 years with confirmed COVID-19 treated in ICU. We enrolled patients in 156 ICUs across 15 European countries between March 2020 and April 2021.The primary endpoint was 30-day mortality. Results Cohort included 3074 patients, most of whom were male (2197/3074, 71.4%) at the mean age of 75.7 years (SD 4.6). NIV frequency was 25.7% and varied from 1.1 to 62.0% between participating countries. Primary NIV failure, defined as need for endotracheal intubation or death within 30 days since ICU admission, occurred in 470/629 (74.7%) of patients. Factors associated with increased NIV failure risk were higher Sequential Organ Failure Assessment (SOFA) score (OR 3.73, 95% CI 2.36–5.90) and Clinical Frailty Scale (CFS) on admission (OR 1.46, 95% CI 1.06–2.00). Patients initially treated with NIV (n = 630) lived for 1.36 fewer days (95% CI − 2.27 to − 0.46 days) compared to primary IMV group (n = 1876). Conclusions Frequency of NIV use varies across European countries. Higher severity of illness and more severe frailty were associated with a risk of NIV failure among critically ill older adults with COVID-19. Primary IMV was associated with better outcomes than primary NIV. Clinical Trial Registration NCT04321265 , registered 19 March 2020, https://clinicaltrials.gov .

Abstract Introduction Fluid challenges are widely adopted in critically ill patients to reverse haemodynamic instability. We reviewed the literature to appraise fluid challenge characteristics in intensive care unit (ICU) patients receiving haemodynamic monitoring and considered two decades: 2000–2010 and 2011–2021. Methods We assessed research studies and collected data regarding study setting, patient population, fluid challenge characteristics, and monitoring. MEDLINE, Embase, and Cochrane search engines were used. A fluid challenge was defined as an infusion of a definite quantity of fluid (expressed as a volume in mL or ml/kg) in a fixed time (expressed in minutes), whose outcome was defined as a change in predefined haemodynamic variables above a predetermined threshold. Results We included 124 studies, 32 (25.8%) published in 2000–2010 and 92 (74.2%) in 2011–2021, overall enrolling 6,086 patients, who presented sepsis/septic shock in 50.6% of cases. The fluid challenge usually consisted of 500 mL (76.6%) of crystalloids (56.6%) infused with a rate of 25 mL/min. Fluid responsiveness was usually defined by a cardiac output/index (CO/CI) increase ≥ 15% (70.9%). The infusion time was quicker (15 min vs 30 min), and crystalloids were more frequent in the 2011–2021 compared to the 2000–2010 period. Conclusions In the literature, fluid challenges are usually performed by infusing 500 mL of crystalloids bolus in less than 20 min. A positive fluid challenge response, reported in 52% of ICU patients, is generally defined by a CO/CI increase ≥ 15%. Compared to the 2000–2010 decade, in 2011–2021 the infusion time of the fluid challenge was shorter, and crystalloids were more frequently used.

Abstract Background Spinal anesthesia (SA) is widely used for anesthetic management of patients undergoing hip surgery, and hypotension is the most common cardiovascular side effect of SA. This paper aims to assess the lowest effective dose of SA that reduces the occurrence of intraoperative hypotension in elderly patients scheduled for major lower limb orthopedic surgery. Methods We conducted a systematic review of randomized controlled trials (RCTs) performed in elderly patients scheduled for surgical hip repair and a meta-analysis with meta-regression on the occurrence of hypotensive episodes at different effective doses of anesthetics. We searched PUBMED®, EMBASE®, and the Cochrane Controlled Clinical trials registered. Results Our search retrieved 2085 titles, and after screening, 6 were finally included in both the qualitative and quantitative analysis, including 344 patients [15% (10–28) males], with a median (25th to 75th interquartile) age of 82 (80–85). The risk of bias assessment reported “low risk” for 5 (83.3%) and “some concerns” for 1 (16.7%) of the included RCTs. The low dose of SA of [mean 6.5 mg (1.9)] anesthetic was associated with a lower incidence of hypotension [OR = 0.09 (95%CI 0.04–0.21); p = 0.04; I 2 = 56.9%], as compared to the high-dose of anesthetic [mean 10.5 mg (2.4)]. Conclusions In the included studies of this meta-analysis, a mean dose of 6.5 mg of SA was effective in producing intraoperative comfort and motor block and associated with a lower incidence of hypotension as compared to a mean dose of 10.5 mg. Trial registration CRD42020193627

