Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

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

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC

: The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC

Effect of a Resuscitation Strategy Targeting Peripheral Perfusion Status vs Serum Lactate Levels on 28-Day Mortality Among Patients With Septic Shock : The ANDROMEDA-SHOCK Randomized Clinical Trial

IMPORTANCE: Abnormal peripheral perfusion after septic shock resuscitation has been associated with organ dysfunction and mortality. The potential role of the clinical assessment of peripheral perfusion as a target during resuscitation in early septic shock has not been established. OBJECTIVE: To determine if a peripheral perfusion–targeted resuscitation during early septic shock in adults is more effective than a lactate level–targeted resuscitation for reducing mortality. DESIGN, SETTING, AND PARTICIPANTS Multicenter, randomized trial conducted at 28 intensive care units in 5 countries. Four-hundred twenty-four patients with septic shock were included between March 2017 and March 2018. The last date of follow-up was June 12, 2018. INTERVENTIONS: Patients were randomized to a step-by-step resuscitation protocol aimed at either normalizing capillary refill time (n = 212) or normalizing or decreasing lactate levels at rates greater than 20% per 2 hours (n = 212), during an 8-hour intervention period. MAIN OUTCOMES AND MEASURES The primary outcome was all-cause mortality at 28 days. Secondary outcomes were organ dysfunction at 72 hours after randomization, as assessed by Sequential Organ Failure Assessment (SOFA) score (range, 0 [best] to 24 [worst]); death within 90 days; mechanical ventilation–, renal replacement therapy–, and vasopressor-free days within 28 days; intensive care unit and hospital length of stay. RESULTS: Among 424 patients randomized (mean age, 63 years; 226 [53%] women), 416 (98%) completed the trial. By day 28, 74 patients (34.9%) in the peripheral perfusion group and 92 patients (43.4%) in the lactate group had died (hazard ratio, 0.75 [95% CI, 0.55 to 1.02]; P = .06; risk difference, −8.5% [95% CI, −18.2% to 1.2%]). Peripheral perfusion–targeted resuscitation was associated with less organ dysfunction at 72 hours (mean SOFA score, 5.6 [SD, 4.3] vs 6.6 [SD, 4.7]; mean difference, −1.00 [95% CI, −1.97 to −0.02]; P = .045). There were no significant differences in the other 6 secondary outcomes. No protocol-related serious adverse reactions were confirmed. CONCLUSIONS AND RELEVANCE: Among patients with septic shock, a resuscitation strategy targeting normalization of capillary refill time, compared with a strategy targeting serum lactate levels, did not reduce all-cause 28-day mortality.Facultad de Ciencias Médica

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

Early mobilisation in critically ill COVID-19 patients: a subanalysis of the ESICM-initiated UNITE-COVID observational study

Background Early mobilisation (EM) is an intervention that may improve the outcome of critically ill patients. There is limited data on EM in COVID-19 patients and its use during the first pandemic wave. Methods This is a pre-planned subanalysis of the ESICM UNITE-COVID, an international multicenter observational study involving critically ill COVID-19 patients in the ICU between February 15th and May 15th, 2020. We analysed variables associated with the initiation of EM (within 72 h of ICU admission) and explored the impact of EM on mortality, ICU and hospital length of stay, as well as discharge location. Statistical analyses were done using (generalised) linear mixed-effect models and ANOVAs. Results Mobilisation data from 4190 patients from 280 ICUs in 45 countries were analysed. 1114 (26.6%) of these patients received mobilisation within 72 h after ICU admission; 3076 (73.4%) did not. In our analysis of factors associated with EM, mechanical ventilation at admission (OR 0.29; 95% CI 0.25, 0.35; p = 0.001), higher age (OR 0.99; 95% CI 0.98, 1.00; p ≤ 0.001), pre-existing asthma (OR 0.84; 95% CI 0.73, 0.98; p = 0.028), and pre-existing kidney disease (OR 0.84; 95% CI 0.71, 0.99; p = 0.036) were negatively associated with the initiation of EM. EM was associated with a higher chance of being discharged home (OR 1.31; 95% CI 1.08, 1.58; p = 0.007) but was not associated with length of stay in ICU (adj. difference 0.91 days; 95% CI − 0.47, 1.37, p = 0.34) and hospital (adj. difference 1.4 days; 95% CI − 0.62, 2.35, p = 0.24) or mortality (OR 0.88; 95% CI 0.7, 1.09, p = 0.24) when adjusted for covariates. Conclusions Our findings demonstrate that a quarter of COVID-19 patients received EM. There was no association found between EM in COVID-19 patients' ICU and hospital length of stay or mortality. However, EM in COVID-19 patients was associated with increased odds of being discharged home rather than to a care facility. Trial registration ClinicalTrials.gov: NCT04836065 (retrospectively registered April 8th 2021)

