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In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.

The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1High, likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output.

STUDY QUESTION What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsies were obtained from 15 heart beating organ donors (age range: 19–85 years) and 24 patients (age range: 19–45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE Increasing age was negatively associated with Leydig cell number (R = −0.49; P LIMITATIONS, REASONS FOR CAUTION In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Background. The increase of the optic nerve sheath diameter (ONSD) is a reliable, noninvasive sonographic marker of intracranial hypertension. Aim of the study was to demonstrate the efficacy of ONSD evaluation, when monitoring neurocritical patients, to early identify malignant intracranial hypertension in patients with brain death (BD).Methods. Data from ultrasound ONSD evaluation have been retrospectively analyzed in 21 sedated critical patients with neurological diseases who, during their clinical course, developed BD. 31 nonneurological controls were used for standard ONSD reference.Results. Patients with neurological diseases, before BD, showed higher ONSD values than control group (CTRL: RT0.45±0.03 cm; LT0.45±0.02 cm; pre-BD: RT0.54±0.02 cm; LT0.55±0.02 cm;p<0.000) even without intracranial hypertension, evaluated with invasive monitoring. ONSD was further significantly markedly increased in respect to the pre-BD evaluation in neurocritical patients after BD, with mean values above 0.7 cm (RT0.7±0.02 cm; LT0.71±0.02 cm;p<0.000), with a corresponding dramatic raise in intracranial pressure. Logistic regression analysis showed a strong correlation between ONSD and ICP (R0,895,p<0.001).Conclusions. ONSD is a reliable marker of intracranial hypertension, easy to be performed with a minimal training. Routine ONSD daily monitoring could be of help in Intensive Care Units when invasive intracranial pressure monitoring is not available, to early recognize intracranial hypertension and to suspect BD in neurocritical patients.

To date, in the human seminiferous epithelium, only six associations of cell types have been distinguished, subdividing the epithelial cycle into six stages of very different duration. This hampers comparisons between studies on human and laboratory animals in which the cycle is usually subdivided into 12 stages. We now propose a new stage classification on basis of acrosomal development made visible by immunohistochemistry (IHC) for (pro)acrosin. IHC for acrosin gives results that are comparable to periodic acid Schiff staining. In the human too, we now distinguish 12 stages that differ from each other in duration by a factor of two at most. B spermatogonia are first apparent in stage I, preleptotene spermatocytes are formed in stage V, leptonema starts in stage VII, and spermiation takes place at the end of stage VI. A similar timing was previously observed in several monkeys. Stage identification by way of IHC for acrosin appeared possible for tissue fixed in formalin, Bouin fixative, diluted Bouin fixative, Cleland fluid, and modified Davidson fixative, indicating a wide applicability. In addition, it is also possible to distinguish the 12 stages in glutaraldehyde/osmium-tetroxide fixed/plastic embedded testis material without IHC for acrosin. The new stage classification will greatly facilitate research on human spermatogenesis and enable a much better comparison with results from work on experimental animals than hitherto possible. In addition, it will enable a highly focused approach to evaluate spermatogenic impairments, such as germ cell maturation arrests or defects, and to study details of germ cell differentiation.

