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Background: Sjögren's syndrome is an autoimmune disease characterised by dry eyes and mouth, systemic features, and reduced quality of life. There are no disease-modifying treatments. A new biologic, ianalumab (VAY736), with two modes of suppressing B cells, has previously shown preliminary efficacy. This dose-finding trial aimed to assess the safety and efficacy of different subcutaneous doses of ianalumab in patients with moderate to severe primary Sjögren's syndrome., Methods: VAY736A2201 was a randomised, parallel, double-blind, placebo-controlled, phase 2b dose-finding study done in 56 centres in 19 countries. Patients aged 18-75 years with primary Sjögren's syndrome with moderate to severe disease activity (European Alliance of Associations for Rheumatology [EULAR] Sjögren's Syndrome Disease Activity Index [ESSDAI] score ≥6) and symptom severity (EULAR Sjögren's Syndrome Patient Reported Index score ≥5) were eligible. Participants were randomly assigned (1:1:1:1) to receive subcutaneous placebo or ianalumab (5 mg, 50 mg, or 300 mg) every 4 weeks for 24 weeks using a secure, online randomisation system. Randomisation was stratified by the ESSDAI score at baseline (≥10 or <10). Study personnel and patients were masked to treatment assignment. The primary outcome was the change in ESSDAI score from baseline to 24 weeks in all randomly assigned patients. Dose-related change in disease activity (ESSDAI) from baseline at week 24 was assessed by multiple comparison procedure with modelling analysis. Safety was measured in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT02962895., Findings: Between June 27, 2017, and Dec 06, 2018, 293 patients were screened, 190 of whom were randomly assigned (placebo n=49, ianalumab 5 mg n=47, ianalumab 50 mg n=47, ianalumab 300 mg n=47). Statistically significant dose-responses were seen for overall disease activity (ESSDAI score) in four of the five dose-response models tested (p<0·025 in four models, p=0·060 in one model). The ESSDAI score decreased from baseline in all ianalumab groups, with the maximal ESSDAI score change from baseline observed in the ianalumab 300 mg group: placebo-adjusted least-squares mean change from baseline -1·92 points (95% CI -4·15 to 0·32; p=0·092). There were four serious adverse events in three patients considered treatment-related (pneumonia [n=1] and gastroenteritis [n=1] in the placebo group; appendicitis plus tubo-ovarian abscess in the same patient in the ianalumab 50 mg group)., Interpretation: The study met its primary objective, showing a dose-related decrease in disease activity as measured by ESSDAI at week 24. Overall, ianalumab was well tolerated and safe, with no increase in infections. To our knowledge, this is the first large, randomised, controlled trial in primary Sjögren's syndrome that met its primary endpoint, and its results mean there is potential for more studies of this mechanism in the future., Funding: Novartis., Competing Interests: Declaration of interests SJB and BAF have received support from the National Institute for Health Research (NIHR) Birmingham Biomedical Research Centre and the NIHR/Wellcome Trust Birmingham Clinical Research Facility. SJB has received consultancy funding from Abbvie, AstraZeneca, Galapagos, and Novartis in the past 36 months. RF has received consultancy funding from Pfizer and Eli Lilly. TD has received grants and consultancy funding from AbbVie, Celgene, Eli Lilly, EMD MerckSerono, GSK, Janssen, Novartis, and Roche; grants from UCB, Sanofi, Deutsche Forschungsgemeinschaft, and EU Horizon2020 HarmonicSS; and consultancy funding from Gilead/Galapagos. XM received consultancy funding from BMS, Galapagos, GSK, Novartis, and Servier and grants from Servier. AP received grants and consultancy funding from Novartis. TG-B has received consultancy fees from Eli Lilly and Novartis. BAF has received consultancy fees from BMS, Galapagos, Novartis, Roche, and Servier. HS-K received grants and consulting fees from Novartis. RJM received grants from Aintree University Hospital and Novartis. GJ, JNW, MAS, W-LL, and WH are employees of Novartis. All other authors declare no competing interests. The views expressed in this publication are those of the authors and not necessarily those of the institutions they are associated with., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectif: Cette étude a pour objectif de déterminer la prévalence du coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) 2019 (COVID-19) chez des patients adultes traités par biothérapies ou inhibiteurs des JAK pour des rhumatismes inflammatoires chroniques, en particulier des arthrites inflammatoires chroniques., Méthodes: Pour cela, une étude basée sur la population, dans la province d'Udine (466 700 habitants d'âge > 15 ans, région du Frioul-Vénétie-Julienne, Italie) a été planifiée. Le critère principal de jugement était la prévalence du COVID-19 durant les deux premiers mois de l'épidémie. Tous les patients de notre province atteints de maladies rhumatismales et traités par biothérapies ou inhibiteurs des JAK au cours des 6 mois précédents ont été inclus ( n  = 1051)., Résultats: Du 29 février au 25 avril 2020, 4 patients adultes (4/1051, 3,8/1000, IC 95 % 1,5-9,7/1000) ont été testés positifs au COVID-19 par RT-PCR et écouvillon. Au total, 47/1051 patients (4,5 %) ont été soumis au test COVID-19 par RT-PCR durant la même période, en raison de symptômes compatibles avec le COVID-19 pour 15 d'entre eux. Dans la population générale, la prévalence était de 937 cas/466700 (2/1000, IC 95 % 1,9-2,1/1000, valeur p  = 0,33, test du Chi 2 ), et 20 179/466 700 (4,3 %) prélèvements COVID-19 sur écouvillon ont été effectués., Conclusion: Le risque de COVID-19 chez les patients atteints de maladies rhumatismales et traités par biothérapies ou inhibiteurs des JAK n'apparaît pas différent de celui observé dans la population générale. Les patients doivent être encouragés à poursuivre en toute sécurité leur traitement et à respecter les mesures de prévention et de protection contre le COVID-19., (© 2021 Société Française de Rhumatologie. Published by Elsevier Masson SAS. All rights reserved.)

