Exploring novel circulating biomarkers for liver cancer through extracellular vesicle characterization with infrared spectroscopy and plasmonics.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with cirrhosis being a major risk factor. Traditional blood markers like alpha-fetoprotein (AFP) demonstrate limited efficacy in distinguishing between HCC and cirrhosis, underscoring the need for more effective diagnostic methodologies. In this context, extracellular vesicles (EVs) have emerged as promising candidates; however, their practical diagnostic application is restricted by the current lack of label-free methods to accurately profile their molecular content. To address this gap, our study explores the potential of mid-infrared (mid-IR) spectroscopy, both alone and in combination with plasmonic nanostructures, to detect and characterize circulating EVs. RESULTS: EVs were extracted from HCC and cirrhotic patients. Mid-IR spectroscopy in the Attenuated Total Reflection (ATR) mode was utilized to identify potential signatures for patient classification, highlighting significant changes in the Amide I-II region (1475-1700 cm-1). This signature demonstrated diagnostic performance comparable to AFP and surpassed it when the two markers were combined. Further investigations utilized a plasmonic metasurface suitable for ultrasensitive spectroscopy within this spectral range. This device consists of two sets of parallel rod-shaped gold nanoantennas (NAs); the longer NAs produced an intense near-field amplification in the Amide I-II bands, while the shorter NAs were utilized to provide a sharp reflectivity edge at 1800-2200 cm-1 for EV mass-sensing. A clinically relevant subpopulation of EVs was targeted by conjugating NAs with an antibody specific to Epithelial Cell Adhesion Molecule (EpCAM). This methodology enabled the detection of variations in the quantity of EpCAM-presenting EVs and revealed changes in the Amide I-II lineshape. SIGNIFICANCE: The presented results can positively impact the development of novel laboratory methods for the label-free characterization of EVs, based on the combination between mid-IR spectroscopy and plasmonics. Additionally, data obtained by using HCC and cirrhotic subjects as a model system, suggest that this approach could be adapted for monitoring these conditions.

Differentiating hepatocellular carcinoma from dysplastic nodules at gadobenate dimeglumine-enhanced hepatobiliary-phase magnetic resonance imaging.

PURPOSE: We evaluated whether the addition of delayed phase imaging (DPI) gadobenate dimeglumine-enhanced MRI to dynamic postcontrast imaging improves the characterization of small hepatocellular carcinoma (HCC) and the differentiation between HCC, high grade dysplastic nodules (HGDN), and low grade dysplastic nodules (LGDN). METHODS: Twenty-five cirrhotic patients with 30 nodules (16 HCC, 8 HGDNs, and 6 LGDNs; maximum size of 3 cm) were included in this retrospective study. The diagnostic reference standard was histology. All the patients underwent MRI both prior to and following intravenous administration of gadobenate dimeglumine. The lesions were classified as hypointense, isointense, hyperintense on DPI for qualitative assessment. In the quantitative analysis the relative tumor-liver contrast to noise ratio (CNR) of the lesions on DPI was calculated. RESULTS: All HCCs were hypointense on DPI while only 8 (57.1%) of 14 DNs were hypointense and only 1 of 6 (16.6%) LGDNs was hypointense. There was a statistically significant difference in the hypointensity on DPI between HCCs and DNs (p = 0.003) in the qualitative analysis but not in the CNR values while there was a strong statistically significant difference in the hypointensity on DPI in the qualitative (p = 0.00001) and quantitative analysis (p < 0.05) between LGDNs and the group obtained by unifying HGDNs and HCCs. CONCLUSION: DPI is helpful in differentiating HCCs and HGDNs from LGDNs. Demonstration of hypointensity on DPI should raise the suspicion of HGDN or hypovascular HCC in the case of nodules with atypical dynamic pattern.

Gastrointestinal symptoms and digestive comorbidities in an Italian cohort of patients with COVID-19.

OBJECTIVE: The Coronavirus Disease 2019 (COVID-19) pandemic mainly involves respiratory symptoms, though gastrointestinal (GI) symptoms are increasingly being recognized. In this context, the presence of comorbidities appears to be associated with adverse outcomes. However, the role of digestive manifestations is not yet well defined. The primary aim of this study was to assess the prevalence of GI symptoms and digestive comorbidities in a cohort of patients with COVID-19 compared to controls. The secondary aim was to determine the association of GI-symptoms and digestive comorbidities with clinical outcomes. PATIENTS AND METHODS: Inpatients with COVID-19 and controls with similar symptoms and/or radiological findings were enrolled. Symptoms at admission and throughout hospitalization were collected as they were comorbidities. The measured clinical outcomes were mortality, intensive care unit admission and cumulative endpoint. RESULTS: A total of 105 patients were included: 34 with COVID-19 and 71 controls. At admission, the prevalence of GI symptoms among COVID-19 patients was 8.8%. During hospitalization, the frequency of GI symptoms was higher in patients with COVID-19 than in controls (p=0.004). Among patients with COVID-19, the mortality and a cumulative endpoint rates of those with GI symptoms were both lower than for those without GI symptoms (p=0.016 and p=0.000, respectively). Finally, we found digestive comorbidities to be associated with a milder course of COVID-19 (p=0.039 for cumulative endpoint). CONCLUSIONS: Our results highlighted the non-negligible frequency of GI symptoms in patients with COVID-19, partly attributable to the therapies implemented. In addition, the presence of GI symptoms and digestive comorbidities is associated with better outcomes. Most likely, digestive comorbidities do not hinder the host's immune response against SARS-COV-2, and the occurrence of GI symptoms might be linked to a faster reduction of the viral load via the faecal route.