To assess the feasibility of remifentanil-based sedation in hypoxemic acute respiratory failure (HARF) patients refusing to continue noninvasive ventilation (NPPV) for intolerance to two different interfaces-helmet and total face mask.Prospective uncontrolled clinical investigation in a 14-bed ICU of an university hospital in Italy.Thirty-six patients with persistent severe HARF who complained of discomfort and asked for interruption of NPPV session.Patients started sedation with remifentanil (0.025 μg kg(-1) min(-1)) and the infusion rate was increased by 0.01 μg kg(-1) min(-1) every minute to a maximum of 0.12 μg kg(-1) min(-1) to obtain patient comfort.Twenty-two out of 36 patients (61%) with median (IQR) SAPS II score of 32 (30, 38) continued the NPPV treatment after the introduction of remifentanil infusion. In this success group, median (IQR) respiratory rate decreased from 34 (31, 37) to 24 (20, 26) min(-1) (p0.0001) and PaO(2)/FiO(2) ratio increased from 156 (144, 176) to 270 (210, 300) mmHg (p0.0001) after 1 h of NPPV with remifentanil-analgosedation either with helmet or total face mask. Fourteen patients failed to continue the noninvasive treatment and were intubated after a mean of 2.5 ± 2.3 h; they showed a respiratory rate decrease from 35 (30, 38) to 27 (25, 35) min(-1) (p = 0.02) and an inability to increase the PaO(2)/FiO(2) ratio above 180 mmHg. The ICU mortality in the failure group patients was 50 versus 14% in the NPPV success group (p0.05). The mean remifentanil dose administered was 0.07 ± 0.03 μg kg(-1) min(-1).This clinical study suggests that a remifentanil-based sedation protocol can decrease the rate of failure in patients with intolerance to NPPV.

Objective: Transcranial Doppler (TCD) is a sensitive technique for circulatory arrest diagnosis in brain death when patterns such as reverberant flow and short systolic spikes are observed. In infants, the nonossified fontanelles compensate for intracranial hypertension. We describe TCD patterns in infants with brain death, different from adults, with the hemodynamic modifications induced by anterior fontanelle compression. Method: TCD was performed in 2 infants with diagnosed brain death admitted to the neonatal intensive care unit. Results: TCD showed a large peak ‘reverberant’ flow, with a high peak systolic velocity and a consistent retrograde component, away from the brain. Compression of the anterior fontanelle induced, at first, a reduction in systolic flow with the subsequent appearance of the characteristic short systolic spikes. Upon compression removal, a brief increase in the systolic flow was observed before the prompt reappearance of the reverberant flow. Conclusion: TCD for brain death diagnosis should be done cautiously in infants. In these cases, reverberating flow may be indicative of circulatory arrest even if with a large peak and with a high peak systolic velocity. Heavy fontanelle compression may reproduce the classical adult TCD patterns of brain death, thus supporting the diagnosis of cerebral circulatory arrest.

Objective. We conducted a randomized prospective study comparing noninvasive positive pressure ventilation (NPPV) with conventional mechanical ventilation via endotracheal intubation (ETI) in a group of patients with chronic obstructive pulmonary disease who failed standard medical treatment in the emergency ward after initial improvement and met predetermined criteria for ventilatory support. Design and setting. Prospective randomized study in a university hospital 13-bed general ICU. Patients. Forty-nine patients were randomly assigned to receive NPPV (n=23) or conventional ventilation (n=26). Results. both NPPV and conventional ventilation significantly improved gas exchanges. The two groups had similar length of ICU stay, number of days on mechanical ventilation, overall complications, ICU mortality, and hospital mortality. In the NPPV group 11 (48%) patients avoided intubation, survived, and had a shorter duration of ICU stay than intubated patients. One year following hospital discharge the NPPV group had fewer patients readmitted to the hospital (65% vs. 100%) or requiring de novo permanent oxygen supplementation (0% vs. 36%). Conclusions. The use of NPPV in patients with chronic obstructive pulmonary disease and acute respiratory failure requiring ventilatory support after failure of medical treatment avoided ETI in 48% of the patients, had the same ICU mortality as conventional treatment and, at 1-year follow-up was associated with fewer patients readmitted to the hospital or requiring for long-term oxygen supplementation. An editorial regarding this article can be found in the same issue (http://dx.doi.org/10.1007/s00134-002-1503-3).

2-deoxy-5-fluorouridine, also known as floxuridine (FUdr) is a fluoropyridine antimetabolite, used in the treatment of metastatic renal cell carcinoma. We report the first case of lesional pulmonary oedema developed after receiving Fudr, recently treated in our unit. The patient refused endotracheal intubation, and was successfully treated associating noninvasive ventilation (NIV) with full-face mask to steroid treatment. The authors conclude that mechanical ventilation via face-mask can be an effective, comfortable, dignified method of support for patients with end-stage disease and acute respiratory failure.