True-T - Improving T-cell response quantification with holistic artificial intelligence based prediction in immunohistochemistry images.

The immune response associated with oncogenesis and potential oncological ther- apeutic interventions has dominated the field of cancer research over the last decade. T-cell lymphocytes in the tumor microenvironment are a crucial aspect of cancer's adaptive immunity, and the quantification of T-cells in specific can- cer types has been suggested as a potential diagnostic aid. However, this is cur- rently not part of routine diagnostics. To address this challenge, we present a new method called True-T, which employs artificial intelligence-based techniques to quantify T-cells in colorectal cancer (CRC) using immunohistochemistry (IHC) images. True-T analyses the chromogenic tissue hybridization signal of three widely recognized T-cell markers (CD3, CD4, and CD8). Our method employs a pipeline consisting of three stages: T-cell segmentation, density estimation from the segmented mask, and prediction of individual five-year survival rates. In the first stage, we utilize the U-Net method, where a pre-trained ResNet-34 is em- ployed as an encoder to extract clinically relevant T-cell features. The segmenta- tion model is trained and evaluated individually, demonstrating its generalization in detecting the CD3, CD4, and CD8 biomarkers in IHC images. In the second stage, the density of T-cells is estimated using the predicted mask, which serves as a crucial indicator for patient survival statistics in the third stage. This ap- proach was developed and tested in 1041 patients from four reference diagnostic institutions, ensuring broad applicability. The clinical effectiveness of True-T is demonstrated in stages II-IV CRC by offering valuable prognostic information that surpasses previous quantitative gold standards, opening possibilities for po- tential clinical applications. Finally, to evaluate the robustness and broader ap- plicability of our approach without additional training, we assessed the universal accuracy of the CD3 component of the True-T algorithm across 13 distinct solid tumors.

Nursing students and COVID-19 vaccination. ESitA Study An Observational Study.

BACKGROUND AND AIM: Vaccine hesitancy is an important problem in terms of health policy. This historical moment leads us to wonder if vaccine hesitancy is also present among nursing students who should be particularly sensitive to the subject Methods: Between February 10 to February 17 2021, 1080 students enrolled in the Bachelor of Science in Nursing in the Department of Medicine and Surgery of the University of Perugia, were invited to reply to an online questionnaire sent to their university email accounts. RESULTS AND CONCLUSIONS: A certain amount of vaccination hesitancy was detected among the students in our study. It can be assumed that the issues surrounding the AstraZeneca vaccine, which occurred at the start of the vaccination campaign, may have led to an increase in people's hesitancy. Boosting vaccination campaigns, including appropriate use of social media, may lead to greater acceptance. Also, it would be useful to assess the cultural basis of the recent anti-Vax controversy, particularly for students of nursing or other health professions, who should be able to evaluate, source and recognize the most validated data.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

The cellular and molecular origins of extracellular vesicles released by the helminth pathogen, Fasciola hepatica.

Parasitic helminths secrete extracellular vesicles (EVs) which have potent immunomodulatory effects. Whilst the cargo of EVs has been characterised for many species, we know little about the mechanisms that govern their biogenesis and release. Using antibodies raised against a panel of Fasciola hepatica EV (FhEV) marker proteins, we have identified multiple sites of EV production in the parasite. Discrete immunofluorescence patterns were observed within the gastrodermal cells and tegumental syncytium for different marker proteins whilst the protonephridial (excretory) system and parenchymal-type 2 cells were identified as additional sites of production (or transit) of FhEVs. Ligation was used to mechanically block the oral sucker, excretory pore, or both, to determine the effect on FhEV release from live adult flukes in vitro. This revealed that FhEVs are predominately derived from the gut, whilst the tegument releases EVs to a lesser extent. The data also suggest that the protonephridial system contributes to the small (120 K) EV sub-population. Sphingomyelinase (SMase) activity is a key driver of EV biogenesis in mammalian cells and we have previously identified SMases in FhEVs by mass spectrometry. SMase activity associated with isolated FhEVs was susceptible to the chemical inhibitor GW4869 and treatment of adult flukes with GW4869 led to a significant reduction in 120 K EV release in vitro, suggesting that a ceramide-dependent mechanism could drive 120 K EV formation. In contrast, the release of the larger 15 K EVs was only moderately impacted, indicating that they form independently of SMase activity. Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma and vacuolation of the tegument, gastrodermal cells and epithelial lining of the excretory ducts. This work establishes that targeted disruption of EV biogenesis and release in helminths is possible, and provides proof-of-concept for future studies investigating EV secretion as a target for parasite control.

Paramphistomosis of Ruminants: An Emerging Parasitic Disease in Europe.

Whilst historically regarded as being of minor importance in European livestock, recent evidence suggests that the prevalence of paramphistomosis is greater than that of fasciolosis in parts of the UK. In order to address this emerging threat to ruminant farming systems, and associated risks for food security posed by rumen fluke infection, it is imperative that we develop a better understanding of the basic biology of this parasite and how it interacts with its ruminant host. In this Opinion article we review recent progress in tracking the spread of rumen fluke infection in Europe, and propose some research questions that should be addressed if we are to develop tools to diagnose and treat paramphistomosis more effectively in the future.