Abstract Background Prone positioning improves survival in moderate-to-severe acute respiratory distress syndrome (ARDS) unrelated to the novel coronavirus disease (COVID-19). This benefit is probably mediated by a decrease in alveolar collapse and hyperinflation and a more homogeneous distribution of lung aeration, with fewer harms from mechanical ventilation. In this preliminary physiological study we aimed to verify whether prone positioning causes analogue changes in lung aeration in COVID-19. A positive result would support prone positioning even in this other population. Methods Fifteen mechanically-ventilated patients with COVID-19 underwent a lung computed tomography in the supine and prone position with a constant positive end-expiratory pressure (PEEP) within three days of endotracheal intubation. Using quantitative analysis, we measured the volume of the non-aerated, poorly-aerated, well-aerated, and over-aerated compartments and the gas-to-tissue ratio of the ten vertical levels of the lung. In addition, we expressed the heterogeneity of lung aeration with the standardized median absolute deviation of the ten vertical gas-to-tissue ratios, with lower values indicating less heterogeneity. Results By the time of the study, PEEP was 12 (10–14) cmH2O and the PaO2:FiO2 107 (84–173) mmHg in the supine position. With prone positioning, the volume of the non-aerated compartment decreased by 82 (26–147) ml, of the poorly-aerated compartment increased by 82 (53–174) ml, of the normally-aerated compartment did not significantly change, and of the over-aerated compartment decreased by 28 (11–186) ml. In eight (53%) patients, the volume of the over-aerated compartment decreased more than the volume of the non-aerated compartment. The gas-to-tissue ratio of the ten vertical levels of the lung decreased by 0.34 (0.25–0.49) ml/g per level in the supine position and by 0.03 (− 0.11 to 0.14) ml/g in the prone position (p

Abstract Background Assessing consciousness in other subjects, particularly in non-verbal and behaviourally disabled subjects (e.g., patients with disorders of consciousness), is notoriously challenging but increasingly urgent. The high rate of misdiagnosis among disorders of consciousness raises the need for new perspectives in order to inspire new technical and clinical approaches. Main body We take as a starting point a recently introduced list of operational indicators of consciousness that facilitates its recognition in challenging cases like non-human animals and Artificial Intelligence to explore their relevance to disorders of consciousness and their potential ethical impact on the diagnosis and healthcare of relevant patients. Indicators of consciousness mean particular capacities that can be deduced from observing the behaviour or cognitive performance of the subject in question (or from neural correlates of such performance) and that do not define a hard threshold in deciding about the presence of consciousness, but can be used to infer a graded measure based on the consistency amongst the different indicators. The indicators of consciousness under consideration offer a potential useful strategy for identifying and assessing residual consciousness in patients with disorders of consciousness, setting the theoretical stage for an operationalization and quantification of relevant brain activity. Conclusions Our heuristic analysis supports the conclusion that the application of the identified indicators of consciousness to its disorders will likely inspire new strategies for assessing three very urgent issues: the misdiagnosis of disorders of consciousness; the need for a gold standard in detecting consciousness and diagnosing its disorders; and the need for a refined taxonomy of disorders of consciousness.

Abstract Background Intensive care unit (ICU) patients age 90 years or older represent a growing subgroup and place a huge financial burden on health care resources despite the benefit being unclear. This leads to ethical problems. The present investigation assessed the differences in outcome between nonagenarian and octogenarian ICU patients. Methods We included 7900 acutely admitted older critically ill patients from two large, multinational studies. The primary outcome was 30-day-mortality, and the secondary outcome was ICU-mortality. Baseline characteristics consisted of frailty assessed by the Clinical Frailty Scale (CFS), ICU-management, and outcomes were compared between octogenarian (80–89.9 years) and nonagenarian (> 90 years) patients. We used multilevel logistic regression to evaluate differences between octogenarians and nonagenarians. Results The nonagenarians were 10% of the entire cohort. They experienced a higher percentage of frailty (58% vs 42%; p

Abstract Background The Clinical Frailty Scale (CFS) is frequently used to measure frailty in critically ill adults. There is wide variation in the approach to analysing the relationship between the CFS score and mortality after admission to the ICU. This study aimed to evaluate the influence of modelling approach on the association between the CFS score and short-term mortality and quantify the prognostic value of frailty in this context. Methods We analysed data from two multicentre prospective cohort studies which enrolled intensive care unit patients ≥ 80 years old in 26 countries. The primary outcome was mortality within 30-days from admission to the ICU. Logistic regression models for both ICU and 30-day mortality included the CFS score as either a categorical, continuous or dichotomous variable and were adjusted for patient’s age, sex, reason for admission to the ICU, and admission Sequential Organ Failure Assessment score. Results The median age in the sample of 7487 consecutive patients was 84 years (IQR 81–87). The highest fraction of new prognostic information from frailty in the context of 30-day mortality was observed when the CFS score was treated as either a categorical variable using all original levels of frailty or a nonlinear continuous variable and was equal to 9% using these modelling approaches (p

Abstract Background Noninvasive respiratory support (NIRS) has been diffusely employed outside the intensive care unit (ICU) to face the high request of ventilatory support due to the massive influx of patients with acute respiratory failure (ARF) caused by coronavirus-19 disease (COVID-19). We sought to summarize the evidence on clinically relevant outcomes in COVID-19 patients supported by NIV outside the ICU. Methods We searched PUBMED®, EMBASE®, and the Cochrane Controlled Clinical trials register, along with medRxiv and bioRxiv repositories for pre-prints, for observational studies and randomized controlled trials, from inception to the end of February 2021. Two authors independently selected the investigations according to the following criteria: (1) observational study or randomized clinical trials enrolling ≥ 50 hospitalized patients undergoing NIRS outside the ICU, (2) laboratory-confirmed COVID-19, and (3) at least the intra-hospital mortality reported. Preferred Reporting Items for Systematic reviews and Meta-analysis guidelines were followed. Data extraction was independently performed by two authors to assess: investigation features, demographics and clinical characteristics, treatments employed, NIRS regulations, and clinical outcomes. Methodological index for nonrandomized studies tool was applied to determine the quality of the enrolled studies. The primary outcome was to assess the overall intra-hospital mortality of patients under NIRS outside the ICU. The secondary outcomes included the proportions intra-hospital mortalities of patients who underwent invasive mechanical ventilation following NIRS failure and of those with ‘do-not-intubate’ (DNI) orders. Results Seventeen investigations (14 peer-reviewed and 3 pre-prints) were included with a low risk of bias and a high heterogeneity, for a total of 3377 patients. The overall intra-hospital mortality of patients receiving NIRS outside the ICU was 36% [30–41%]. 26% [21–30%] of the patients failed NIRS and required intubation, with an intra-hospital mortality rising to 45% [36–54%]. 23% [15–32%] of the patients received DNI orders with an intra-hospital mortality of 72% [65–78%]. Oxygenation on admission was the main source of between-study heterogeneity. Conclusions During COVID-19 outbreak, delivering NIRS outside the ICU revealed as a feasible strategy to cope with the massive demand of ventilatory assistance. Registration PROSPERO, https://www.crd.york.ac.uk/prospero/ , CRD42020224788, December 11, 2020.

Abstract Since the lockdown because of the pandemic, family members have been prohibited from visiting their loved ones in hospital. While it is clearly complicated to implement protocols for the admission of family members, we believe precise strategic goals are essential and operational guidance is needed on how to achieve them. Even during the pandemic, we consider it a priority to share strategies adapted to every local setting to allow family members to enter intensive care units and all the other hospital wards.

Abstract Background Postoperative complications impact on early and long-term patients’ outcome. Appropriate perioperative fluid management is pivotal in this context; however, the most effective perioperative fluid management is still unclear. The enhanced recovery after surgery pathways recommend a perioperative zero-balance, whereas recent findings suggest a more liberal approach could be beneficial. We conducted this trial to address the impact of restrictive vs. liberal fluid approaches on overall postoperative complications and mortality. Methods Systematic review and meta-analysis, including randomised controlled trials (RCTs). We performed a systematic literature search using MEDLINE (via Ovid), EMBASE (via Ovid) and the Cochrane Controlled Clinical trials register databases, published from 1 January 2000 to 31 December 2019. We included RCTs enrolling adult patients undergoing elective abdominal surgery and comparing the use of restrictive/liberal approaches enrolling at least 15 patients in each subgroup. Studies involving cardiac, non-elective surgery, paediatric or obstetric surgeries were excluded. Results After full-text examination, the metanalysis finally included 18 studies and 5567 patients randomised to restrictive (2786 patients; 50.0%) or liberal approaches (2780 patients; 50.0%). We found no difference in the occurrence of severe postoperative complications between restrictive and liberal subgroups [risk difference (95% CI) = 0.009 (− 0.02; 0.04); p value = 0.62; I 2 (95% CI) = 38.6% (0–66.9%)]. This result was confirmed also in the subgroup of five studies having a low overall risk of bias. The liberal approach was associated with lower overall renal major events, as compared to the restrictive [risk difference (95% CI) = 0.06 (0.02–0.09); p value = 0.001]. We found no difference in either early (p value = 0.33) or late (p value = 0.22) postoperative mortality between restrictive and liberal subgroups Conclusions In major abdominal elective surgery perioperative, the choice between liberal or restrictive approach did not affect overall major postoperative complications or mortality. In a subgroup analysis, a liberal as compared to a restrictive perioperative fluid policy was associated with lower overall complication renal major events, as compared to the restrictive. Trial Registration CRD42020218059; Registration: February 2020, https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=218059 .

Abstract Background The COVID-19 pandemic has led highly developed healthcare systems to the brink of collapse due to the large numbers of patients being admitted into hospitals. One of the potential prognostic indicators in patients with COVID-19 is frailty. The degree of frailty could be used to assist both the triage into intensive care, and decisions regarding treatment limitations. Our study sought to determine the interaction of frailty and age in elderly COVID-19 ICU patients. Methods A prospective multicentre study of COVID-19 patients ≥ 70 years admitted to intensive care in 138 ICUs from 28 countries was conducted. The primary endpoint was 30-day mortality. Frailty was assessed using the clinical frailty scale. Additionally, comorbidities, management strategies and treatment limitations were recorded. Results The study included 1346 patients (28% female) with a median age of 75 years (IQR 72–78, range 70–96), 16.3% were older than 80 years, and 21% of the patients were frail. The overall survival at 30 days was 59% (95% CI 56–62), with 66% (63–69) in fit, 53% (47–61) in vulnerable and 41% (35–47) in frail patients (p

Abstract Background Adenylosuccinate lyase deficiency (ADSLD) is an ultrarare neurometabolic recessive disorder caused by loss-of-function mutations in the ADSL gene. The disease is characterized by wide clinical variability. Here we provide an updated clinical profiling of the disorder and discuss genotype–phenotype correlations. Results Data were collected through "Our Journey with ADSL deficiency Association" by using a dedicated web survey filled-in by parents. Clinical and molecular data were collected from 18 patients (12 males, median age 10.9 years ± 7.3), from 13 unrelated families. The age at onset ranged from birth to the first three years (median age 0.63 years ± 0.84 SD), and age at diagnosis varied from 2 months to 17 years, (median age 6.4 years ± 6.1 SD). The first sign was a psychomotor delay in 8/18 patients, epilepsy in 3/18, psychomotor delay and epilepsy in 3/18, and apneas, hypotonia, nystagmus in single cases. One patient (sibling of a previously diagnosed child) had a presymptomatic diagnosis. The diagnosis was made by exome sequencing in 7/18 patients. All patients were definitively diagnosed with ADSL deficiency based on pathogenic variants and/or biochemical assessment. One patient had a fatal neonatal form of ADSL deficiency, seven showed features fitting type I, and nine were characterized by a milder condition (type II), with two showing a very mild phenotype. Eighteen different variants were distributed along the entire ADSL coding sequence and were predicted to have a variable structural impact by impairing proper homotetramerization or catalytic activity of the enzyme. Six variants had not previously been reported. All but two variants were missense. Conclusions The study adds more details on the spectrum of ADSLD patients’ phenotypes and molecular data.

Abstract Background Appropriate perioperative fluid management is of pivotal importance to reduce postoperative complications, which impact on early and long-term patient outcome. The so-called perioperative goal-directed therapy (GDT) approach aims at customizing perioperative fluid management on the individual patients’ hemodynamic response. Whether or not the overall amount of perioperative volume infused in the context of GDT could influence postoperative surgical outcomes is unclear. Methods We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) comparing the efficacy of GDT approach between study population and control group in reducing postoperative complications and perioperative mortality, using MEDLINE, EMBASE and the Cochrane Controlled Clinical trials register. The enrolled studies were grouped considering the amount infused intraoperatively and during the first 24 h after the admission in the critical care unit (perioperative fluid). Results The metanalysis included 21 RCTs enrolling 2729 patients with a median amount of perioperative fluid infusion of 4500 ml. In the studies reporting an overall amount below or above this threshold, the differences in postoperative complications were not statically significant between controls and GDT subgroup [43.4% vs. 34.2%, p value = 0.23 and 54.8% vs. 39.8%; p value = 0.09, respectively]. Overall, GDT reduced the overall rate of postoperative complications, as compared to controls [pooled risk difference (95% CI) = − 0.10 (− 0.14, − 0.07); Chi2 = 30.97; p value

Abstract The current coronavirus pandemic has impacted heavily on ICUs worldwide. Although many hospitals and healthcare systems had plans in place to manage multiple casualties as a result of major natural disasters or accidents, there was insufficient preparation for the sudden, massive influx of severely ill patients with COVID-19. As a result, systems and staff were placed under immense pressure as everyone tried to optimize patient management. As the pandemic continues, we must apply what we have learned about our response, both good and bad, to improve organization and thus patient care in the future.

Abstract Background There is little evidence to support the management of severe COVID-19 patients. Methods To document this variation in practices, we performed an online survey (April 30–May 25, 2020) on behalf of the European Society of Intensive Care Medicine (ESICM). A case vignette was sent to ESICM members. Questions investigated practices for a previously healthy 39-year-old patient presenting with severe hypoxemia from COVID-19 infection. Results A total of 1132 ICU specialists (response rate 20%) from 85 countries (12 regions) responded to the survey. The survey provides information on the heterogeneity in patient’s management, more particularly regarding the timing of ICU admission, the first line oxygenation strategy, optimization of management, and ventilatory settings in case of refractory hypoxemia. Practices related to antibacterial, antiviral, and anti-inflammatory therapies are also investigated. Conclusions There are important practice variations in the management of severe COVID-19 patients, including differences at regional and individual levels. Large outcome studies based on multinational registries are warranted.