Four undescribed and 17 known diterpenoids were isolated from the roots of Zhumeria majdae Rech.f. & Wendelbo. Using 1D and 2D NMR spectroscopy, ECD spectroscopy, and HRESIMS data analysis, the structures of the undescribed compounds were elucidated. The anti-proliferative activity of isolated compounds was evaluated against HeLa and MCF7 cancer cell lines. The binding affinity of all compounds to HSP90, one of the targets for the modern anticancer therapy, was investigated using surface plasmon resonance. The results demonstrated that lanugon Q interacted with the chaperone. To explain its mechanism of action, experimental and computational tests were also conducted., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Salivary gland ultrasonography (SGUS) has proven to be a promising tool for diagnosing various diseases manifesting with abnormalities in salivary glands (SGs), including primary Sjögren's syndrome (pSS). At present, the major obstacle for establishing SUGS as a standardized tool for pSS diagnosis is its low inter/intra observer reliability. The aim of this study was to address this problem by proposing a robust deep learning-based solution for the automated segmentation of SGUS images. For these purposes, four architectures were considered: a fully convolutional neural network, fully convolutional "DenseNets" (FCN-DenseNet) network, U-Net, and LinkNet. During the course of the study, the growing HarmonicSS cohort included 1184 annotated SGUS images. Accordingly, the algorithms were trained using a transfer learning approach. With regard to the intersection-over-union (IoU), the top-performing FCN-DenseNet (IoU = 0.85) network showed a considerable margin above the inter-observer agreement (IoU = 0.76) and slightly above the intra-observer agreement (IoU = 0.84) between clinical experts. Considering its accuracy and speed (24.5 frames per second), it was concluded that the FCN-DenseNet could have wider applications in clinical practice. Further work on the topic will consider the integration of methods for pSS scoring, with the end goal of establishing SGUS as an effective noninvasive pSS diagnostic tool. To aid this progress, we created inference (frozen models) files for the developed models, and made them publicly available., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background and Aims: Calcium is an essential element for human health, with key roles in the prevention and therapy of multifactorial conditions. Calcium dietary intake is often insufficient in the general population. The aim of this study was to perform a clinical audit for general practitioners (GPs) to understand the efficacy of training intervention on doctors' awareness about dietary calcium and supplements., Methods and Results: General practice outpatients were enrolled (Before Clinical Audit, BCA) from the same sanitary district, and calcium dietary intake was evaluated with a validated questionnaire, also collecting information about the consumption of calcium and vitamin D supplements. Then, a training intervention with a frontal lesson and discussion with GPs involved was performed. After one month of this intervention, a second outpatient enrolment was performed (Post Clinical Audit, PCA) in the same general practices to evaluate differences in nutritional suggestions and supplement prescription by GPs. In BCA, the calcium dietary intake was low, with nobody reaching 1000 mg as suggested by the guidelines. Only 6.6% and 24.5% took calcium and vitamin D supplements, respectively; in the PCA, these percentages increased to 28% and 78% for calcium and vitamin D supplements, respectively (p < 0.01 PCA vs BCA). There were no differences in calcium dietary intake between BCA and PCA., Conclusion: Training intervention on GPs was successful to sensitize them regarding calcium intake problems; GPs tended to increase the prescription of supplements but not to suggest changes in dietary habits., Competing Interests: Declaration of competing interest No one of the Authors have conflicts of interest., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Objective: Some of the manifestations of mixed cryoglobulinemia syndrome (MCS) can be severe or life-threatening, and should be rapidly contained but, as the therapeutic approaches to such conditions are largely based on anecdotal data, a consensus conference was organised by the Italian Group for the Study of Cryoglobulinemia (GISC) with the aim of providing a set of recommendations based on an in-depth survey of the available data and expert opinion., Methods: The consensus panel, which included specialists working in different medical fields involved in the management of MCS patients, was first asked to divide the manifestations of MCS into severe or life-threatening conditions on the basis of their own experience, after which a complete literature review was carried out in accordance with the Cochrane guidelines for systematic reviews., Results: Therapeutic plasma exchange (TPE) was considered the elective first-line treatment in the case of life-threatening manifestations of MCS (LT-MCS) and patients with severe clinical symptoms (S-MCS) who fail to respond to (or who are ineligible for) other treatments. The data supporting the combined use of cyclophosphamide and TPE were considered limited and inconclusive. High-dose pulsed glucocorticoid (GCS) therapy can be considered the first-line treatment of severe MCS, generally in association with TPE. Rituximab (RTX)-based treatments should be considered in patients with skin ulcers, peripheral neuropathy or glomerulonephritis, and in patients with persistent LT-MCS after TPE. In patients with hepatitis C virus-related MCS with S-MCS, viral eradication should be attempted as soon as a patient's condition allows the use of direct-acting antivirals., (Copyright © 2019. Published by Elsevier B.V.)

Data quality assessment has gained attention in the recent years since more and more companies and medical centers are highlighting the importance of an automated framework to effectively manage the quality of their big data. Data cleaning, also known as data curation, lies in the heart of the data quality assessment and is a key aspect prior to the development of any data analytics services. In this work, we present the objectives, functionalities and methodological advances of an automated framework for data curation from a medical perspective. The steps towards the development of a system for data quality assessment are first described along with multidisciplinary data quality measures. A three-layer architecture which realizes these steps is then presented. Emphasis is given on the detection and tracking of inconsistencies, missing values, outliers, and similarities, as well as, on data standardization to finally enable data harmonization. A case study is conducted in order to demonstrate the applicability and reliability of the proposed framework on two well-established cohorts with clinical data related to the primary Sjögren's Syndrome (pSS). Our results confirm the validity of the proposed framework towards the automated and fast identification of outliers, inconsistencies, and highly-correlated and duplicated terms, as well as, the successful matching of more than 85% of the pSS-related medical terms in both cohorts, yielding more accurate, relevant, and consistent clinical data., (Copyright © 2019. Published by Elsevier Ltd.)

