The treatment of HCV and its sequelae are used to be predominantly based on Interferon (IFN). However, this was associated with significant adverse events as a result of its immunostimulant capabilities. Since their introduction, the directly acting antiviral drugs (DAAs), have become the standard of care to treat of HCV and its complications including mixed cryoglobulinemic vasculitis (MCV). In spite of achieving sustained viral response (SVR), there appeared many reports describing unwelcome complications such as hepatocellular and hematological malignancies as well as relapses. Prolonged inflammation induced by a multitude of factors, can lead to DNA damage and affects BAFF and APRIL, which serve as markers of B-cell proliferation. We compared, head-to-head, three antiviral protocols for HCV-MCV treatment As regards the treatment response and relapse, levels of BAFF and APRIL among pegylated interferon α-based and free regimens (Sofosbuvir + Ribavirin; SOF-RIBA, Sofosbuvir + Daclatasvir; SOF-DACLA). Regarding clinical response HCV-MCV and SVR; no significant differences could be identified among the 3 different treatment protocols, and this was also independent form using IFN. We found no significant differences between IFN-based and free regimens DNA damage, markers of DNA repair, or levels of BAFF and APRIL. However, individualized drug-to-drug comparisons showed many differences. Those who were treated with IFN-based protocol showed decreased levels of DNA damage, while the other two IFN-free groups showed increased DNA damage, being the worst in SOF-DACLA group. There were increased levels of BAFF through follow-up periods in the 3 protocols being the best in SOF-DACLA group (decreased at 24 weeks). In SOF-RIBA, CGs relapsed significantly during the follow-up period. None of our patients who were treated with IFN-based protocol had significant clinico-laboratory relapse. Those who received IFN-free DAAs showed a statistically significant relapse of constitutional manifestations. Our findings suggest that IFN-based protocols are effective in treating HCV-MCV similar to IFN-free protocols. They showed lower levels of DNA damage and repair. We believe that our findings may offer an explanation for the process of lymphoproliferation, occurrence of malignancies, and relapses by shedding light on such possible mechanisms.

BACKGROUND: Hepatitis C virus (HCV) chronic infection is frequently associated to autoimmune manifestations. The aim of this study was to prospectively evaluate the occurrence of clinical and/or laboratory features of autoimmunity in a cohort of 140 consecutive HCV chronically infected patients treated with direct-acting antiviral agents (DAAs) and followed-up for 96 weeks.
METHODS: All patients were screened for cryoglobulins, rheumatoid factor (RF), C3, C4, antinuclear antibody (ANA), anti-smooth muscle (ASMA), anti-liver kidney microsome type 1 (anti-LKM1), anti-mitochondrial antibodies (AMA), anti-neutrophil cytoplasmic antibodies (ANCA), and anti-liver cytosol type 1/soluble liver antigen (anti-LC1/SLA) autoantibodies before therapy and 12, 48 and 96 weeks after treatment. They were then grouped according to the expression of laboratory findings and related autoimmune diseases.
RESULTS: At baseline, autoimmune manifestations were found in 70 patients: 83% of them were cryoglobulinemic, whereas ANA, AMA, perinuclear ANCA (pANCA) and LKM/LC1 autoantibodies were found in the remaining 17%. An autoimmune disease was diagnosed in 9 cases, two of them featuring an autoimmune liver disease (AILD). At the end of follow-up, despite viral clearance and regression of vasculitis, cryoglobulins persisted in 12 patients (21%), and autoantibodies disappeared or decreased in most of cases but, with the exception of the 2 patients diagnosed as AILD, associated autoimmune diseases remained stable. In one patient with relapsing cryoglobulinemia and ANA positivity, type-1 autoimmune hepatitis was defined. Conversely, autoantibodies first appeared after viral clearance in 5 patients, of whom one was diagnosed with type-1 autoimmune hepatitis and one with pANCA+ primary sclerosing cholangitis.
CONCLUSIONS: Following DAA-induced viral clearance, cryoglobulins may persist or reappear. Autoantibodies changed dynamically in step with the disappearance of a previously diagnosed or the occurrence of a new AILD. A longer follow-up will be necessary to establish the possible diagnosis of a newly onset AILD, the reactivation of cryoglobulinemic vasculitis and even its progression to non-Hodgkin lymphoma.

Chronic hepatitis C virus (HCV) infection is characterized by a variety of extra-hepatic manifestations; peripheral neuropathy (PN) is one of the most common, especially when mixed cryoglobulinemia (MCG) is present. The prevalence and risk factors of HCV-related PN in the absence of MCG are largely unknown. We conducted a prospective, single-center study, examining the prevalence and reversibility of HCV-associated neuropathy in the absence of MCG. Nerve fiber density in the epidermis was evaluated through skin biopsy and electroneurography (ENG) before HCV-treatment initiation and 1 year post sustained virological remission (SVR). Forty HCV-infected individuals (nine HIV co-infected) with no other neuron-harming factors were included; four other HCV mono- and three HIV co-infected individuals were excluded due to presence of diabetes, B12 insufficiency, or neurotoxic drugs. Twelve consecutive controls with no neuron-harming conditions were also recruited; eight more were excluded due to meeting exclusion criteria. Four patients had ENG signs of polyneuropathy (two with HCV mono- and two with HIV co-infection), while seven more (five with HCV mono- and two with HIV co-infection) had signs of mono-neuropathy, leading to PN prevalences of 22.5% and 44% for mono- and co-infection, respectively (p value 0.179). The two patients with HCV mono-infection and polyneuropathy and the one with ulnar nerve damage showed ENG improvement 1 year post SVR. Regarding intraepidermal nerve density, HCV infection, irrespective of HIV co-infection, was correlated with a lower intraepidermal neuron density that improved 1 year post SVR (p value 0.0002 for HCV and 0.0326 for HCV/HIV co-infected patients). PN is common in HCV infection; successful eradication of HCV leads to PN improvement.

Twenty-nine patients with HCV infection (HCV+) and mixed cryoglobulinemia (MC+) were retrospectively selected and matched for age and sex with 31 HCV+ MC- patients. Biomarkers of cholestasis (direct bilirubin, alkaline phosphatase, and gamma-glutamyl transferase), HCV-RNA and genotype, and plasma cryoprecipitates were measured before and after virus eradication; liver histology and plasma cells (aggregation and distribution), observed blinded by two pathologists, were analyzed. Sixty participants (mean age: 56.5; range: 35-77, males: 50%) with HCV infection were enrolled. Cholestasis (≥2 pathologically increased cholestasis biomarkers) was significantly higher in the MC group (p = 0.02) and correlated with cryoglobulinemia (OR 6.52; p = 0.02). At liver histological assessment, plasma cells were significantly increased in the MC+ group (p = 0.004) and tended to form aggregates more than the control group (p = 0.05). At multivariate analysis with MC, age, HCV-RNA, HBV diabetes, and cirrhosis, cholestasis was only significantly correlated to MC (OR 8.30; p < 0.05). In 25% patients, MC persisted after virus eradication with new antiviral treatment. Our study identified for the first time an association between MC, cholestasis, and an increased number of intrahepatic plasma cells in chronic hepatitis C (CHC) patients before virus eradication. Future studies are required to understand how MC contributes to liver damage and how its persistence affects the patients\' follow-up after antiviral therapies.