Ischaemic jejunal vasculitis during treatment with pegylated interferon-alpha 2b and ribavirin for hepatitis C virus related cirrhosis.

A 53-year-old male with compensated cirrhosis (Child-Pugh class A5) and mixed cryoglobulinaemia (cryocrit: 2.0%), both hepatitis C virus-related, was treated with pegylated interferon-alpha 2b and ribavirin. After three months of therapy, he developed segmental jejunal vasculitis requiring emergency resection of an ischaemic intestinal loop 60cm long. Pathological examination of the surgical specimen revealed signs of ischaemic injury with haemorrhagic infarction due to arteritis and arterial occlusion. The postoperative course was complicated by progressive liver and renal failure that led to the patient's death six months after surgery. To our knowledge, ischaemic jejunal vasculitis has never been reported during interferon therapy, but the latter treatment may have played causative roles.

Paradoxical psoriasis in a large cohort of patients with inflammatory bowel disease receiving treatment with anti-TNF alpha: 5-year follow-up study.

BACKGROUND: Psoriasis is an emerging paradoxical side effect in patients with inflammatory bowel disease (IBD) when treated with anti-TNF alpha. Patients with severe skin lesions unresponsive to topical therapy need to withdraw from treatment. AIM: To estimate the incidence of paradoxical psoriasis in a large cohort of IBD patients treated with anti-TNF alpha and to analyse its clinical correlates. METHODS: A retrospective cohort study on all IBD patients who started anti-TNF alpha at our IBD Centre from January 2008 to December 2013 was performed. Proportional hazards regression models were used to estimate the association between each predictor and time to the development of psoriasis. Time-dependent predictors were updated at each available time point. RESULTS: Four hundred and two patients were included. Participants contributed a total of 839 person-years of follow-up, during which 42 incident cases of psoriasis were recorded, with an incidence rate of five per 100 person-years. Cox-regression survival analysis revealed smoking as independent predictor of psoriasis (HR: 2.37, 95% CI: 1.36-4.48; P = 0.008). Conversely, concomitant immunosuppressive therapy was inversely related to psoriasis (HR: 0.33, 95% CI: 0.12-0.92; P = 0.03). CONCLUSIONS: Paradoxical psoriasis is a relevant side effect of anti-TNF alpha therapy, with an incidence rate of five per 100 person-years. Smoking is confirmed as the main risk factor for developing lesions. The combination therapy with anti-TNF alpha plus immunosuppressants is associated with a reduced risk of paradoxical psoriasis.

Increased expression of Aquaporin 4 in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration.

Trimethyltin chloride (TMT) is a neurotoxicant producing neuronal degeneration and reactive astrogliosis in the mammalian central nervous system, especially the hippocampus. A previous magnetic resonance imaging investigation in TMT-treated rats evidenced dilation of lateral ventricles, also suggesting alterations in blood-brain barrier permeability and brain edema. Aquaporin 4 (AQP4), a glial water channel protein expressed mainly in the nervous system, is considered a specific marker of vascular permeability and thought to play an important role in brain edema (conditions). We studied AQP4 expression in the hippocampus and cerebral cortex of TMT-treated rats in order to explore the molecular mechanisms involved in brain edema occurring in these experimental conditions. Real-time PCR and western blotting data showed significant up-regulation of both AQP4 mRNA and protein levels starting 14 days after TMT treatment in the hippocampus and cortex. Parallel immunofluorescence studies indicated intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting 14 days after TMT intoxication. In order to study the effects of TMT on vascular integrity, double-label immunofluorescence experiments for rat immunoglobulin G (IgG) and rat endothelial cell antigen-1 (RECA-1) or neuronal nuclei (NeuN) (endothelial and neuronal markers respectively) were performed. The results indicated, at 21 and 35 days after treatment, the presence of rat IgG in paravasal parenchyma and in some neuronal cells of the hippocampus and cortex. The extravasated IgG staining was temporally correlated with over-expression of neuronal vascular endothelial growth factor (VEGF) and the active phosphorylated form of its neuronal receptor (VEGFR-2P), suggesting that these factors may cooperate in mediating vascular leakage.

Cathepsin D plays a crucial role in the trimethyltin-induced hippocampal neurodegeneration process.

Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA(1)/CA(3) subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca(2+) cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca(2+) chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA(3) neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon.