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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identified in China, in December 2019 determines COronaVIrus Disease 19 (COVID-19). Whether or not the virus was present in Italy earlier the first autochthonous COVID-19 case was diagnosed is still uncertain. We aimed to identify anti-SARS-CoV-2 antibodies in sera collected from 4th November 2019 to 9th March 2020, in order to assess the possible spread of the virus in Italy earlier than the first official national diagnosis.Anti-SARS-CoV-2 antibodies were evaluated in retrospective serum samples from 234 patients with liver diseases (Hep-patients) and from 56 blood donors (BDs). We used two rapid serologic tests which were confirmed by a validated chemoluminescence assay.Via rapid tests, we found 10/234 (4.3%) IgG-positive and 1/234 (0.4%) IgM-positive cases in the Hep-patient group. Two/56 (3.6%) IgG-positive and 2/56 (3.6%) IgM-positive cases were detected in BD group. Chemoluminescence confirmed IgG-positivity in 3 Hep-patients and 1 BD and IgM-positivity in 1 Hep-patient. RNAemia was not detected in any of the subjects, rendering the risk of transfusion transmission negligible.Our results suggest an early circulation of SARS-CoV-2 in Italy, before the first COVID-19 cases were described in China. Rapid tests have multiple benefits; however, a confirmation assay is required to avoid false positive results.

BackgroundMixed cryoglobulinaemic vasculitis (MCV) is an immune-complex-mediated systemic vasculitis characterized by heterogeneous clinical manifestations mainly involving skin, kidney and peripheral nervous system.Despite reassuring safety data from EULAR Coronavirus Vaccine (COVAX) physician-reported registry, a significant proportion of patients with autoimmune diseases reported unwillingness to get vaccinated against SARS-CoV-2 infection in the preliminary results of the COVAD study, due to concerns about the lack of long-term safety data, and fear of associated side effects and disease flare.ObjectivesAims of this multicentre Italian study were to investigate the prevalence of vaccination against SARS-CoV-2 in Italian population of MCV patients, to explore the reason for the missed vaccination, and to investigate short and long-term side effects of the vaccine, including vasculitis flare.MethodsAll MCV patients referring to 12 Italian centres were investigated about vaccination and possible both short- (within 48 hours) and long-term (within 30 days) adverse events (AE), classified according to FDA Toxicity Grading Scale for preventive vaccine clinical trials, and possible disease flares. Patients with MCV related to lymphoproliferative disorders or connective tissue diseases were excluded from the study.The baseline variables were expressed as percentages or mean±standard deviation. The differences between continuous variables were analysed using the Mann–Whitney nonparametric test. The chi-squared test, or Fischer’s exact when appropriate, were used for categorical variables (absolute numbers and percentages) regarding baseline characteristics.ResultsA total of 416 patients, 69.2% females and 30.8% males, with a mean age of 70.4±11.7 years, were included in the study.Only 7.7% of patients were not vaccinated, mainly for fear of adverse events (50%) or for medical decision (18.8%). Corminaty was the vaccine most frequently used (80.5%). Interestingly, 6 patients (1.44%) were with a heterologous vaccination (usually AstraZeneca-Corminaty).Considering ongoing treatment, not vaccinated subjects were more frequently treated with chronic glucocorticoid therapy and/or Rituximab (p=0.049 and p=0.043 respectively).AE were recorded in 31.7% of cases, mainly mild and self-limiting (grade 1). More severe adverse events, such as flare of vasculitis, were observed in 5.3% of cases.AE were not associated with the kind of vaccine used and with the clinical manifestations of vasculitis. Patients with active MCV showed a lower frequency of short-term (within 48 hours) adverse events, but patients affected by peripheral neuropathies or skin vasculitis frequently showed a flare of their symptoms, recorded in 40% and 25% of cases, respectively. Finally, patients under glucocorticoid treatment were more prone to develop a vasculitis flare within a month after vaccination.ConclusionVaccination in MCV patients has been performed in a high percentage of patients showing a good safety. Other than patients’ fear, treatments with rituximab and glucocorticoids are the main reasons for delaying vaccination, and it should be considered by the physician before starting therapy. Vasculitis flares were observed in about 5% of cases, in line with that observed in other autoimmune diseases. Specific attention should be reserved to people with purpura or peripheral neuropathy, for the increased risk of exacerbation of their symptoms.References[1]Visentini M et al Flares of mixed cryoglobulinaemia vasculitis after vaccination against SARS-CoV-2 2021[2]Machado PM et al Safety of vaccination against SARS-CoV-2 in people with rheumatic and musculoskeletal diseases: results from the EULAR Coronavirus Vaccine COVAX physician-reported registry 2021[3]Sen P et al COVAD Study Group. COVID-19 vaccination in autoimmune disease COVAD survey protocol 2022[4]Scarpato S et al Italian Group for the Study of Cryoglobulinaemia GISC. Provisional recommendations for SARS-CoV-2 vaccination in patients with cryoglobulinaemic vasculitis 2021Disclosure of InterestsNone declared

BackgroundPatients with autoimmune systemic diseases (ASDs) can be counted among frail populations as regards the predisposition to COVID-19 due to the frequent visceral organ involvement and comorbidities, as well as the ongoing immunomodulating treatments.ObjectivesOur long-term multicenter telephone survey prospectively investigated the prevalence, prognostic factors, and outcomes of COVID-19 in Italian ASD patients during the first 3 pandemic waves.MethodsA large series of 3,918 ASD patients (815 M, 3103 F; mean age 59±12SD years) was consecutively recruited at the 36 referral centers of COVID-19 & ASD Italian Study Group. In particular, ASD series encompassed the following conditions: rheumatoid arthritis (n: 981), psoriatic arthritis (n: 471), ankylosing spondylitis (n: 159), systemic sclerosis (n: 1,738), systemic lupus (172), systemic vasculitis (n: 219), and a miscellany of other ASDs (n: 178). The development of COVID-19 was recorded by means of telephone survey using standardized symptom-assessment questionnaire (1).ResultsA significantly increased prevalence of COVID-19 (8.37% vs 6.49%; pInterestingly, a significantly higher COVID-19-related death rate was observed in systemic sclerosis patients compared to the Italian general population (6.29% vs 2.95%; p=0.018). Other adverse prognostic factors to develop COVID-19 were the patients’ older age, male gender, pre-existing ASD-related interstitial lung involvement, and chronic steroid treatment. Conversely, patients treated with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) showed a significantly lower prevalence of COVID-19 compared to those without (3.58% vs 46.99%; p=0.000), as well as the chronic administration of low dose aspirin in a subgroup of SSc patients (with 5.57% vs without 27.84%; p=0.000).ConclusionThe cumulative impact of COVID-19 on ASD patients after the first 3 pandemic waves revealed less severe than that observed during the first phase of pandemic (1), especially with regards to the death rate that was comparable to the Italian general population in spite of the increased prevalence of complicating COVID-19 in the same ASD series.Ongoing long-term treatments, mainly csDMARDs, might usefully contribute to generally positive outcomes of in this frail patients’ population.Of note, a significantly increased COVID-19-related mortality was recorded in only SSc patients’ subgroup, possibly favored by pre-existing lung fibrosis. Among different ASD, SSc deserves special attention, since it shares the main pathological alterations with COVID-19, namely the interstitial lung involvement and the endothelial injury responsible for diffuse microangiopathy.Besides SSc, the patients’ subgroups characterized by older age, chronic steroid treatment, pre-existing interstitial lung disease, and/or impaired COVID-19 vaccine response (1-3), may deserve well-designed prevention and management strategies.References[1]Ferri C, et al. Ann Rheum Dis. 2020 Oct 14 doi: 10.1136/annrheumdis-2020-219113.[2]Ferri C et al. J Autoimmun. 2021 Dec;125:102744. doi: 10.1016/j.jaut.2021.102744.[3]Visentini M et al. Ann Rheum Dis. 2021 Nov 24. doi: 10.1136/annrheumdis-2021-221248Disclosure of InterestsNone declared

Chondrocytes are responsible for maintaining the cartilage that helps joints like the knee and hip bear load and move smoothly. These cells typically respond to physiological compression with pathways consistent with matrix synthesis, and chondrocyte mechanotransduction is essential for tissue and joint homeostasis. In osteoarthritis (OA), chondrocyte mechanotransduction appears to be dysregulated, yet many pathways and mechanisms of osteoarthritic chondrocyte mechanotransduction remain poorly understood. The objective of this study is to document the phosphoproteomic responses of primary osteoarthritic chondrocytes to physiological sinusoidal compression. Here we show that OA chondrocytes respond to physiological compression by first activating proteins consistent with cytoskeletal remodeling and decreased transcription, and then later activating proteins for transcription. These results show that several microtubule-related proteins respond to compression, as well as proteins related to calcium signaling, which has previously been extensively shown in chondrocytes. Our results demonstrate that compression is a relevant physiological stimulus for osteoarthritic chondrocytes. We anticipate these data to be a starting point for more sophisticated analysis of both normal and osteoarthritic chondrocyte mechanotransduction. For example, finding differences in compression-induced phosphoproteins between normal and OA cells may lead to druggable targets to restore homeostasis to diseased joints.

Monoclonal plasma cell proliferative disorders comprise a wide spectrum of diseases associated to clonal B-cell expansion. Serum protein electrophoretic profile (SPEP) and circulating free light chains (FLCs) levels are the mainstay of diseases management. Recently, soluble (s) Syndecan-1 (SDC1, CD138) produced by myeloma plasma cells has been suggested in the monitoring and follow-up of patients with myeloma. The aim of our study is to evaluate sCD138 in addition with FLCs and SPEP for the screening of patients with different evolutive disease pathways.Sera from 73 patients with monoclonal gammopathy of undetermined significance (MGUS), 120 smoldering and 42 multiple myeloma (SMM and MM, respectively), 70 HCV-related mixed cryoglobulinemia (MC), 35 B-cell non-Hodgkin's lymphoma (B-NHL) and sera from 50 healthy donors (HD), were tested for sCD138, FLCs (assessed by means of ELISA and turbidimetric assay, respectively) and electrophoresis pattern (performed on Capillarys system) for the generation of a novel biomarker score (BS).Our results were grouped according to the two main lines of disease progression (vs. MM or B-NHL): in one group we found BS mean values of 0.2, 3.4, 5.3, 7.1 for HD, MGUS, SMM and MM, respectively; in the other group of 0.2, 4.4, 6.7 for HD, MC and B-NHL.We showed that BS mean values follow the ingravescence disease status towards the two main lines of progression to cancerous conditions; it could represent an additional useful tool in the management of screening and/or follow-up.

Osteoarthritis (OA) is the most common degenerative joint disease, and joint injury increases the risk of OA by 10-fold. Although the injury event itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), the production of inflammatory cytokines, and we hypothesize, changes to the joint metabolome. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Flavopiridol is a potent and selective inhibitor of Cdk9 kinase activity, which prevents the transcriptional activation of early response genes. To model post-traumatic osteoarthritis, we subjected mice to non-invasive ACL-rupture joint injury. Following injury, mice were treated with flavopiridol to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 hour after injury. We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism and others. Importantly, we found that inhibition of primary response gene activation at the time of injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites, which may offer novel targets for cell-mediated secondary joint damage. Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses, and these data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury.Significance StatementJoint injury is an excellent predictor of future osteoarthritis. It is increasingly apparent that the acute cellular responses to injury contribute to the initiation and pathogenesis of OA. Although changes to the joint transcriptome have been extensively studied in the context of joint injury, little is known about changes to small-molecule metabolites. Here we use a non-invasive ACL rupture model of joint injury in mice to identify injury-induced changes to the global metabolomic profiles. In one experimental group we prevented the activation of primary response gene transcription using the Cdk9 inhibitor flavopiridol. Through this comparison, we identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not.