Abstract Background Fluid boluses are administered to septic shock patients with the purpose of increasing cardiac output as a means to restore tissue perfusion. Unfortunately, fluid therapy has a narrow therapeutic index, and therefore, several approaches to increase safety have been proposed. Fluid responsiveness (FR) assessment might predict which patients will effectively increase cardiac output after a fluid bolus (FR+), thus preventing potentially harmful fluid administration in non-fluid responsive (FR−) patients. However, there are scarce data on the impact of assessing FR on major outcomes. The recent ANDROMEDA-SHOCK trial included systematic per-protocol assessment of FR. We performed a post hoc analysis of the study dataset with the aim of exploring the relationship between FR status at baseline, attainment of specific targets, and clinically relevant outcomes. Methods ANDROMEDA-SHOCK compared the effect of peripheral perfusion- vs. lactate-targeted resuscitation on 28-day mortality. FR was assessed before each fluid bolus and periodically thereafter. FR+ and FR− subgroups, independent of the original randomization, were compared for fluid administration, achievement of resuscitation targets, vasoactive agents use, and major outcomes such as organ dysfunction and support, length of stay, and 28-day mortality. Results FR could be determined in 348 patients at baseline. Two hundred and forty-two patients (70%) were categorized as fluid responders. Both groups achieved comparable successful resuscitation targets, although non-fluid responders received less resuscitation fluids (0 [0–500] vs. 1500 [1000–2500] mL; p 0.0001), exhibited less positive fluid balances, but received more vasopressor testing. No difference in clinically relevant outcomes between FR+ and FR− patients was found, including 24-h SOFA score (9 [5–12] vs. 8 [5–11], p = 0.4), need for MV (78% vs. 72%, p = 0.16), need for RRT (18% vs. 21%, p = 0.7), ICU-LOS (6 [3–11] vs. 6 [3–16] days, p = 0.2), and 28-day mortality (40% vs. 36%, p = 0.5). Only thirteen patients remained fluid responsive along the intervention period. Conclusions Systematic assessment allowed determination of fluid responsiveness status in more than 80% of patients with early septic shock. Fluid boluses could be stopped in non-fluid responsive patients without any negative impact on clinical relevant outcomes. Our results suggest that fluid resuscitation might be safely guided by FR assessment in septic shock patients. Trial registration ClinicalTrials.gov identifier, NCT03078712. Registered retrospectively on March 13, 2017.

Abstract Background Bedside functional hemodynamic assessment has gained in popularity in the last years to overcome the limitations of static or dynamic indexes in predicting fluid responsiveness. The aim of this systematic review and metanalysis of studies is to investigate the reliability of the functional hemodynamic tests (FHTs) used to assess fluid responsiveness in adult patients in the intensive care unit (ICU) and operating room (OR). Methods MEDLINE, EMBASE, and Cochrane databases were screened for relevant articles using a FHT, with the exception of the passive leg raising. The QUADAS-2 scale was used to assess the risk of bias of the included studies. In-between study heterogeneity was assessed through the I 2 indicator. Bias assessment graphs were plotted, and Egger’s regression analysis was used to evaluate the publication bias. The metanalysis determined the pooled area under the receiving operating characteristic (ROC) curve, sensitivity, specificity, and threshold for two FHTs: the end-expiratory occlusion test (EEOT) and the mini-fluid challenge (FC). Results After text selection, 21 studies met the inclusion criteria, 7 performed in the OR, and 14 in the ICU between 2005 and 2018. The search included 805 patients and 870 FCs with a median (IQR) of 39 (25–50) patients and 41 (30–52) FCs per study. The median fluid responsiveness was 54% (45–59). Ten studies (47.6%) adopted a gray zone analysis of the ROC curve, and a median (IQR) of 20% (15–51) of the enrolled patients was included in the gray zone. The pooled area under the ROC curve for the end-expiratory occlusion test (EEOT) was 0.96 (95%CI 0.92–1.00). The pooled sensitivity and specificity were 0.86 (95%CI 0.74–0.94) and 0.91 (95%CI 0.85–0.95), respectively, with a best threshold of 5% (4.0–8.0%). The pooled area under the ROC curve for the mini-FC was 0.91 (95%CI 0.85–0.97). The pooled sensitivity and specificity were 0.82 (95%CI 0.76–0.88) and 0.83 (95%CI 0.77–0.89), respectively, with a best threshold of 5% (3.0–7.0%). Conclusions The EEOT and the mini-FC reliably predict fluid responsiveness in the ICU and OR. Other FHTs have been tested insofar in heterogeneous clinical settings and, despite promising results, warrant further investigations.