The infection caused by the hepatitis C virus (HCV) is a significant global health concern. The prevailing genotype of HCV in Egypt is 4a, commonly referred to as GT-4a. A significant proportion exceeding 50% of patients infected with HCV experience extrahepatic manifestations (EHMs), encompassing a diverse range of clinical presentations. These manifestations, including essential mixed cryoglobulinemia (MC), can serve as initial and solitary indicators of the disease. The complete understanding of the pathogenesis of EHM remains unclear, with autoimmune phenomena being recognized as the primary causative factor. In this study, we examined the predictive significance of T-cell subpopulations in relation to the occurrence and prognosis of cryoglobulinemia in HCV patients. A total of 450 CHC genotype four treatment naïve patients were enrolled in this analytic cross-sectional study after thorough clinical, laboratory, and radiological examinations. All patients underwent laboratory investigations, including testing for cryoglobulin antibodies and measurements of CD4 and CD8 levels; two groups were described according to their test results: Group 1 consists of patients who have tested positive for cryoglobulin antibodies and Group 2 consists of patients who have tested negative for cryoglobulin antibodies. The exclusion criteria encompassed individuals with HIV infection or chronic HBV infection. Additionally, pelvi-abdominal ultrasonography was performed. Our study included 450 treatment naïve CHC patients (59% male, mean age 50.8 years). The patients were categorized according to their cryoglobulin antibodys test results into two groups: group A, CHC patients with cryoglobulin antibodies (Abs) negative (364 patients), and group B, CHC patients with cryoglobulin Ab positive (86 patients). Group B demonstrated a higher average age, elevated international normalized ratio, more prolonged duration of HCV infection, lower albumin, higher alanine aminotransferase, higher aspartate aminotransferase, higher bilirubin, lower CD8, lower CD4, and lower CD4:CD8 ratio. In contrast, 27 out of 86 (31.40%) patients in group B had symptoms; 85.8% had purpura and arthralgia, 74.3% had paresthesias, 86.7% had weakness, and 12.2% had non-Hodgkin\'s lymphoma. The levels of CD4 and CD8 were found to be decreased in chronic HCV patients with MC. T-cell subpopulation serves as a reliable indicator for assessing the prevalence and prognosis of MC in individuals with genotype 4 chronic hepatitis C. However, additional research is needed to further understand the development and spread of various emerging infectious diseases. Nevertheless, it is noteworthy that a critical threshold may exist beyond which EHM reaches a point of no return.

Type I cryoglobulinemia (CG) accounts for 10%-15% of all cryoglobulinemias and are exclusively seen in clonal proliferative hematologic conditions. In this multicenter nationwide cohort study, we analyzed the prognosis and long-term outcomes of 168 patients with type I CG (93 (55.4%) IgM and 75 [44.6%] IgG). Five- and 10-year event-free survivals (EFS) were 26.5% (95% CI 18.2%-38.4%) and 20.8% (95% CI 13.1%-33.1%), respectively. In multivariable analysis, factors associated with poorer EFS were renal involvement (HR: 2.42, 95% CI 1.41-4.17, p = .001) and IgG type I CG (HR: 1.96, 95% CI 1.13-3.33, p = 0.016), regardless of underlying hematological disorders. IgG type I CG patients had higher cumulative incidence of relapse (94.6% [95% CI 57.8%-99.4%] vs. 56.6% [95% CI 36.6%-72.4%], p = .0002) and death at 10 years (35.8% [19.8%-64.6%] vs. 71.3% [54.0%-94.2%], p = .01) as compared to IgM CG, respectively. Overall, complete response of type I CG at 6 months was 38.7%, with no significant difference between Igs isotypes. In conclusion, renal involvement and IgG CG were identified as independent poor prognostic factors of type I CG.

OBJECTIVE:  Cryofibrinogens (CFs) and cryoglobulins (CGs) are cryoproteins responsible for obstructive vasculopathy and vasculitis. The aim of this study was to compare the characteristics of CF and CG, and to define the conditions of their association.
RESULTS:  This retrospective study was conducted at the Lyon University Hospitals, and included patients with at least one sample tested for CF and/or CG between September 2013 and April 2021. Serum and plasma samples were analyzed in very strict conditions of temperature. After cold precipitation, CF and CG were characterized and quantified in the cryoprecipitates. CRP and plasma fibrinogen levels were also investigated. Over this 7-year period, 1,712 samples for CF detection and 25,650 samples for CG detection were sent to the laboratory. Simultaneous testing of CF and CG was performed in 1,453/1,712 samples (85%). CF was less often positive than CG (8.3 vs. 13.5%, p < 0.0001). In positive CF samples, CG was associated in 28.9% of cases. In CF, fibrinogen was associated with fibronectin in 98/142 (69%) samples, especially in highly concentrated CF. CF concentration was independent of C-reactive protein and plasma fibrinogen concentrations.
CONCLUSIONS:  The simultaneous detection of CF and CG is essential for the diagnosis of vasculitis or thromboembolic events and their treatment.

Mixed cryoglobulinemia syndrome (MCs) is a rare immunoproliferative systemic disorder with cutaneous and multiple organ involvement. Our multicenter survey study aimed to investigate the prevalence and outcome of COVID-19 and the safety and immunogenicity of COVID-19 vaccines in a large MCs series.
The survey included 430 unselected MCs patients (130 M, 300 F; mean age 70 ± 10.96 years) consecutively collected at 11 Italian referral centers. Disease classification, clinico-serological assessment, COVID-19 tests, and vaccination immunogenicity were carried out according to current methodologies.
A significantly higher prevalence of COVID-19 was found in MCs patients compared to Italian general population (11.9% vs 8.0%, p < 0.005), and the use of immunomodulators was associated to a higher risk to get infected (p = 0.0166). Moreover, higher mortality rate was recorded in MCs with COVID-19 compared to those without (p < 0.01). Patients\' older age (≥ 60 years) correlated with worse COVID-19 outcomes. The 87% of patients underwent vaccination and 50% a booster dose. Of note, vaccine-related disease flares/worsening were significantly less frequent than those associated to COVID-19 (p = 0.0012). Impaired vaccination immunogenicity was observed in MCs patients compared to controls either after the first vaccination (p = 0.0039) and also after the booster dose (p = 0.05). Finally, some immunomodulators, namely, rituximab and glucocorticoids, hampered the vaccine-induced immunogenicity (p = 0.029).
The present survey revealed an increased prevalence and morbidity of COVID-19 in MCs patients, as well an impaired immunogenicity even after booster vaccination with high rate of no response. Therefore, MCs can be included among frail populations at high risk of infection and severe COVID-19 manifestations, suggesting the need of a close monitoring and specific preventive/therapeutical measures during the ongoing pandemic.

Cryoglobulinemic vasculitis (CV) or mixed cryoglobulinemic syndrome (MCS) is a systemic small-vessel vasculitis characterized by the proliferation of B-cell clones producing pathogenic immune complexes, called cryoglobulins. It is often secondary to hepatitis C virus (HCV), autoimmune diseases, and hematological malignancies. CV usually has a mild benign clinical course, but severe organ damage and life-threatening manifestations can occur. Recently, evidence in favor of rituximab (RTX), an anti-CD 20 monoclonal antibody, is emerging in CV: nevertheless, questions upon the safety of this therapeutic approach, especially in HCV patients, are still being issued and universally accepted recommendations that can help physicians in MCS treatment are lacking. A Consensus Committee provided a prioritized list of research questions to perform a systematic literature review (SLR). A search was made in Medline, Embase, and Cochrane library, updated to August 2021. Of 1227 article abstracts evaluated, 27 studies were included in the SLR, of which one SLR, 4 RCTs, and 22 observational studies. Seventeen recommendations for the management of mixed cryoglobulinemia with rituximab from the Italian Study Group of Cryoglobulinemia (GISC) were developed to give a valuable tool to the physician approaching RTX treatment in CV.

The clinical value of cryoglobulinemia (CG) in systemic lupus erythematosus (SLE) is largely unknown. The aim of this retrospective study was to describe the characteristics of CG in SLE, its impact on SLE phenotype, and the features associated with cryoglobulinemic vasculitis (CryoVas) in SLE patients.
This retrospective study conducted in a French university hospital reviewed the data from 213 SLE patients having been screened for CG between January 2013 and December 2017. SLE patients positive for CG were compared to SLE patients without CG. Patients were classified as CryoVas using the criteria of De Vita et al. RESULTS: Of the 213 SLE patients included (mean age 29.2 years, female sex 85%), 142 (66%) had at least one positive CG in their history, 67% of them having a persistent CG at follow-up. CG was type III in 114 (80%) cases and type II in 27 (19%) cases. The mean concentration of the cryoprecipitate was 40mg/L (range 0-228). Patients with CG had significantly more C4 consumption. Among patients with CG, 21 (15%) developed a CryoVas. The clinical manifestations of patients with CryoVas were mainly cutaneous (purpura, ulcers, digital ischemia) and articular, without any death at follow-up. Severe manifestations of CG included glomerulonephritis in 1/21 (5%) patients and central nervous system involvement in 4/21 (19%) patients. A response to first-line treatments was observed in 12/13 (92%) patients, but relapses were observed for 3 of them.
CG is frequent in SLE, but mostly asymptomatic. CryoVas features involve mostly joints, skin, and general symptoms. CryoVas in SLE appears to be a specific condition, with a low prevalence of neuropathy, membranoproliferative glomerulonephritis, and severe manifestations.