Chondrocytes are the sole cell type found in articular cartilage and are repeatedly subjected to mechanical loading in vivo. We hypothesized that physiological dynamic compression results in changes in energy metabolism to produce proteins for maintenance of the pericellular and extracellular matrices. The objective of this study was to develop an in-depth understanding for the short term (

Chondrocytes are responsible for maintaining the cartilage that helps joints bear load and move smoothly. These cells typically respond to physiological compression with pathways consistent with matrix synthesis, and chondrocyte mechanotransduction is essential for homeostasis. In osteoarthritis (OA), chondrocyte mechanotransduction appears to be dysregulated, yet the mechanisms remain poorly understood. The objective of this study is to document the phosphoproteomic responses of primary osteoarthritic chondrocytes to physiological sinusoidal compression. We show that OA chondrocytes respond to physiological compression by first activating proteins consistent with cytoskeletal remodeling and decreased transcription, and then later activating proteins for transcription. These results show that several microtubule-related proteins respond to compression. Our results demonstrate that compression is a relevant physiological stimulus for osteoarthritic chondrocytes. Future analyses may build on these results to find differences in compression-induced phosphoproteins between normal and OA cells that lead to druggable targets to restore homeostasis to diseased joints.

In this paper, the tearing behavior of thin polymer sheets was studied experimentally and numerically. First, the specific essential work of fracture for thin biaxially oriented polyethylene terephthalate (Mylar) was measured experimentally. Double-edged notched tensile specimens of Mylar with three different thicknesses and varying ligament lengths were tested. Load-displacement graphs were formed, and the total internal energy was evaluated, from which the specific fracture energy was determined. Next, a finite element analysis was performed to predict the tearing response of polymer sheets. The finite element model incorporated a user-defined cohesive element to properly represent the structural behavior of a thin polymer sheet in the presence of damage, including the propensity of the sheet to wrinkle. The results indicated that the measured specific essential work-of-fracture values were nearly identical for the three separate thicknesses, which confirmed that it is indeed a material property of the ...

Cells sense and respond to mechanical loads in a process called mechanotransduction. These processes are disrupted in the chondrocytes of cartilage during joint disease. A key driver of cellular mechanotransduction is the stiffness of the surrounding matrix. Many cells are surrounded by extracellular matrix that allows for tissue mechanical function. Although prior studies demonstrate that extracellular stiffness is important in cell differentiation, morphology and phenotype, it remains largely unknown how a cell’s biological response to cyclical loading varies with changes in surrounding substrate stiffness. Understanding these processes is important for understanding cells that are cyclically loaded during daily in vivo activities (e.g. chondrocytes and walking). This study uses high-performance liquid chromatography - mass spectrometry to identify metabolomic changes in primary chondrocytes under cyclical compression for 0–30 minutes in low- and high- stiffness environments. Metabolomic analysis reveals metabolites and pathways that are sensitive to substrate stiffness, duration of cyclical compression, and a combination of both suggesting changes in extracellular stiffness in vivo alter mechanosensitive signaling. Our results further suggest that cyclical loading minimizes matrix deterioration and increases matrix production in chondrocytes. This study shows the importance of modeling in vivo stiffness with in vitro models to understand cellular mechanotransduction.

Mouse models are common tools for examining post-traumatic osteoarthritis (OA), which involves cartilage deterioration following injury or stress. One challenge to current mouse models is longitudinal monitoring of the cartilage deterioration in vivo in the same mouse during an experiment. The objective of this study was to assess the feasibility for using a novel transgenic mouse for non-invasive quantification of cartilage. Chondrocytes are defined by expression of the matrix protein aggrecan, and we developed a novel mouse containing a reporter luciferase cassette under the inducible control of the endogenous aggrecan promoter. We generated these mice by crossing a Cre-dependent luciferase reporter allele with an aggrecan creERT2 knockin allele. The advantage of this design is that the targeted knockin retains the intact endogenous aggrecan locus and expresses the tamoxifen-inducible CreERT2 protein from a second IRES-driven open reading frame. These mice display bioluminescence in the joints, tail, and trachea, consistent with patterns of aggrecan expression. To evaluate this mouse as a technology for non-invasive quantification of cartilage loss, we characterized the relationship between loss of bioluminescence and loss of cartilage after induction with (i) ex vivo collagenase digestion, (ii) an in vivo OA model utilizing treadmill running, and (iii) age. Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence. In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease. We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age. These results indicate that the bioluminescent signal from this mouse may be used as a non-invasive quantitative measure of cartilage. Future studies may use this reporter mouse to advance basic and preclinical studies of murine experimental OA with applications in synovial joint biology, disease pathogenesis, and drug delivery.