PACAP in the reproductive system

The production of a mature oocyte that is capable of being fertilized and undergoing embryo development is the major function of the female reproductive system. This process depends on the close association between germ cells and somatic cells, i.e., granulosa and theca cells. The growth of the mammalian oocyte is finely regulated by the coordinate expression of autocrine and paracrine factors. The initial phases of folliculogenesis are independent from gonadotropins, but at later stages, it is strictly dependent on follicle-stimulating hormone (FSH) and luteinizing hormone (LH). However, increasing amounts of paracrine/autocrine growth factors participate in the modulation of gonadotropin effects. Pituitary Adenylate Cyclase-Activating Peptide (PACAP) is an extremely conserved regulatory peptide that was initially found in the central nervous system. However, PACAP has been found together with its receptors in many organs, tissues and cell types, including lung, testis, adrenal gland and ovary. In the ovary, PACAP expression has been found mainly in preovulatory follicles after the LH surge. However, PACAP is constantly expressed through the ovarian cycle in the nerve fibres in the hilus region and in the interstitial tissue in the proximity of the primary follicles. In the ovary, PACAP promotes steroidogenesis and cAMP production, reduces the rate of apoptosis in granulosa cells, and accelerates oocyte meiotic maturation

WNT ligands control initiation and progression of human papillomavirus-driven squamous cell carcinoma

Human papillomavirus (HPV)-driven cutaneous squamous cell carcinoma (cSCC) is the most common cancer in immunosuppressed patients. Despite indications suggesting that HPV promotes genomic instability during cSCC development, the molecular pathways underpinning HPV-driven cSCC development remain unknown. We compared the transcriptome of HPV-driven mouse cSCC with normal skin and observed higher amounts of transcripts for Porcupine and WNT ligands in cSCC, suggesting a role for WNT signaling in cSCC progression. We confirmed increased Porcupine expression in human cSCC samples. Blocking the secretion of WNT ligands by the Porcupine inhibitor LGK974 significantly diminished initiation and progression of HPV-driven cSCC. Administration of LGK974 to mice with established cSCC resulted in differentiation of cancer cells and significant reduction of the cancer stem cell compartment. Thus, WNT/β-catenin signaling is essential for HPV-driven cSCC initiation and progression as well as for maintaining the cancer stem cell niche. Interference with WNT secretion may thus represent a promising approach for therapeutic intervention

Zoonotic tick-borne bacteria among wild boars (Sus scrofa) in Central Italy

The objective of this investigation was to estimate the occurrence of infections by the three zoonotic bacteria Anaplasma phagocytophilum (A. phagocytophilum), Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) and Coxiella burnetii in wild boars (Sus scrofa) in Central Italy. The spleen samples from 100 hunted wild boars were submitted to DNA extraction and PCR assays were carried out to detect the three agents. One (1%) animal was positive for A. phagocytophilum, and three (3%) for B. burgdorferi s.l. No positive reactions were observed for Coxiella burnetii. Wild boars did not seem to play an important role in the epidemiology of the three investigated agents. However, the detection of A. phagocytophilum and B. burgdorferi s.l. in the spleen of the tested animals showed that wild boars can harbor these pathogens, thus ticked that feeding on infected wild boars are likely to become infected, too, which represents a source of infection for other animals and humans. This is the first detection of A. phagocytophilum in wild boars in Italy

Monocytes promote UV‐induced epidermal carcinogenesis

Mononuclear phagocytes consisting of monocytes, macrophages, and DCs play a complex role in tumor development by either promoting or restricting tumor growth. Cutaneous squamous cell carcinoma (cSCC) is the second most common nonmelanoma skin cancer arising from transformed epidermal keratinocytes. While present at high numbers, the role of tumor-infiltrating and resident myeloid cells in the formation of cSCC is largely unknown. Using transgenic mice and depleting antibodies to eliminate specific myeloid cell types in the skin, we investigated the involvement of mononuclear phagocytes in the development of UV-induced cSCC in K14-HPV8-E6 transgenic mice. Although resident Langerhans cells were enriched in the tumor, their contribution to tumor formation was negligible. Equally, dermal macrophages were dispensable for the development of cSCC. In contrast, mice lacking circulating monocytes were completely resistant to UV-induced cSCC, indicating that monocytes promote tumor development. Collectively, these results demonstrate a critical role for classical monocytes in the initiation of skin cancer