Abstract Background Ethical approval (EA) must be obtained before medical research can start. We describe the differences in EA for an pseudonymous, non-interventional, observational European study. Methods Sixteen European national coordinators (NCs) of the international study on very old intensive care patients answered an online questionnaire concerning their experience getting EA. Results N = 8/16 of the NCs could apply at one single national ethical committee (EC), while the others had to apply to various regional ECs and/or individual hospital institutional research boards (IRBs). The time between applying for EA and the first decision varied between 7 days and 300 days. In 9/16 informed consent from the patient was not deemed necessary; in 7/16 informed consent was required from the patient or relatives. The upload of coded data to a central database required additional information in 14/16. In 4/16 the NCs had to ask separate approval to keep a subject identification code list to de-pseudonymize the patients if questions would occur. Only 2/16 of the NCs agreed that informed consent was necessary for this observational study. Overall, 6/16 of the NCs were satisfied with the entire process and 8/16 were (very) unsatisfied. 11/16 would welcome a European central EC that would judge observational studies for all European countries. Discussion Variations in the process and prolonged time needed to get EA for observational studies hampers inclusion of patients in some European countries. This might have a negative influence on the external validity. Further harmonization of ethical approval process across Europe is welcomed for low-risk observational studies. Conclusion Getting ethical approval for low-risk, non-interventional, observational studies varies enormously across European countries.

Abstract Background Assessment of fluid responsiveness is problematic in intensive care unit (ICU) patients, in particular for those undergoing modes of partial support, such as pressure support ventilation (PSV). We propose a new test, based on application of a ventilator-generated sigh, to predict fluid responsiveness in ICU patients undergoing PSV. Methods This was a prospective bi-centric interventional study conducted in two general ICUs. In 40 critically ill patients with a stable ventilatory PSV pattern and requiring volume expansion (VE), we assessed the variations in arterial systolic pressure (SAP), pulse pressure (PP) and stroke volume index (SVI) consequent to random application of 4-s sighs at three different inspiratory pressures. A radial arterial signal was directed to the MOSTCARE™ pulse contour hemodynamic monitoring system for hemodynamic measurements. Data obtained during sigh tests were recorded beat by beat, while all the hemodynamic parameters were averaged over 30 s for the remaining period of the study protocol. VE consisted of 500 mL of crystalloids over 10 min. A patient was considered a responder if a VE-induced increase in cardiac index (CI) ≥ 15% was observed. Results The slopes for SAP, SVI and PP of were all significantly different between responders and non-responders (p

Abstract Background Maximal left ventricular (LV) pressure rise (LV dP/dtmax), a classical marker of LV systolic function, requires LV catheterization, thus surrogate arterial pressure waveform measures have been proposed. We compared LV and arterial (femoral and radial) dP/dtmax to the slope of the LV end-systolic pressure-volume relationship (Ees), a load-independent measure of LV contractility, to determine the interactions between dP/dtmax and Ees as loading and LV contractility varied. Methods We measured LV pressure-volume data using a conductance catheter and femoral and radial arterial pressures using a fluid-filled catheter in 10 anesthetized pigs. Ees was calculated as the slope of the end-systolic pressure-volume relationship during a transient inferior vena cava occlusion. Afterload was assessed by the effective arterial elastance. The experimental protocol consisted of sequentially changing afterload (phenylephrine/nitroprusside), preload (bleeding/fluid bolus), and contractility (esmolol/dobutamine). A linear-mixed analysis was used to assess the contribution of cardiac (Ees, end-diastolic volume, effective arterial elastance, heart rate, preload-dependency) and arterial factors (total vascular resistance and arterial compliance) to LV and arterial dP/dtmax. Results Both LV and arterial dP/dtmax allowed the tracking of Ees changes, especially during afterload and contractility changes, although arterial dP/dtmax was lower compared to LV dP/dtmax (bias 732 ± 539 mmHg⋅s− 1 for femoral dP/dtmax, and 625 ± 501 mmHg⋅s− 1 for radial dP/dtmax). Changes in cardiac contractility (Ees) were the main determinant of LV and arterial dP/dtmax changes. Conclusion Although arterial dP/dtmax is a complex function of central and peripheral arterial factors, radial and particularly femoral dP/dtmax allowed reasonably good tracking of LV contractility changes as loading and inotropic conditions varied.

Abstract Background Sepsis-induced myocardial dysfunction is associated with poor outcomes, but traditional measurements of systolic function such as left ventricular ejection fraction (LVEF) do not directly correlate with prognosis. Global longitudinal strain (GLS) utilizing speckle-tracking echocardiography (STE) could be a better marker of intrinsic left ventricular (LV) function, reflecting myocardial deformation rather than displacement and volume changes. We sought to investigate the prognostic value of GLS in patients with sepsis and/or septic shock. Methods We conducted a systematic review (PubMed and Embase up to 26 October 2017) and meta-analysis to investigate the association between GLS and mortality at longest follow up in patients with severe sepsis and/or septic shock. In the primary analysis, we included studies reporting transthoracic echocardiography data on GLS according to mortality. A secondary analysis evaluated the association between LVEF and mortality including data from studies reporting GLS. Results We included eight studies in the primary analysis with a total of 794 patients (survival 68%, n = 540). We found a significant association between worse LV function and GLS values and mortality: standard mean difference (SMD) − 0.26; 95% confidence interval (CI) − 0.47, − 0.04; p = 0.02 (low heterogeneity, I 2 = 43%). No significant association was found between LVEF and mortality in the same population of patients (eight studies; SMD, 0.02; 95% CI − 0.14, 0.17; p = 0.83; no heterogeneity, I 2 = 3%). Conclusions Worse GLS (less negative) values are associated with higher mortality in patients with severe sepsis or septic shock, while such association is not valid for LVEF. More critical care research is warranted to confirm the better ability of STE in demonstrating underlying intrinsic myocardial disease compared to LVEF.