In articular cartilage, chondrocytes reside within a gel-like pericellular matrix (PCM). This matrix provides a mechanical link through which joint loads are transmitted to chondrocytes. The stiffness of the PCM decreases in the most common degenerative joint disease, osteoarthritis. To develop a system for modeling the stiffness of both the healthy and osteoarthritic PCM, we determined the concentration–stiffness relationships for agarose. We extended these results to encapsulate chondrocytes in agarose of physiological stiffness. Finally, we assessed the relevance of stiffness for chondrocyte mechanotransduction by examining the biological response to mechanical loading for cells encapsulated in low- and high-stiffness gels. We achieved agarose equilibrium stiffness values as large as 51.3 kPa. At 4.0% agarose, we found equilibrium moduli of 34.3 ± 1.65 kPa, and at 4.5% agarose, we found equilibrium moduli of 35.7 ± 0.95 kPa. Cyclical tests found complex moduli of ~100–300 kPa. Viability was >96% for all studies. We observed distinct metabolomic responses in >500 functional small molecules describing changes in cell physiology, between primary human chondrocytes encapsulated in 2.0 and 4.5% agarose indicating that the gel stiffness affects cellular mechanotransduction. These data demonstrate both the feasibility of modeling the chondrocyte pericellular matrix stiffness and the importance of the physiological pericellular stiffness for understanding chondrocyte mechanotransduction.

Chondrocyte mechanotransduction is the process by which cartilage cells transduce mechanical loads into biochemical and biological signals. Previous studies have identified several pathways by which chondrocytes transduce mechanical loads, yet a general understanding of which signals are activated and in what order remains elusive. This study was performed to identify candidate mediators of chondrocyte mechanotransduction using SW1353 chondrocytes embedded in physiologically stiff agarose. Dynamic compression was applied to cell-seeded constructs for 0–30 minutes, followed immediately by whole-cell metabolite extraction. Metabolites were detected via LC-MS, and compounds of interest were identified via database searches. We found several metabolites which were statistically different between the experimental groups, and we report the detection of 5 molecules which are not found in metabolite databases of known compounds indicating potential novel molecules. Targeted studies to quantify the response of central energy metabolites to compression found a transient increase in the ratio of NADP+ to NADPH and a continual decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. These data are consistent with the remodeling of cytoskeletal components by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ∼25-200 kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live-dead imaging following 24 and 72 hours in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ∼1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.

Hepatitis C virus infection and diabetes mellitus are two worldwide, major public health problems with increasing complication and mortality rates. Type 1 diabetes mellitus (T1D) is characterized by an autoimmune process leading to pancreatic beta cell destruction; only when the major part of pancreatic beta cells have been destroyed the diabetes become clinically manifest. At the basis of the development of the T1D there is an interplay among environmental factors, pancreatic beta cells, the innate and adaptive immune system, the genetic background and the comorbidities of the patient. Viral infections, including hepatitis C virus infection, may be one of the factors that can almost accelerate progression to diabetes, through different mechanisms.

Psychiatric symptoms are commonly identified in patients with viral hepatitis. They may have been present prior to the onset of disease and may include symptoms related to addiction issues. Furthermore, the virus and antiviral therapy, in particular interferon, may induce or modify psychiatric symptoms. Recent data support chronic hepatitis C replication in the brain and subsequent changes of cerebral metabolite spectra and magnetic resonance alterations. In chronic viral hepatitis and in other chronic inflammatory diseases, an alteration of the neuro-endocrine-immune system response has been observed. Catecholamines and glucocorticoids modulate this immune/inflammatory reaction. Psychiatric assessment and monitoring before, during and after antiviral therapy can identify patients whose psychiatric symptoms preclude therapy, and those who may benefit from psychopharmacological therapy and counselling, thereby improving therapeutic results. This review will discuss current insights into the complex interplay between cytokines, liver and brain in chronic viral hepatitis closely associated with psychiatric issues, especially in the case of antiviral therapy, with the aim of indicating future research and possible treatments.

The specific essential work of fracture for thin biaxially-oriented polyethylene terephthalate, (Mylar), is measured using double-edge notched (DENT) specimens loaded in a uniaxial tension test. Experiments were performed on DENT specimens with varying ligament lengths. Three different thicknesses of Mylar were tested: 25.4 µm (1 mil), 50.8 µm (2 mil), and 76.2 µm (3 mil) Mylar. Load vs. displacement graphs were formed, and from them the area under the curve, or strain energy, was determined. Energy terms were normalized, and then plotted versus ligament length. Specific essential work of fracture values were determined to be 37.027 kJ/m 2 for the 25.4 µm specimens, 36.974 kJ/m 2 for the 50.8 µm specimens, and 36.853 kJ/m 2 for the 76.2 µm specimens. The specific essential work of fracture values were nearly identical for the three separate thicknesses, and therefore is indeed a material property of the Mylar itself. In a companion paper [1], the specific essential work of fracture is then used in a finite element model to predict the tearing response of polymer sheets.

This study was done to evaluate whether and which of the magnetic resonance diffusion-weighted imaging (MR-DWI) parameters - apparent diffusion coefficient (ADC), diffusion (D) or perfusion fraction (f) - correlates with the degree of chronic liver disease progression.Twenty-eight patients were evaluated with abdominal MR-DWI from March to November 2010: seven healthy volunteers, seven patients with chronic liver disease F0-F2 (METAVIR score), seven F3-F4 Child-Pugh A, and seven F4 Child-Pugh BC, classified as groups 1-4, respectively. DWI acquisitions were performed during breath-holding (b = 0-150 s/mm(2) and 1,000) and free breathing (multi-b = 0-200-400-600-800-1,000 s/mm(2)). Using a double-blind control procedure, two observers estimated ADC, D, and f by applying a region of interest (ROI) in 4/12 sections in the middle-lower portion of the right hepatic lobe. Statistical analysis was done with analysis of variance (ANOVA).A reduction in the mean value of f, ADC(150) and, to a lesser extent, ADC(1,000) is shown to progress from healthy volunteers (group 1) to cirrhosis patients (group 4), with wide overlap among groups. There were no statistically significant changes of D.Our results indicate that stratifying patients with chronic liver disease for clinical purposes cannot be done with DWI. However, there is a tendency among groups for reduced perfusion-related parameters as chronic liver disease progresses.