Autoimmune rheumatic diseases are characterised by an abnormal immune system response, complement activation, cytokines dysregulation and inflammation. In last years, despite many progresses in managing these patients, it has been shown that clinical remission is reached in less than 50% of patients and a personalised and tailored therapeutic approach is still lacking resulting in a significant gap between guidelines and real-world practice. In this context, the need for biomarkers facilitating early diagnosis and profiling those individuals at the highest risk for a poor outcome has become of crucial interest. A biomarker generally refers to a measured characteristic which may be used as an indicator of some biological state or condition. Three different types of medical biomarkers has been suggested: i. mechanistic markers; ii. clinical disease markers; iii. therapeutic markers. A combination of biomarkers from these different groups could be used for an ideal more accurate diagnosis and treatment. However, although a growing body of evidence is focused on improving biomarkers, a significant amount of this information is not integrated on standard clinical care. The overarching aim of this work was to clarify the meaning of specific biomarkers during autoimmune diseases; their possible role in confirming diagnosis, predicting outcome and suggesting specific treatments., (Copyright © 2018. Published by Elsevier B.V.)

The main aim of this study was to define and apply a multidisciplinary and multi-criteria approach to sustainability in evaluating alternative end-uses for disused areas. Taking into account the three pillars of sustainability (social, economic and environmental dimension) as well as the need for stakeholders to have new practical instruments, the innovative approach consists of four modules stated (i) sociological, (ii) economic, (iii) environmental and (iv) multi-criteria assessment. By means of a case study on a small Municipality in Southern Italy, three end-uses alternatives, representing three essential services for citizens, were selected: Municipal gym; Market area; Municipal Solid Waste (MSW) separate collection centre. The sociological module was useful to select the most socially sound alternative by means of a consultative referendum, simulated with the use of a structured questionnaire administered to a sample of the population. The economic evaluation was conducted defining the bill of quantities with regarding to six main items (soil handling, landfill disposal tax, public services, structure and services, completion work, equipment and furnishings). The environmental evaluation was performed applying the Delphi method with local technicians who were involved in a qualitative-quantitative evaluation of the three alternatives with regarding to eight possible environmental impacts (landscape impact, soil handling, odour, traffic, noise, atmospheric pollution, wastewater, waste). Finally, the Simple Additive Weighting was used as multi-criteria technique to define alternatives priorities. The obtained results showed how the multi-criteria analysis is a useful decision support tool able to identify transparently and efficiently the most sustainable solutions to a complex social problem., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a common form of dementia mainly characterized by the deposition of neurofibrillary tangles and β-amyloid (Aβ) peptides in the brain. Additionally, increasing evidence demonstrates that a neuro-inflammatory state plays a key role in the development of this disease. Beside synthetic drugs, the use of natural compounds represents an alternative for the development of new potential drugs for the treatment of AD. Among these, the root of Salvia miltiorhiza Bunge (also known as Danshen) used for the treatment of cardiovascular, cerebrovascular disease and CNS functional decline in Chinese traditional medicine is one of the most representative examples. We therefore evaluated the effects of tanshinone IIA (TIIA) and cryptotanshinone (CRY) (the two major lipophilic compounds of Danshen) in a non-genetic mouse model of β-amyloid (Aβ)-induced AD, which is mainly characterized by reactive gliosis and neuro-inflammation in the brain. To this aim, mice were injected intracerebroventricularly (i.c.v.) with Aβ 1-42 peptide (3μg/3μl) and after with TIIA and CRY (1, 3, or 10mg/kg) intraperitoneally (i.p.) 3 times weekly for 21days following the induction of experimental AD. Spatial working memory was assessed as a measure of short-term memory in mice, whereas the level of GFAP, S100β, COX-2, iNOS and NF-kBp65 monitored by western blot and ELISA assay, were selected as markers of reactive gliosis and neuro-inflammation. Finally, by docking studies, the modulation of key pro-inflammatory enzymes and pathways involved in the AD-related neuro-inflammation were also investigated. Results indicate that TIIA and CRY alleviate memory decline in Aβ 1-42 -injected mice, in a dose dependent manner. Moreover, the analysis of gliosis-related and neuro-inflammatory markers in the hippocampal tissues reveal a remarkable reduction in the expression of GFAP, S100β, COX-2, iNOS and NF-kBp65 after CRY (10mg/kg) treatment. These effects were less evident, but still significant, after TIIA (10mg/kg). Finally, in silico analysis also revealed that both compounds were able to interact with the binding sites of NF-kBp65 endorsing the data from biochemical analysis. We conclude that TIIA and CRY display anti-inflammatory and neuroprotective effect in a non-genetic mouse model of AD, thus playing a role in slowing down the course and onset of AD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Autoimmune diseases are a complex set of diseases characterized by immune system activation and, although many progresses have been done in the last 15years, several unmet needs in the management of these patients may be still identified. Recently, a panel of international Experts, divided in different working groups according to their clinical and scientific expertise, were asked to identify, debate and formulate a list of key unmet needs within the field of rheumatology, serving as a roadmap for research as well as support for clinicians. After a systematic review of the literature, the results and the discussions from each working group were summarised in different statements. Due to the differences among the diseases and their heterogeneity, a large number of statements was produced and voted by the Experts to reach a consensus in a plenary session. At all the steps of this process, including the initial discussions by the steering committee, the identification of the unmet needs, the expansion of the working group and finally the development of statements, a large agreement was attained. This work confirmed that several unmet needs may be identified and despite the development of new therapeutic strategies as well as a better understanding of the effects of existing therapies, many open questions still remain in this field, suggesting a research agenda for the future and specific clinical suggestions which may allow physicians to better manage those clinical conditions still lacking of scientific clarity., (Copyright © 2017. Published by Elsevier B.V.)

Objective: This study aims to compare clinical and laboratory features of patients who, while satisfying the American European Consensus Group (AECG) criteria for primary Sjögren's syndrome (SS), do not present the positivity for anti-Ro/SSA and/or anti-La/SSB, with patients that meet the AECG criteria and are positive for anti-Ro/SSA and/or anti-La/SSB., Methods: 548 patients were selected based on the following criteria, and exclusion of patients negative for histopathology but positive for anti-Ro/SSA and anti-La/SSB: 1. Fulfilment of the AECG criteria, 2. Performance of minor salivary gland biopsy, 3. Search for anti-Ro/SSA and anti-La/SSB, 4. Absence of hepatitis C virus infection. Univariate and multivariate analyses were performed., Results: Two groups were compared: 342 patients were positive for both the histopathology and for anti-Ro/SSA and/or anti-La/SSB (H-only) and 206 patients were positive for histopathology, but negative for autoantibodies (H+SSA/SSB). The following variables were statistically found to be associated with H+SSA/SSB: younger age at diagnosis (p<0.0001), glandular swelling (p=0.01), purpura (p=0.04), leucopoenia (p=0.0001), lymphoma (p=0.002), low C3 (p=0.04), low C4 (p=0.01), hypergammaglobulinemia (p<0.0001), ANA (p<0.0001), rheumatoid factor (p<0.0001), and serum cryoglobulins (p=0.039). ANA positivity (OR 6.9), hypergammaglobulinemia (OR 5.1), positive rheumatoid factor (OR 2.3), and age at diagnosis (OR 0.97) were also selected by multivariate analyses as associated with H+SSA/SSB., Conclusion: Primary SS negative for anti-Ro/SSA and anti-La/SSB antibodies appears to be characterized by a lower risk of lymphoma and by a lower level of B-cell expansion., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mixed cryoglobulinaemia syndrome (MCS) is associated with a number of infectious, autoimmune and lymphoproliferative disorders, particularly chronic hepatitis C infection. Although circulating mixed cryoglobulins (cMCGs) are a frequent finding in HCV-infected patients, only a minority of them develop a frank MCS. The only available data concerning the prevalence of MCS, which is generally considered a rare disease, come from hospital records. The aim of this investigation was to estimate the prevalence of cMCGs and MCS in a population-based study. All of the adult residents in Origgio, a town of about seven thousand inhabitants in northern Italy, were mailed a validated questionnaire, and a randomly selected sample of respondents was invited to undergo a clinical examination and laboratory tests including the determination of cMCGs. The 1594 respondents to the questionnaire (54.3% women, 64.5% aged >49years) accounted for 26.4% of the total adult population. Forty-nine (3.1%) positively responded to at least two questions, including a disproportionately high number of people aged >70years (p=0.001). Of the 266 randomly selected subjects invited to undergo a clinical examination and laboratory tests, 147 accepted, 30 (20.4%) of whom had asymptomatic type III cMCGs and four MCS. The risk of cMCG positivity was independently associated with C4 levels of <16mg/dL (adjusted odds ratio [AOR] 4.40, 95% confidence interval [CI] 1.07-18.08; p=0.040) and HCV positivity (AOR 6.87, 95% CI 1.16-40.79; p=0.034). No co-morbidities known to be related to cMCG production could be detected in more than 50% of the positive cases. After including the other positive respondents who agreed to undergo a clinical examination, the number of diagnosed MCS increased to seven: five HCV-related, one HBV-related, and one essential MCS. In conclusion, MCS seems to be more frequent than expected for a 'rare' disease, and the unexpectedly high prevalence of cMCGs raises questions about the frequency with which they are triggered, the spectrum of diseases involved in triggering them, and their real role as disease indicators., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

Identification of genetic biomarkers of response to biologics in rheumatoid arthritis (RA) is a relevant issue. Being IL-6 a key cytokine for B cell survival, the interleukin-6 (IL-6) -174G>C and the IL-6 receptor (IL-6R) D358A gene polymorphisms were investigated in 158 RA patients treated with rituximab (RTX). One hundred and twenty-eight (81.0%) were RF positive and 126 (79.7%) were anti-CCP positive. Response to therapy was evaluated at the end of the sixth month after the first RTX infusion, by using both the EULAR and the ACR criteria. The possible relationship with IL-6 serum levels was also studied. By univariate analysis, lack of response by the EULAR criteria was more prevalent in RA patients with the IL-6 -174 CC genotypes (39.1%), than in the GC/GG patients (18.5%) (OR 2.83; 95%CI=1.10-7.27; p=0.031). A good response was noticed in only one patient (4.3%) with the IL-6 -174 CC genotype, while it was present in 24.4% of GG/GC cases (p=0.06). By stepwise multivariate analysis (including RA duration, baseline DAS28, baseline HAQ, RF status, anti-CCP status and IL-6 genotype as covariates), the IL-6 -174CC genotype was selected as an independent predictor of no response to RTX by both EULAR and ACR≥50 criteria, while the IL-6R polymorphism resulted as not associated. No definite association between gene polymorphisms and IL-6 serum levels was noticed. Present results suggest a possible role for IL-6 genotyping to better plan treatment with RTX in RA, and larger studies are worthwhile., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Hematopoietic stem cells (HSCs) interact with osteoblastic, stromal, and vascular components of the BM hematopoietic microenvironment (HM) that are required for the maintenance of long-term self-renewal in vivo. Osteoblasts have been reported to be a critical cell type making up the HSC niche in vivo. Rac1 GTPase has been implicated in adhesion, spreading, and differentiation of osteoblast cell lines and is critical for HSC engraftment and retention. Recent data suggest a differential role of GTPases in endosteal/osteoblastic versus perivascular niche function. However, whether Rac signaling pathways are also necessary in the cell-extrinsic control of HSC function within the HM has not been examined. In the present study, genetic and inducible models of Rac deletion were used to demonstrate that Rac depletion causes impaired proliferation and induction of apoptosis in the OP9 cell line and in primary BM stromal cells. Deletion of Rac proteins caused reduced trabecular and cortical long bone growth in vivo. Surprisingly, HSC function and maintenance of hematopoiesis in vivo was preserved despite these substantial cell-extrinsic changes. These data have implications for therapeutic strategies to target Rac signaling in HSC mobilization and in the treatment of leukemia and provide clarification to our evolving concepts of HSC-HM interactions.

Objective: Mixed cryoglobulinemia syndrome (MCs) is a systemic vasculitis characterized by multiple organ involvement due to the vascular deposition of immune-complexes, mainly the cryoglobulins. B-lymphocyte expansion represents the underlying pathological alteration frequently triggered by hepatitis C virus (HCV) infection. The treatment of MCs syndrome is generally based on antiviral drugs and/or immunosuppressors, among which rituximab, an anti-CD20 monoclonal antibody, has been usefully employed for both cutaneous and visceral MCs organ involvement. This multicenter study retrospectively evaluated the effects of rituximab in a large series of patients with active MCs. The observed results were compared to those emerging from the updated review of the literature on this topic., Methods: The study included 87 patients (male/female 19/68, mean age 62.3±11.4SD years, mean disease duration 9±6.2SD years, HCV infection in 92% of cases) with active cryoglobulinemic vasculitis evaluated before rituximab monotherapy and after 6-month follow-up by means of main clinico-serological parameters. A PubMed search up to May 31, 2011, was done to find published clinical studies, including case reports of MCs treated with rituximab., Results: A significant clinical improvement was observed in a relevant percentage of cases, regardless the presence/absence of associated HCV infection; namely, complete/partial remission of pre-treatment active manifestations was observed in 74% of skin purpuric lesions, up to 87% of non-healing vasculitic leg ulcers, and 44% of the peripheral neuropathy, mainly paresthesias (patient's visual analogical scale from 62±25 to 37±27; p≤.0001). Moreover, cryoglobulinemic nephropathy, observed in 38 patients, significantly improved in 95% of cases (serum creatinine from 1.8±1.1SD to 1.4±0.8SD mg/dl, p≤.0001; 24-hour proteinuria from 2.2±2.1SD to 0.9±1.7SD g/24h, p≤.0001), with complete remission in the 50%. Among 6 patients with complicating non-Hodgkin's B-cell lymphoma a complete or partial remission was observed in 5/6. A complete remission of abdominal vasculitis was also observed in one patient. These beneficial effects were mirrored by the improvement of cryoglobulinemic serological hallmarks, namely cryocrit and low complement C4, in half cases. The safety of rituximab was confirmed by the small number of side effects recorded during the 6-month follow-up. On the whole, the results of the present study are in keeping with those reported in 39 papers present in world literature, including a total of 279 MCs patients., Conclusions: Rituximab may be regarded as useful and safe pathogenetic treatment of cryoglobulinemic vasculitis. The actual role of this drug should be definitely confirmed by randomized controlled trials, as well as its position in the therapeutical strategy, mainly with respect to antiviral treatment in HCV-associated MCs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: The objective of this review was to define a core set of recommendations for the treatment of HCV-associated mixed cryoglobulinemia syndrome (MCS) by combining current evidence from clinical trials and expert opinion., Methods: Expert physicians involved in studying and treating patients with MCS formulated statements after discussing the published data. Their attitudes to treatment approaches (particularly those insufficiently supported by published data) were collected before the consensus conference by means of a questionnaire, and were considered when formulating the statements., Results: An attempt at viral eradication using pegylated interferon plus ribavirin should be considered the first-line therapeutic option in patients with mild-moderate HCV-related MCS. Prolonged treatment (up to 72 weeks) may be considered in the case of virological non-responders showing clinical and laboratory improvements. Rituximab (RTX) should be considered in patients with severe vasculitis and/or skin ulcers, peripheral neuropathy or glomerulonephritis. High-dose pulsed glucocorticoid (GC) therapy is useful in severe conditions and, when necessary, can be considered in combination with RTX; on the contrary, the majority of conference participants discouraged the chronic use of low-medium GC doses. Apheresis remains the elective treatment for severe, life-threatening hyper-viscosity syndrome; its use should be limited to patients who do not respond to (or who are ineligible for) other treatments, and emergency situations. Cyclophosphamide can be considered in combination with apheresis, but the data supporting its use are scarce. Despite the limited available data, colchicine is used by many of the conference participants, particularly in patients with mild-moderate MCS refractory to other therapies. Careful monitoring of the side effects of each drug, and its effects on HCV replication and liver function tests is essential. A low-antigen-content diet can be considered as supportive treatment in all symptomatic MCS patients. Although there are no data from controlled trials, controlling pain should always be attempted by tailoring the treatment to individual patients on the basis of the guidelines used in other vasculitides., Conclusion: Although there are few controlled randomised trials of MCS treatment, increasing knowledge of its pathogenesis is opening up new frontiers. The recommendations provided may be useful as provisional guidelines for the management of MCS., (Copyright © 2011. Published by Elsevier B.V.)

Elevated B-Lymphocyte Stimulator (BLyS) and April (a proliferation-inducing ligand) expressions characterize several autoimmune diseases. We here analysed the possible role of BLyS and April in autoimmune thyroid diseases (AITD), comprising Hashimoto's thyroiditis (HT) and Graves' disease (GD). Seventy-seven patients with AITD and 77 blood donors (HBD) were enrolled in the study. Serum BLyS and April levels were assessed by ELISA. Results indicated a significant upregulation of BLyS in AITD patients (1.12+/-0.39 ng/ml versus 0.666+/-0.240 ng/ml in HBD; p<0.0001), with GD patients presenting higher BLyS levels than HT patients (1.22+/-0.42 ng/ml versus 1.07+/-0.38 ng/ml; p=0.0393). In contrast, April levels were downregulated, but only in HT patients [9.9+/-36.6 (median 0) ng/ml versus 7.4+/-22.1 (median 1.16) ng/ml in HBD; p=0.003; and versus 4.2+/-5.9 ng/ml (median 0.9) ng/ml in GD; p=0.0353]. In HT patients, Levo-thyroxine supplementation further increased BLyS and tended to normalize April levels. Neither BLyS nor April did correlate with the levels of the pathognomonic autoantibodies (TPOAb, TgAb, TRAb). Data are preliminary, but, for the first time, we provide the analyses of BLyS and April levels in AITD patients, suggesting new tools for the diagnosis, prognosis and possible therapeutic management of AITD., (Copyright 2009 Elsevier B.V. All rights reserved.)