Abstract This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2018. Other selected articles can be found online at https://www.biomedcentral.com/collections/annualupdate2018. Further information about the Annual Update in Intensive Care and Emergency Medicine is available from http://www.springer.com/series/8901.

Abstract Background Intensive care medicine is a relatively young discipline that has rapidly grown into a full-fledged medical subspecialty. Intensivists are responsible for managing an ever-increasing number of patients with complex, life-threatening diseases. Several factors may influence their performance, including age, training, experience, workload, and socioeconomic context. The aim of this study was to examine individual- and work-related aspects of the Latin American intensivist workforce, mainly with academic appointments, which might influence the quality of care provided. In consequence, we conducted a cross-sectional study of intensivists at public and private academic and nonacademic Latin American intensive care units (ICUs) through a web-based electronic survey submitted by email. Questions about personal aspects, work-related topics, and general clinical workflow were incorporated. Results Our study comprised 735 survey respondents (53% return rate) with the following country-specific breakdown: Brazil (29%); Argentina (19%); Chile (17%); Uruguay (12%); Ecuador (9%); Mexico (7%); Colombia (5%); and Bolivia, Peru, Guatemala, and Paraguay combined (2%). Latin American intensivists were predominantly male (68%) young adults (median age, 40 [IQR, 35–48] years) with a median clinical ICU experience of 10 (IQR, 5–20) years. The median weekly workload was 60 (IQR, 47–70) h. ICU formal training was between 2 and 4 years. Only 63% of academic ICUs performed multidisciplinary rounds. Most intensivists (85%) reported adequate conditions to manage patients with septic shock in their units. Unsatisfactory conditions were attributed to insufficient technology (11%), laboratory support (5%), imaging resources (5%), and drug shortages (5%). Seventy percent of intensivists participated in research, and 54% read scientific studies regularly, whereas 32% read no more than one scientific study per month. Research grants and pharmaceutical sponsorship are unusual funding sources in Latin America. Although Latin American intensivists are mostly unsatisfied with their income (81%), only a minority (27%) considered changing to another specialty before retirement. Conclusions Latin American intensivists constitute a predominantly young adult workforce, mostly formally trained, have a high workload, and most are interested in research. They are under important limitations owing to resource constraints and overt dissatisfaction. Latin America may be representative of other world areas with similar challenges for intensivists. Specific initiatives aimed at addressing these situations need to be devised to improve the quality of critical care delivery in Latin America.

Abstract Background The fluid challenge is considered the gold standard for diagnosis of fluid responsiveness. The objective of this study was to describe the fluid challenge techniques reported in fluid responsiveness studies and to assess the difference in the proportion of ‘responders,’ (PR) depending on the type of fluid, volume, duration of infusion and timing of assessment. Methods Searches of MEDLINE and Embase were performed for studies using the fluid challenge as a test of cardiac preload with a description of the technique, a reported definition of fluid responsiveness and PR. The primary outcome was the mean PR, depending on volume of fluid, type of fluids, rate of infusion and time of assessment. Results A total of 85 studies (3601 patients) were included in the analysis. The PR were 54.4% (95% CI 46.9–62.7) where 500 ml was administered (p = 0.71). The PR was not affected by type of fluid. The PR was similar among patients administered a fluid challenge for 10 minutes after the fluid challenge. The proportions of responders were 53.9%, 57.7% and 52.3%, respectively (p = 0.47). Conclusions The PR decreases with a long infusion time. A standard technique for fluid challenge is desirable.

Abstract Background Adenylosuccinate lyase (ADSL) deficiency is a defect of purine metabolism affecting purinosome assembly and reducing metabolite fluxes through purine de novo synthesis and purine nucleotide recycling pathways. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The most severe form is characterized by neonatal encephalopathy, absence of spontaneous movement, respiratory failure, intractable seizures, and early death within the first weeks of life. More commonly, ADSL presents purely neurologic clinical picture characterized by severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features (type I) or a more slowly progressing form with later onset, and major features including slight to moderate psychomotor retardation, and transient contact disturbances (type II). Diagnostic markers are the presence of succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (SAdo) in extracellular fluids. ADSL is a rare disorder, although its prevalence remains unknown. Of note, the wide range of essentially nonspecific manifestations and lack of awareness of the condition often prevent diagnosis. Case presentation We present here the case of particularly mild, late onset ADSL that has been unsuccessfully investigated until whole exome sequencing (WES) was performed. Conclusions Besides emphasizing the valuable diagnostic value of WES, this report provides new data further documenting the relatively wide clinical manifestation of ADSL.