Many patients chronically infected by hepatitis C virus (HCV) experience symptoms like fatigue, dyspnea and reduced physical activity. However, in many patients, these symptoms are not proportional to the liver involvement and could resemble symptoms of chronic heart failure. To our knowledge, no study evaluated serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) in a large series of patients with HCV chronic infection (HCV+). Serum NT-proBNP was assayed in 50 patients HCV+ and in 50 sex- and age-matched controls. HCV+ patients showed significantly higher mean NT-proBNP level than controls (P = 0.001). By defining high NT-proBNP level as a value higher than 125 pg/mL (the single cut-off point for patient under 75 years of age), 34% HCV+ and 6% controls had high NT-proBNP (Fisher exact test; P0.001). With a cut-off point of 300 pg/mL (used to rule out chronic heart failure in patients under 75 years of age) 10% HCV+ and 0 controls had high NT-proBNP (Fisher exact test; P = 0.056). With a cut-off point of 900 pg/mL (used for ruling in chronic heart failure in patients with age 50-75) 8% HCV+ patients and 0 controls had high NT-proBNP (Fisher exact test; P = 0.12). The study demonstrates high levels of circulating NT-proBNP in HCV+ patients compared to healthy controls. The increase of NT-proBNP may indicate the presence of a sub-clinical cardiac dysfunction. Further prospective studies quantifying these symptoms in correlation with echocardiography are needed to confirm this association.

To compare the clinico-serological features of arthritis from two HCV+ patient groups characterized by mixed cryoglobulinemia (MC) or chronic hepatitis (CH).We retrospectively studied 157 MC patients (119 females, mean age 64.8 yrs, range 36-88) and 155 CH patients (103 females, mean age 58.8 yrs, range 30-81). Patients with HBV and/or HIV co-infections and a follow-up shorter than 1 year were excluded. MC was classified according to standard criteria, while CH classification was based on Desmet's criteria.No differences in epidemiology were demonstrated between the two series of patients. Although significantly prevalent in MC patients, extra-hepatic manifestations including nephropathy, neuropathy, pneumopathy, mixed cryoglobulins, RF positivity and hypocomplementemia were detected in both patient groups. Arthritis was present in 23 CH (15%) and 12 MC (8%) patients. A symmetrical polyarthritis was observed in 87% of 23 CH patients, while cryoglobulinemic arthritis was invariably asymmetrical and pauciarticular. Unlike MC patients, who had a constantly non-erosive arthritis, in 7/23 CH patients arthritis was erosive. Steroids and/or hydroxycloroquine or D-penicillamine were safe and useful in controlling the arthritis over the short-medium time, although clinical response was more evident in MC patients. Instead, in 5/23 CH and 3/12 MC patients, interferon-alpha treatment was able to trigger or exacerbate the arthritis despite a good control of liver function.HCV infection seems to be, possibly in genetically predisposed patients, responsible for arthritis at times similar to rheumatoid arthritis. In these patients a careful assessment of the interferon-alpha treatment is mandatory owing to the potential "arthritogenic effect" due to its immunoregulatory properties.

Haemodialysis patients are at great risk for HCV infection, and a strict relationship is clear between anti-HCV positivity and dialysis age or hospital dialysis, irrespective of previous blood transfusions. Notwithstanding that, the precise root of its nosocomial nontransfusional diffusion among haemodialysis patients is not clear yet. As isolation is a very expensive policy, we evaluated whether simpler measures such as the observance of the Universal Precautions (UP), and the use of anti-HCV positive patient dedicated monitors can stop the diffusion of HCV infection in a hospital haemodialysis centre. Since January 1990 to December 1991 (1st phase), the patients shared the monitors irrespective of their serological status for HCV, and training of the dialysis care staff was not performed with regard to the UP. Since January 1991 to June 1996 (2nd phase), according to the UP, strictly personal dialysis-tools were used for all patients, anti-HCV positive patients were assigned to dedicated monitors in defined (not separated) areas of the dialysis rooms and the dialysis care staff was trained to the strict observance of the UP. In the first phase of the follow-up 5 seroconversions occurred; none occurred in the second one. Our study shows that isolation is not required for such patients. We believe that measures such as the application of UP, dedicated machines and continuous training of the care staff, instead of the isolation of positive patients, result in the same efficacy and are cheaper than isolation of positive patients. Therefore they are mandatory for all haemodialysis centres.

Knowledge about vertical transmission of HCV is still limited. In this study we followed up the virological status of a series of offspring born to anti-HCV positive, anti-HIV negative mothers.Between January 1993 and January 1995, 5000 consecutive, anti-HIV negative pregnant women were screened for anti-HCV (ELISA III) and all positive samples were confirmed by RIBA III and analyzed for HCV-RNA by polymerase chain reaction (PCR). Babies born to anti-HCV positive mothers were followed from birth to two years by testing for ALT levels, anti-HCV antibodies and HCV-RNA.Of 5000 mothers 80 (1.6%) were anti-HCV positive (ELISA III) and RIBA III positive. Of these, 56 (70%) were HCV-RNA positive. We examined 80 babies, born to anti-HCV positive mothers: 56 with HCV-RNA positive mothers and 24 with HCV-RNA negative mothers. Two babies (3.6%) of 56 were anti-HCV and HCV-RNA positive, with normal liver function tests. Seventy-eight babies (97.5%) of 80 (54 with HCV-RNA positive mothers and 24 with HCV-RNA negative mothers) were HCV-RNA negative, with normal liver function tests and detectable levels of anti-HCV antibodies that gradually disappeared between 8-12 months.Mother-to-infant transmission of HCV is possible only in the case of HCV-RNA positive mothers. In our study the rate of transmission is 3.6%, (2/56 babies with HCV-RNA positive mothers). HCV transmission may occur without evident association with breast-feeding or vaginal delivery.

The issue of infection of peripheral blood mononuclear cells (PBMC) by the hepatitis C virus (HCV) has potentially important implications, but is still debated. We have used the severe combined immunodeficiency (SCID) mouse model to test for the persistence of HCV in PBMC. Hematopoietic cells isolated from 14 subjects infected by HCV were inoculated intraperitoneally into SCID mice. Serum and blood cell samples from these mice were obtained with a mean follow-up of 8 weeks. As controls, human fibroblasts and sheep PBMC, preincubated with a human HCV-positive serum, were inoculated concomitantly into mice and analyzed. HCV-RNA positive strands were detected in 7 of 26 serum samples and 8 of 26 cell fractions from SCID mice inoculated with HCV-positive PBMC, after 8 weeks of follow-up. In contrast, no HCV RNA was detectable in the 10 control mice. HCV-RNA negative strands were detected in only 2 of 10 tested samples from 2 mice, and both positive mice had been inoculated with PBMC from HCV-positive subjects with malignant hematopoietic syndrome. Our study offers strong evidence for the persistence of HCV infection in mononuclear cells. Our results are also consistent with a low rate of HCV multiplication. This SCID mouse model might therefore be useful in analyzing the mechanisms of HCV persistence in mononuclear cells.

In this study we aimed to correlate liver histology and the presence of hepatitis C virus (HCV) viremia, genotype, and quantity of HCV genome in 19 positive and 11 RIBA II indeterminate patients presenting persistently normal ALT values over 24 months before biopsy. In addition, after biopsy serum ALT values were monitored monthly for a mean follow-up period of 24.8 months, after which patients were reevaluated for RIBA II and the presence of viremia. Sixteen patients (53%) were serum HCV-RNA-positive; 13 of them (68%) were confirmed positive and 3 (27%) indeterminate on RIBA II. Histology of the HCV-RNA-positive patients showed eight cases of CPH (one case of genotype 1a; four cases type 1b; three cases type 2), six cases of CAH (three cases type 1b, three cases type 2), one case of CLH (type not determined), and one case of normal liver (NL) (type 1b). Histology of the HCV-RNA-negative patients showed four cases of CPH, one case of CAH, two cases of CLH, and seven cases of NL. During the follow-up period nine patients (30%) presented slight increases in ALT values (2 x N), and in particular, flares of ALT were observed four times in the CAH and five times in the CPH patients, who were all viremic, but never in the NL subjects. These results indicate that subjects positive on RIBA II, but with persistently normal ALT values, had a high probability of being serum HCV-RNA-positive and that almost all these viremic subjects presented histologic signs of liver disease. In contrast, RIBA II indeterminate subjects had a moderate probability of being HCV-RNA-positive, but a number of these may present signs of liver disease. In both cases there was no association with genotype or HCV-RNA serum levels. The other nonviremic cases included subjects with hepatic changes going toward resolution or with normal liver in whom hepatic biopsy can be avoided. Only one case was a true carrier since he was viremic with normal liver and persistently normal ALT values.

Chronic infection by hepatitis B virus (HBV) and hepatitis C virus (HCV) is now recognized as a major cause of liver cirrhosis. This study was aimed at evaluating the natural history of the disease in a large series of Italian patients with HBV- and HCV-related cirrhosis without portal hypertension at entry.The clinical records of 405 patients (233 males, mean age 54 +/- 9 yr) with histologically proven cirrhosis (321 with HCV-related and 84 with HBV-related cirrhosis) and no clinical evidence of portal hypertension at entry were retrospectively examined to evaluate the occurrence of complications and the cumulative mortality rate during follow-up.Patients had a mean follow-up of 8 +/- 3 yr. The cumulative survival rate was 99.1% at 5 yr, 76.8% at 10 yr, and 49.4% at 15 yr. The age-adjusted death rate was 3.14 and 2.84 times higher than in the general Italian population in men and women, respectively. Only the bilirubin level was an independent indicator of survival. Esophageal varices, ascites, jaundice, hemorrhage, hepatic encephalopathy, and hepatocellular carcinoma significantly reduced the survival rate (major complications), whereas thrombocytopenia, diabetes, and cholelithiasis did not affect survival (minor complications). The incidence of hepatocellular carcinoma was similar in patients with either HBV- or HCV-related disease and was quite frequent, especially in males.This study demonstrates that the course of virus-induced liver cirrhosis is not influenced by the etiology of the disease and that the occurrence of complications significantly shortens life expectancy. The longer survival rate observed in this study is probably due to the fact that cirrhosis was here recognized by liver biopsy in the absence of clinical evidence of portal hypertension.

The role of hepatitis B virus (HBV) and hepatitis C virus (HCV) as a major cause of chronic liver disease is now accepted worldwide. This study was aimed at evaluating the natural history of the disease in patients with virus-induced chronic active hepatitis or cirrhosis, and the influence played by age, sex and etiology, liver function tests and by the occurrence of different complications. We retrospectively examined the clinical records of 506 inpatients: 194 were affected by chronic active hepatitis (125 males, 69 females, mean age 45 +/- 11 years, 146 HCV- and 48 HBV-related), and 312 by cirrhosis without clinical evidence of portal hypertension (178 males, 134 females, mean age 53 +/- 9 years, 249 HCV- and 63 HBV-related). The occurrence of cirrhosis in the chronic active hepatitis group was then calculated, together with the occurrence of complications and the cumulative mortality rate of established cirrhosis. During follow-up 93 patients with chronic hepatitis developed cirrhosis. The cumulative probability of developing cirrhosis in this group was 6.64% at 5 years, 56.1% at 10 years and 86.8% at 15 years. These patients were therefore included in the cirrhosis group for the final analysis, so that a total of 405 cirrhotic patients were evaluated: these patients had a cumulative survival rate of 99.1% at 5, 76.8% at 10 and 49.4% at 15 years. Comparing the age-adjusted death rate of our patients with the general Italian population, we observed that in patients with liver cirrhosis it was 3.14 and 2.84 times higher in men and women, respectively. Bilirubin was an independent indicator of survival. Several complications, such as esophageal varices, ascites, jaundice, hemorrhage, hepatic encephalopathy and hepatocellular carcinoma significantly reduced the survival rate and were indicated as major complications, while thrombocytopenia, cholelithiasis and diabetes did not affect survival and thus were called minor complications. Incidence of hepatocellular carcinoma was very high especially in males, without correlation with etiology. In conclusion, the progression of virus-induced chronic active hepatitis to cirrhosis is not influenced by sex and etiology. Similarly, the different etiology does not modify the natural history of cirrhosis while the occurrence of one or more major complications significantly shortens survival. The longer survival rate observed in patients with cirrhosis included in this study is probably due to the selective inclusion of patients with early disease and no evidence of portal hypertension.

Liver transplantation for liver diseases related to hepatitis B virus (HBV) and hepatitis delta virus (HDV) remains problematic because of the risk of viral recurrence. We report here the long-term virological outcome of patients transplanted for HDV-related liver cirrhosis (HDV cirrhosis). From December 1984 to December 1990, 76 patients with HDV cirrhosis underwent liver transplantation. Before transplantation, all the patients were HBsAg-positive/anti-HDV positive, and all but one were HBV DNA-negative by dot blot hybridization. HDV RNA was detected by HDV RT-PCR and liver HDAg by fluorescent HDV Ab. After transplantation, all the patients except four received continuous long-term anti-HBs passive immunoprophylaxis. The actuarial 5-year survival was 88%. All patients who did not receive anti-HBs immunoprophylaxis remained HBsAg-positive and developed hepatitis. Among the 68 patients receiving antiHBs immunoprophylaxis with a minimum follow-up of 2 months, HBsAg reappeared in 7 (10.3%) after a mean of 17 months. These seven patients developed hepatitis, with simultaneous HBV and HDV replication; and four cleared later HBsAg. Patients without HBV reinfection were studied for HDV reinfection: liver HD Ag or serum HDV RNA were present in 88% of the patients during the first year, without developing hepatitis; however, they were no longer detectable after 2 years in 95% of the patients. In conclusion, liver transplantation for HDV cirrhosis gives good results, with a 5-year actuarial survival of 88%.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C virus (HCV) infection has been found in the majority of patients with mixed cryoglobulinemia (MC) in studies conducted in different countries. In our series of 110 MC patients the frequency of HCV markers was significantly high (91%) compared with other rheumatic diseases (6.4%) and with healthy Italian controls (1.2%). Moreover, HCV RNA was detected in 81% of the peripheral lymphocytes from MC patients. Comparable percentages of HCV infection were detectable in other disorders, i.e. porphyria cutanea tarda (77%) and autoimmune hepatitis type 1 (77%). The HCV infection of peripheral lymphocytes suggests that this virus could be the triggering factor for the lymphoproliferation underlying MC. In a number of patients with MC the evolution from a benign lymphoproliferation to frank B-cell lymphoma was observed. In these subjects HCV RNA in the sera and in fresh and cultured peripheral lymphocytes was constantly detected. The same phenomenon has been observed in patients with long-lasting type C chronic hepatitis. Interestingly, HCV infection has also been recorded in 32% of idiopathic B-cell non-Hodgkin's lymphomas. Taken together, the above findings suggest that HCV can cause benign B-cell proliferation with the consequent production of various autoantibodies, including rheumatoid factor and mixed cryoglobulins. These serological abnormalities characterise different clinical disorders, including the appearance of lymphoma in a not negligible number of individuals.

Hepatitis C virus has been proven to be the major cause of NANB hepatitis, cirrhosis and hepatocellular carcinoma worldwide. Based on the genome similarities between HCV and flavivirus or pestivirus, this agent has been included within the family Flaviviridae as a separate genus. Among the analogies between HCV and the other members of the same family there is the possibility of infecting blood cells. In particular, significant evidence obtained through studies performed in vivo and in vitro support the concept that HCV is not only a hepatotropic but also a lymphotropic virus. This suggests that, in addition to playing a role in inducing hepatic diseases (both of a non-tumoral and a neoplastic nature), HCV infection may also play a role in extrahepatic pathologies. The striking association observed between HCV infection and some autoimmune-lymphoproliferative disorders of either benign or neoplastic nature is consistent with this hypothesis. However, in analogy with what has been observed in the case of liver disease, the mechanisms involved in the pathogenesis of HCV-related extra-hepatic manifestations have to be more deeply analysed and clarified.

Viral recurrence is the limiting factor in orthotopic liver transplantation (OLT) for hepatitis B virus (HBV) related liver disease. In fact, high rates of HBV infection of the transplanted liver are reported, followed by the recurrence of liver disease in a high percentage of cases. The importance of reinfection stimulates the study of its modalities and mechanisms in order to better identify preventive measures and better select patients for OLT. In HBV and HDV positive patients, the outcome of liver transplantation appears significantly better than in patients that are solely HBV positive, in spite of a high rate of HDV reinfection. Long-term analysis (5 years) of HBV and HDV infection, using the PCR technique, in 15 patients transplanted for an HBV/HDV positive liver disease and treated with anti-HBs immunoglobulin (HBIG), revealed that all patients experienced an HDV reinfection, but only about 7 were still harboring the virus after four years of follow-up. HDV reinfection was either associated to HBV reinfection or isolated whereas no cases of HBV isolated reinfection was observed. Isolated HDV reinfection was frequent and transient in all but one case that was superinfected by HBV. Infected peripheral blood mononuclear cells seem to be implicated in HBV superinfection of HDV infected liver. Liver damage was observed only in cases of HBV/HDV co-infection, suggesting that, in vivo, HBV is necessary to produce liver damage although it is not essential for HDV absorption to target cells, HDV penetration of these cells or HDV genomic replication. In addition, in isolated HDV infection, transient HDV viraemia and its low levels suggest that, perhaps in these patients HDV uses a very limited presence of HBV or alternative ways which are not efficient enough for envelope production. These data suggest that, particularly in HDV positive patients, antiHBs Ig administration, which has previously been proven to significantly reduce HBV reinfection in HBsAg-positive patients, may be useful in changing the natural history of repetition of the original viral infection and liver disease after OLT.

The effects of interferon-alpha (IFN-alpha) on clinical and serologic manifestations of mixed cryoglobulinemia (MC) were investigated by randomized, crossover-controlled trial in 26 patients. The trial alternated 6 months with and 6 months without IFN-alpha therapy (2 x 10(6) IU daily for a month, then every other day for 5 months). In 22 patients, pretreatment steroid dosage remained unchanged during the study. Six patients dropped out (three because of side effects), whereas another 20 patients experienced a significant improvement of purpura (P.02) and serum transaminases (P.005) during IFN-alpha treatment. The presence of clinical improvement was supported by the outcome measurements of several immunologic parameters. In particular, serum cryoglobulins were significantly reduced (P.04) during IFN-alpha therapy. A rebound phenomenon of clinical and serologic parameters was observed after IFN-alpha discontinuation. In addition, no variations were recorded during 6 months without therapy. Hepatitis C virus (HCV) RNA was detected in 91% (20/22) of our MC patients; in 2/13 cases HCV RNA was no longer detectable in serum samples after IFN-alpha therapy. Thus, IFN-alpha could be considered as treatment for MC in patients with HCV seropositivity.

A recently introduced method for the detection of hepatitis B viral (HBV) DNA in the serum, using solution hybridization, was assessed for its sensitivity and specificity in 242 sera from patients with chronic HBV infection, and in controls. In 87 sera the results were compared with the classical dot-blot technique. The new method gave positive results in 82% (56/68) of all dot-blot-positive sera and in 97% (35/36) of those with strong positive dot-blot reactions. All 30 hepatitis B surface antigen (HBsAg) carriers positive for hepatitis B e antigen (HBeAg) were positive in the solution hybridization assay, with a mean HBV-DNA level of 172 +/- SE 36 pg/ml. Negative results were obtained in all 20 subjects negative for all HBV markers and in all 36 HBsAg+/anti-HBe+ carriers with normal aminotransferase activity, negative for hepatitis B core antigen (HBcAg) expression in the liver. By contrast 93% (66/71) of the anti-HBe+ patients with positive HBcAg expression in the liver were positive for serum HBV-DNA in the solution hybridization assay, with a mean level of 30.5 +/- SE 6.7 pg/ml. Of 14 sera with discrepant HBV-DNA results in the two assays, 12 were solution hybridization-negative/dot-blot-positive and 2 were solution hybridization-positive/dot-blot-negative. These results indicate that the new, simple method is specific, and suitable for the determination of serum HBV-DNA in clinical practice.(ABSTRACT TRUNCATED AT 250 WORDS)

A randomized controlled trial of recombinant interferon alfa-2b has been initiated in patients with chronic active hepatitis who were negative for serum hepatitis B e antigen but positive for serum hepatitis B virus DNA and hepatitis B core antigen expression in the liver. Twenty-five patients received interferon alfa-2b 3 million units thrice weekly for 14-16 weeks and 25 served as untreated controls. Seventeen patients in the treatment and 18 in the control group have already completed a 12-month period of observation. Interferon alfa-2b was well tolerated by all patients. At the end of therapy, complete responses, defined as disappearance of hepatitis B virus DNA from serum and return of alanine aminotransferase to normal, were observed in 10 (59%) of the 17 treated patients compared to none in the control group (p less than 0.01). Twelve months after the onset of interferon alfa-2b therapy, 11 (65%) of the 17 treated patients were complete responders compared to 2 (11%) of 18 in the control group (p less than 0.01). Fifty per cent (4/8) of complete responders to interferon alfa-2b therapy, followed for 16-24 months, experienced reactivations of hepatitis B virus replication with reappearance of serum hepatitis B virus DNA and a return of serum alanine aminotransferase activity. The response to interferon alfa-2b therapy appeared to be independent of pre-treatment serum alanine aminotransferase and hepatitis B virus DNA levels.

To the Editor. —A number of reports have suggested a role for hepatitis C virus (HCV) in a variety of hepatic and extrahepatic disorders (ie, chronic hepatitis, autoimmune hepatitis, porphyria cutanea tarda, Waldenstrom's macroglobulinemia, and mixed cryoglobulinemia).1This latter condition is a "benign" lymphoproliferative disorder that in some subjects can switch over to a malignant B-cell non-Hodgkin's lymphoma (NHL).1Moreover, the demonstration of the HCV lymphotropism2suggested a pathogenetic role for this virus in B-cell expansion underlying some lymphoproliferative disorders. We investigated the prevalence of HCV infection in 24 unselected patients with B-cell NHL (14 men, 10 women; mean±SD age [range], 61±10 years [35 to 74 years]). Diagnosis of B-cell NHL was done by lymph node biopsy specimen evaluated according to the Working Formulation classification3and by immunophenotypic analysis for surface T- and B-lymphocyte markers. All patients were Italian-born heterosexuals and had no history of drug