Mixed cryoglobulinemia syndrome (MCsn) is a systemic vasculitis prevalently mediated by immune complexes, i.e., mixed cryoglobulins, and characterized by non-neoplastic B-cell lymphoproliferation favouring the progression into frank B-cell non-Hodgkin lymphoma (NHL) in 5-10% of patients. The hepatitis C virus (HCV) infection is the etiologic agent in the large majority of MCsn cases and chronic antigenic stimulation by HCV is considered a key mechanism sustaining the proliferation of the RF-secreting B-cell clones. Besides chronic antigenic stimulation, cytokines and growth factors may also play a key role in sustaining B-cell overactivation. B-lymphocyte stimulator (BlyS) was recently described as a critical survival factor for B cells, promoting their activation and maturation. Abnormal production of BLyS alters immune tolerance by allowing the survival of autoreactive B cells, thus triggering autoimmune disorders. BLyS inhibits B-cell apoptosis, and B-cell apoptosis is implicated in the pathogenesis of MCsn, as well as of other autoimmune diseases. Both antiviral therapy and B- cell depletive therapy in MCsn may influence BlyS expression. Antiviral therapy, monotherapy against biologic targets downstream viral infection, or the combination of the two, should be optimized in the single patient and stage of the disease, based on disease pathobiology, efficacy and safety issues.

Type II mixed cryoglobulinemia syndrome (MCsn) is a systemic vasculitis mainly linked to immune complex deposition in several organs and to hepatitis C virus (HCV) infection. Therapeutic strategies can target either the viral trigger HCV if present, or pathogenic events downstream the triggering infection, e.g, the proliferating B-cells directly. Antiviral therapy should be considered as first-line treatment in many HCV-positive patients. However, it may prove ineffective, contraindicated, or poorly tolerated. On the other hand, the other available treatments (such as cytotoxic agents, plasma exchange and steroids) may lead to life-threatening complications and may be difficult to manage in the long term. Given the good safety profile in lymphomas, rituximab (RTX) has been used off-label for numerous patients suffering from a variety of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis/ polymyositis, and anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis. Efficacy and safety of RTX in MCsn and in particular in MCsn-related glomerulonephritis was recently described. Based on these results, a multicentre, controlled, randomised, clinical trial is now ongoing to compare RTX versus the best available treatments in some severe MCsn manifestations (i.e. skin ulcers, sensory and/or motor neuropathy and active glomerulonephritis).

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in beta 9 and its polymorphism is connected with preferred anchors at beta 9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

Based on new biologic and clinical insights, the number of drugs blocking different biologic targets in rheumatoid arthritis (RA) [e.g., tumor necrosis factor alpha (TNFalpha), CTLA4, interleukin (IL)-1, IL-6, IL-15, IL-18, B lymphocyte stimulator (BLyS), CD20] has increased considerably over the last decade. Rituximab, a chimeric monoclonal antibody that was developed for the treatment of B-cell lymphomas, has been used in different autoimmune diseases where B-cells are thought to play a pivotal role. However, blinded randomised controlled trials have been completed only for RA so far, indicating the clear efficacy of B-cell blockade in RA and highlighting the pathogenetic B-cell in rheumatoid synovitis. The use of rituximab in RA is herein updated, from early preliminary studies to more recent presentations in International Conferences. Key clinical and biologic issues are discussed, i.e., efficacy and safety of rituximab, role of concomitant therapies, use in the long term and retreatment strategies, differences with anti-TNFalpha therapy. The possible indications in RA are finally discussed, also on the ground of personal experience with rituximab in RA and other rheumatic diseases associated with B-cell lymphoproliferation. Further clinical research should go hand in hand with laboratory research, and tissue studies are now needed.

An increased prevalence of hematologic malignancies is often described in patients with Sjögren's syndrome (SS). Viruses have been proposed as possible etiologic or triggering agents of systemic autoimmune diseases (SADs), with hepatitis C virus (HCV) being one of the viruses most frequently associated with autoimmune features and with systemic autoimmune diseases such as mixed cryoglobulinemia or SS. Moreover, the association between HCV infection and hematologic malignancies, mainly non-Hodgkin's lymphoma (NHL), is supported by several studies. For these reasons, the recognized association of specific systemic autoimmune diseases (mainly SS and mixed cryoglobulinemia) with HCV infection, added to the possible evolution of any one of these entities into a B-cell NHL, suggests the possibility of a close relationship among SS, HCV and B-cell lymphoproliferative disorders, especially in patients with type II mixed cryoglobulinemia.

The best treatment of type II mixed cryoglobulinemia (MC) has still to be defined. Antiviral treatment for the frequent underlying infectious trigger hepatitis C virus (HCV) may be ineffective, contraindicated, or not tolerated in a fraction of cases, whereas current immunosuppressive treatments may lead to relevant complications. Selective B-cell blockade with rituximab was used in this study, based on favorable results in preliminary experience. Fifteen consecutive patients with type II MC (HCV-related in 12 of 15) were treated with rituximab, 375 mg/m(2) intravenously weekly for 4 weeks. Only medium- to low-dose steroids were allowed, if already administered at the time of recruitment. All patients had active disease, poorly controlled or difficult to manage with previous treatments, including corticosteroids in all. Efficacy and safety of rituximab therapy were evaluated in the following 6 months. The overall follow-up after rituximab treatment ranged from 9 to 31 months. Rituximab proved effective on skin vasculitis manifestations (ulcers, purpura, or urticaria), subjective symptoms of peripheral neuropathy, low-grade B-cell lymphoma, arthralgias, and fever. Nephritis of recent onset went into remission in one case. Laboratory features, that is, significantly decreased serum rheumatoid factor and cryoglobulins and increased C4, were consistent with the clinical efficacy. Treatment was well tolerated, with no infectious complications. Thrombosis of retinal artery or self-limiting panniculitis occurred in one patient each. Rituximab may represent a safe and effective alternative to standard immunosuppression in type II MC. Controlled studies are needed to better define drug indications and the cost-efficacy profile in the different systemic manifestations.

Study Objective: To evaluate the effects of beta-blockers on ventilation in heart failure patients. Indeed, beta-blockers ameliorate the clinical condition and cardiac function of heart failure patients, but not exercise capacity. Because ventilation is inappropriately elevated in heart failure patients due to overactive reflexes from ergoreceptors and chemoreceptors, we hypothesized that beta-blockers can elicit their positive clinical effects through a reduction of ventilation., Design: This was a double-blind, randomized, placebo-controlled study., Setting: University hospital heart failure unit., Patients and Interventions: While receiving placebo (2 months) and a full dosage of carvedilol (4 months), 15 chronic heart failure patients were evaluated by quality-of-life questionnaire, pulmonary function tests, cardiopulmonary exercise tests with constant workload, and a ramp protocol., Results: Therapy with carvedilol did not affect resting pulmonary function and exercise capacity. However, carvedilol improved the results of the quality-of-life questionnaire, reduced the mean (+/- SD) slope of the minute ventilation (E)/carbon dioxide output (CO(2)) ratio (from 36.4 +/- 8.9 to 31.7 +/- 3.8; p < 0.01) and reduced ventilation at the following times: at peak exercise (from 60 +/- 14 to 48 +/- 15 L/min; p < 0.05); during the intermediate phases of a ramp-protocol exercise; and during the steady-state phase of a constant-workload exercise (from 42 +/- 14 to 34 +/- 13 L/min; p < 0.05, at third min). The end-expiratory pressure for carbon dioxide increased as ventilation decreased. The reduction in the E/CO(2) ratio was correlated with improvement in quality of life (r = 0.603; p < 0.02)., Conclusions: Improvement in the clinical conditions of heart failure patients treated with carvedilol is associated with reductions in the inappropriately elevated ventilation levels observed during exercise.

Analysis of the immunoglobulin receptor (IGR) variable heavy- and light-chain sequences on 17 hepatitis C virus (HCV)-associated non-Hodgkin lymphomas (NHLs) (9 patients also had type II mixed cryoglobulinemia [MC] syndrome and 8 had NHL unrelated to MC) and analysis of intraclonal diversity on 8 of them suggest that such malignant lymphoproliferations derive from an antigen-driven pathologic process, with a selective pressure for the maintenance of a functional IgR and a negative pressure for additional amino acid mutations in the framework regions (FRs). For almost all NHLs, both heavy- and light-chain complementarity-determining regions (CDR3) showed the highest similarity to antibodies with rheumatoid factor (RF) activity that have been found in the MC syndrome, thus suggesting that a common antigenic stimulus is involved in MC syndrome and in HCV-associated lymphomagenesis. Moreover, because HCV is the recognized pathologic agent of MC and the CDR3 amino acid sequences of some HCV-associated NHLs also present a high homology for antibody specific for the E2 protein of HCV, it may be reasonable to speculate that HCV E2 protein is one of the chronic antigenic stimuli involved in the lymphomagenetic process. Finally, the use of specific segments, in particular the D segment, in assembling the IgH chain of IgR seems to confer B-cell disorders with the property to produce antibody with RF activity, which may contribute to the manifestation of an overt MC syndrome.

A pathogenetic role of the hepatitis C virus (HCV) has been hypothesized for a subset of B-cell non-Hodgkin's lymphomas (NHLs). However, the preliminary characterization of B-cell NHLs in HCV-infected individuals has been poorly addressed. In the present study, we report detailed information on 35 consecutive patients with overt B-cell NHL of recent onset and HCV infection; all patients referred to a single oncological center in Northeast Italy. Histopathologic evaluation was performed by a single reference hemopathologist, and the link with the two relevant autoimmune diseases predisposing to B-cell NHL and in which HCV has been implied, ie, "essential" mixed cryoglobulinemia (EMC) and Sjogren's syndrome, was investigated. Control groups included 122 consecutive HCV-negative patients with B-cell NHL and 464 consecutive histopathologic cases of B-cell NHL referred to the same center, as well as 127 consecutive patients with HCV infection and without lymphoma referred to a different center in the same geographical area. B-cell NHLs in HCV-infected patients frequently presented at onset (1) an extranodal localization with peculiar target organs of HCV infection (ie, the liver and major salivary glands) being significantly overrepresented; (2) a diffuse large cell histotype without any prior history of low-grade B-cell malignancy or bone marrow involvement; and (3) a weak association with a full-blown predisposing autoimmune disease, although serum autoimmune features were common and cryoglobulins were always present. Therefore, the HCV-related B-cell NHLs in this oncological series presented distinctive features compared with B-cell NHLs in HCV-negative patients, and they differed from bone marrow low-grade NHLs frequently diagnosed in HCV-positive patients with EMC. Such novel information may be relevant for future research aimed at clarifying the possible link between HCV infection, autoimmunity, nonmalignant B-cell lymphoproliferation, and overt B-cell malignancy.

Six Epstein-Barr virus (EBV)-related lymphoproliferative disorders were investigated to verify whether the EBV strain harbored by neoplastic cells had the same EBNA-2 and latent membrane protein-1 (LMP-1) DNA sequences of the virus carried by normal lymphocytes of the same patients. Within each case, the analysis of neoplastic lymph nodes, reactive lymphadenopathies, and/or EBV+ spontaneous lymphoblastoid cell lines gave concordant results with respect to type-specific EBNA-2 region and LMP-1 gene. In particular, five cases showed the same deletion in the 3' end of the LMP-1 gene in both normal and neoplastic cells. We also determined the prevalence of LMP-1 deletions in a large series of normal peripheral blood mononucleated cells (PBMCs) from Italian individuals. The analysis showed that 50% (9 of 18) of PBMCs from human immunodeficiency virus (HIV)-seronegative donors carried a 30-bp deletion in the C-terminal portion of the LMP-1 gene, whereas a nondeleted fragment was amplified in about 44% (8 of 18) of the cases. Only one sample (5.6%) showed the amplification of a full-length LMP-1 band together with a deleted fragment. Similarly, PBMCs from HIV-infected patients showed an almost equivalent prevalence of full-length (17 of 37, 46%) and deleted (16 of 37, 43.2%) LMP-1 fragments, whereas about 11% of samples (4 of 37) showed evidence of double infections. Of note, deletions in the LMP-1 gene were detected with similar prevalence values in EBV+ Hodgkin's disease (HD) (13 of 30, 43.3%) and non-Hodgkin's lymphoma (NHL) (2 of 5, 40%) cases from HIV-seronegative patients and in HIV-related, EBV+ NHLs (4 of 7, 57.1%). Conversely, a 30-bp LMP-1 deletion was found in 10 of 12 HIV-associated HD cases (83%), a prevalence significantly higher than that detected in HIV-unrelated HD (P = .01). These findings indicate that: (1) the same EBV strain carrying LMP-1 deletions is harbored by normal and neoplastic cells of patients with EBV+ disorders, ruling out that these mutations might result from immunoselection phenomena; (2) in the Italian population, the prevalence of LMP-1 deletion mutants is comparable to that of EBV strains with full-length LMP-1; (3) HIV-induced immunosuppression is not associated with an increased prevalence of LMP-1 deletions in PBMCs; and (4) HIV-related HD cases, but not those of HIV-seronegative Italian patients, are closely correlated with the presence of LMP-1 deletions, suggesting that infection with these strains may increase the risk of developing HD in the HIV setting.

The role of hepatitis C virus (HCV) in the pathogenesis of type II mixed cryoglobulinemia (MC) has been strongly emphasized in the last few years. Although MC is a benign lymphoproliferative disorder, the risk of overt B-cell malignancy greatly increases during its course. The occurrence of HCV infection in 10% to 30% of patients with non-Hodgkin's lymphoma (NHL) suggests that this virus may have a role in the development of MC-associated B-cell malignancies. We identified 2 patients with hyperplastic reactive lymphadenopathy (HRL) and 12 with NHL in two series of MC patients chronically infected with HCV collected over a 5-year period. Structural and nonstructural HCV-related proteins were investigated in lymph node sections by immunohistochemistry and their location and distribution were correlated with clinical and histologic findings, viremic state, and HCV genotypes. In HRL, HCV proteins were found in the cytoplasm of lymphoid cells, mainly in interfollicular areas. However, occasional positive cells were found in the mantle zone and in the germinal centers of follicles. In addition, strong reactivity was found in the circulating mononuclear cells of capsular blood vessels. HCV immunodeposits were found in 3 of 12 (25%) NHL cases. Positive cells were frequently restricted to the cortex; if not, they were randomly diffused in the neoplastic tissue. Positivity was related to the low-grade type of NHL; in the 2 composite cases, HCV immunodetection was found in the small cells, whereas large anaplastic cells were regularly negative. Other viruses previously involved in lymphoproliferation, ie, human herpes virus-6 and Epstein-Barr virus, were absent in all tissues. These data emphasize that lymphoid organs may be a site of HCV infection. The demonstration of HCV-related proteins in a nonmalignant condition, namely HRL, indicates that HCV infection precedes the neoplastic transformation and possibly plays a major role in lymphomagenesis in MC.

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study, we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs), since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity, but 9-cis-retinoic acid (RA), 13-cis-RA, and all-trans-RA (ATRA) were markedly more efficacious than Ro40-8757, Ro13-6298, and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs, thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1, a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems, the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact, retinoid-induced modifications of cell morphology, phenotype (downregulation of CD19, HLA-DR, and s-Ig, and increased expression of CD38 and c-Ig), and IgM production were late events, highly heterogeneous, and often slightly relevant, being therefore only partially indicative of a drug-related differentiative process. Moreover, EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids, indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients.

Hepatitis C virus (HCV) has been implicated as the major etiologic factor sustaining B-cell clonal expansion in type II mixed cryoglobulinemia (MC). A putative pathogenetic role of HCV in the development of MC-associated B-cell malignancies has also been speculated. We report for the first time the localization of HCV within a parotid non-Hodgkin's lymphoma (NHL) lesion in the course of HCV-related type II essential MC, an important step to implicate any infectious agent in the lymphomagenesis. Plus and minus strand HCV RNA was first demonstrated by polymerase chain reaction on the whole RNA from the lesion. Further immunohistochemical studies localized HCV c22 proteins in the residual ductal or acinar parotid structures, which also abnormally expressed HLA-DR antigens. Weak c22 signals were inconstantly detected in cells strictly confined around the residual epithelium, while all the remaining infiltrating cells in the parotid lesion stained c-22-negative. Staining for c33 and c100 HCV antigens was negative. In situ hybridization (ISH) studies again identified the residual parotid epithelial cells as the site of HCV infection and replication in the NHL lesion. Sialotropic viruses previously involved in lymphoproliferation, ie, Epstein-Barr virus and human herpesvirus-6, were absent in the same tissue lesion. According to the current models of B-cell lymphomagenesis, a role of HCV as an exogenous antigenic stimulus should be considered for NHL development in the present case, whereas malignant B cells do not appear permissive of active HCV replication. Further efforts would be worthwhile to clarify a role of HCV infection in the development of some B-cell malignancies.

The authors present the problems that have been raised by psychologically interviewing 740 couples who ask for A.I.D. They describe the procedure that they adopted and the features that came up during the interview and particularly in the way the couples experienced their sterility. They conclude by pointing out how important a consultation is, both from the point of view of evaluating the couple psychologically in regard to the treatment and from the point of view of clarifying the needs, the doubts and the expectations that the two partners have when they decide to undertake A.I.D.