Abstract Background Chronic cerebrospinal venous insufficiency (CCSVI) has recently been reported to be associated with multiple sclerosis (MS). However, its actual prevalence, possible association with specific MS phenotypes, and potential pathophysiological role are debated. Method We analysed the clinical data of 710 MS patients attending six centres (five Italian and one Canadian). All were submitted to venous Doppler sonography and diagnosed as having or not having CCSVI according to the criteria of Zamboni et al. Results Overall, CCSVI was diagnosed in 86% of the patients, but the frequency varied greatly between the centres. Even greater differences were found when considering singly the five diagnostic criteria proposed by Zamboni et al. Despite these differences, significant associations with clinical data were found, the most striking being age at disease onset (about five years greater in CCSVI-positive patients) and clinical severity (mean EDSS score about one point higher in CCSVI-positive patients). Patients with progressive MS were more likely to have CCSVI than those with relapsing-remitting MS. Conclusion The methods for diagnosing CCSVI need to be refined, as the between-centre differences, particularly in single criteria, were excessively high. Despite these discrepancies, the strong associations between CCSVI and MS phenotype suggest that the presence of CCSVI may favour a later development of MS in patients with a lower susceptibility to autoimmune diseases and may increase its severity.

Abstract Background In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles. Methods The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH. Results The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Conclusions It is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.

Abstract Background In 1997 Vagus Nerve Stimulation (VNS) received approval from the US Food and Drug Administration (FDA) as an adjunctive therapy in the treatment of medically intractable partial epilepsy in people aged 12 years and older who are ineligible for resective epilepsy surgery. Although the exact mechanisms of action are unknown, the use of VNS with children has increased, including those younger than 12 years of age, or those with generalized epilepsy. Methods We describe the outcome for the first group of nine patients, aged 8-28 years, who had pharmaco-resistant epilepsy and were treated with VNS. During the follow up, we gradually and slowly increased the parameters of the stimulation in order to assess the efficacy of VNS even at parameters which would usually be considered "non-therapeutic", along with possible side effects and changes in quality of life. Results At the last follow, up 1 patient was "seizures free", 3 were "very good responders", 3 were "good responders" and 2 were "non responders". We obtained an initial seizure reduction with low stimulation parameters, the highest current reached being 2.00 mA. This observation supports the possibility that, for younger patients, lower stimulation intensities than those commonly used in clinical practice for adults can be therapeutic. We also wanted to underline the reduction in seizure frequency (~91,7%) and the reduction in seizure duration (> 50%) in the patients affected by drug-resistant absence epilepsy. Adverse effects were mild, tolerable and, in most of cases, easily resolved by adjusting the stimulation parameters. Hoarseness of voice was the most frequent side effect. The improvements in the quality of life are relevant and seem to be independent of the VNS effect in controlling seizures. Conclusions Our small experience seems to confirm the efficacy and safety of VNS in drug resistant partial and generalized epilepsy in developing age groups.

Abstract Background Intermittent measurement of cardiac output may be performed using a lithium dilution technique (LiDCO). This can then be used to calibrate a pulse power algorithm of the arterial waveform which provides a continuous estimate of this variable. The purpose of this study was to examine the duration of accuracy of the pulse power algorithm in critically ill patients with respect to time when compared to measurements of cardiac output by an independent technique. Methods Pulse power analysis was performed on critically ill patients using a proprietary commercial monitor (PulseCO). All measurements were made using an in-dwelling radial artery line and according to manufacturers instructions. Intermittent measurements of cardiac output were made with LiDCO in order to validate the pulse power measurements. These were made at baseline and then following 1, 2, 4 and 8 hours. The LiDCO measurement was considered the reference for comparison in this study. The two methods of measuring cardiac output were then compared by linear regression and a Bland Altman analysis. An error rate for the limits of agreement (LOA) between the two techniques of less than 30% was defined as being acceptable for this study. Results 14 critically ill medical and surgical patients were enrolled over a three month period. At baseline patients showed a wide range of cardiac output (median 7.5 L/min, IQR 5.1 -9.0 L/min). The bias and limits of agreement between the two techniques was deemed acceptable for the first four hours of the study with percentage errors being 29%, 22%, and 285 respectively. The percentage error at eight hours following calibration increased to 36%. The ability of the PulseCo to detect changes in cardiac output was assessed with a similar analysis. The PulseCO tracked the changes in cardiac output with adequate accuracy for the first four hours with percentage errors being 20%, 24% and 25%. However at eight hours the error had increased to 43%. Conclusion The agreement between lithium dilution cardiac output and the pulse power algorithm in the PulseCO monitor remains acceptable for up to four hours in critically ill patients.

Abstract Background MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cell responses at single cell level. The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population. Results We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig) with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir), both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR. Conclusion It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent.