Mixed cryoglobulinemia.

Disease name and synonyms

Mixed cryoglobulinemia (type II or type III), cryoglobulinemic vasculitis.

Definition

The term cryoglobulinemia refers to the presence in the serum of one (monoclonal cryoimmunoglobulinemia) or more immunoglobulins (mixed cryoglobulinemia), which precipitate at temperatures below 37°C and re-dissolve on re-warming [ 1 , 2 ]. This is an in vitro phenomenon (Fig. 1 ), the actual mechanism(s) of cryoprecipitation remains obscure, it could be secondary to intrinsic characteristics of both mono- and polyclonal immunoglobulin (Ig) components, it can be caused as well by the interaction among single components of the cryoprecipitate [ 1 - 14 ].

Cryoglobulinemia is usually classified into three subgroups [ 4 ] according to Ig composition (Table 1 ): type I cryoglobulinemia is composed of only one isotype or subclass of immunoglobulin. Both type II and type III mixed cryoglobulins are immune complexes composed of polyclonal IgGs, the autoantigens, and mono- or polyclonal IgMs, respectively; the IgMs are the corresponding autoantibodies with rheumatoid factor (RF) activity [ 3 - 6 ]. With more sensitive methodologies, i.e . immunoblotting or 2-dimensional polyacrylamide gel electrophoresis, type II mixed cryoglobulins frequently shows a microheterogeneous composition; in particular, oligoclonal IgM or a mixture of polyclonal and monoclonal IgM can be detected [ 3 ]. This particular serological subset, termed type II-III mixed cryoglobulinemia (MC), could represent an intermediate, evolutive state from type III to type II MC. Moreover, type II-III MC could fit together the most recent molecular studies showing the presence of oligoclonal B-lymphocyte proliferation in the liver and bone marrow biopsies from MC patients [ 3 ]. In two third of type II MC, a cross-idiotype WA monoclonal RF (firstly isolated from the serum of a patient with Waldenström's macroglobulinemia) has been demonstrated [ 14 ].

Type I cryoglobulinemia is almost invariably associated with well-known hematological disorders and is frequently asymptomatic per se ; similarly, circulating mixed cryoglobulins are commonly detected in a great number of infectious or systemic disorders [ 1 - 14 ]. On the contrary, 'essential' MC represents a distinct disorder [ 3 - 6 ], which can be classified among systemic vasculitides [ 3 - 10 ]. Cryoglobulinemic vasculitis (Fig. 2 ) is secondary to vascular deposition of circulating immune-complexes, mainly cryoglobulins, and complement, with the possible contribution of both hemorheological and local factors [ 3 - 6 ]. Due to its clinical and histological features, MC is classified in the subgroup of small vessel systemic vasculitides, which also includes cutaneous leukocytoclastic vasculitis and Henoch-Schonlein purpura [ 3 , 10 ].

The leukocytoclastic vasculitis is the histopathological hallmark of MC (Fig. 2 ). It may involve small- and medium-sized vessels and may be responsible for multiple organ involvement. The term 'cryoglobulinemic vasculitis' is frequently used as synonym; it better focus on the typical histopathological alterations responsible for cutaneous and visceral organ involvement [ 3 , 10 ].

Epidemiology

The prevalence of MC presents great geographic heterogeneity; the disease is more common in Southern Europe than in Northern Europe or Northern America [ 3 - 13 ]. It is considered to be a rare disorder, however, yet there are no adequate epidemiological studies regarding its overall prevalence. Given its clinical polymorphism, a single manifestation (skin vasculitis, hepatitis, nephritis, peripheral neuropathy, etc .) is often the only apparent or clinically predominant feature, so that MC patients are often referred to different specialties (Fig. 3 ). A correct diagnosis might thus be delayed or overlooked entirely. Consequently, the actual prevalence of MC might be underestimated.

For the same reasons, the clinical pattern of the MC syndrome may vary largely among patients series referred to different tertiary care facilities [ 3 , 6 , 8 - 14 ] (Fig. 3 ).

It has been estimated that low levels of circulating mixed cryoglobulins can be detected in over 50% of HCV-infected individuals, while overt cryoglobulinemic syndrome develops in about 5% [ 3 , 15 , 16 ]. Because of the wide diffusion of HCV infection worldwide, a growing incidence of HCV-related MC can be expected, especially in underdeveloped countries where HCV in the general population is rather prevalent [ 3 , 17 ].

The prevalence of 'essential' MC is reported as approximately 1:100,000 but this term is now referred to a minority of MC patients. The female-to-male ratio is 3:1.

Clinical description

According to its first description [ 5 ], MC syndrome is characterized clinically by a triad -purpura, weakness, arthralgias- and by a series of pathological conditions, including chronic hepatitis, membranoproliferative glomerulonephritis (MPGN), peripheral neuropathy, skin ulcers, diffuse vasculitis, and, less frequently, by lymphatic and hepatic malignancies [ 3 - 12 , 17 - 23 ]. The clinical pattern of cryoglobulinemic vasculitis is comparable in patients with type II or type III MC [ 3 ]. The prevalence of MC manifestations reported in Table 2 regards an Italian patient population referred to our university-based division of rheumatology. Patients' recruitment at different specialist centers, as well as the racial composition among patients' series may be responsible for the variable prevalence of MC symptoms reported in the literature [ 3 - 10 , 14 ].

The presenting symptoms of MC largely vary among patients with cryoglobulinemia. At the initial observation, MC shows different clinico-serological patterns, varying from apparently isolated serum mixed cryoglobulins to complete cryoglobulinemic syndrome (Fig. 4 ). This is a combination of serological findings (mixed cryoglobulins with RF activity and frequent low C4) and clinico-pathological features (purpura, leukocytoclastic vasculitis with multiple organ involvement) [ 6 - 10 , 14 , 24 , 25 ]. Asymptomatic serum mixed cryoglobulins can be found in some chronically HCV-infected individuals [ 3 , 15 , 16 ]; a condition that may precede for years or decades the clinical onset of the disease. On the other hand, some patients show typical cryoglobulinemic syndrome, without serum cryoglobulins, the hallmark of the disease (Fig. 4 ). This is generally a transient phenomenon due to the wide variability of the percentage of cryoprecipitable immune-complex [ 3 , 26 ]. Repeated cryoglobulin determinations are necessary for a correct diagnosis in these subjects.

Skin lesions represent the most frequent manifestations of the MC [ 6 - 10 , 14 , 24 , 25 ]. Orthostatic purpura is generally intermittent, the dimension and diffusion of purpuric lesions largely varied, from sporadic isolated petechias to severe vasculitic lesions, often complicated by torpid ulcers of the legs and malleolar areas (Fig. 2 ). In a significant proportion of patients, repeated episodes of purpura may lead to stable, often confluent areas of ochreous coloration on the legs (Fig. 2 ). Cutaneous manifestations, in particular orthostatic purpura and ulcers, are the direct consequence of vasculitic alterations with the possible contributions of various co-factors, in particular chronic venous insufficiency, physical stress, mainly the prolonged standing, and/or muggy weather. In addition, the contribution of hemorheological disturbances due to high cryocrit levels may also be taken in account [ 27 ]. In this respect, the purpuric outbreaks are frequently observed late in the afternoon when highest cryocrit levels are generally observed, often in concurrence with prolonged standing [ 24 ].

MC patients commonly report arthralgias , while clear clinical signs of arthritis (usually mild, non-erosive oligoarthritis) are relatively rare [ 11 , 12 , 25 , 28 ]

Almost half MC patients complain of xerostomia and xerophthalmia , however, only a few cases meet the current criteria for the classification of primary Sjogren's syndrome (see 'differential diagnosis') [ 11 , 12 , 25 ].

Peripheral neuropathy may frequently complicate the clinical course of the MC, in the majority of cases as mild sensory neuritis [ 24 , 25 , 29 , 30 ]. The common symptoms are paresthesias with painful and/or burning sensations in the lower limbs, often with nocturnal exacerbation. The patient quality of life may be severely compromised because of the chronicity of these symptoms along with their scarce sensibility to therapeutic attempts. In a minority of cases the peripheral neuropathy may be complicated by severe sensory-motor manifestations, which usually appeared abruptly, often as asymmetric mononeuritis; in some patients, it may complicate the alpha-interferon treatment, possibly in predisposed subjects [ 29 - 33 ]. Dysarthria and hemiplegia expression of central nervous system involvement are rarely reported [ 25 , 34 ]; it is often difficult to distinguish these symptoms from the most common atherosclerotic manifestations.

Since HCV infection represents the underlying disorder of the MC in the vast majority of cases, overt chronic hepatitis , generally with mild-to-moderate clinical course, can be observed at any time during the natural history of the disease [ 24 , 25 ]. Chronic hepatitis was found in a great number of patients (Table 2 ), evolving to cirrhosis in 1/4, while only 7 patients developed hepatocellular carcinoma. In few cases, especially in combination with renal involvement, liver involvement became a life-threatening complication. On the whole, the clinical course and the prognostic value of this manifestation seem to be less severe if compared to HCV-related chronic hepatitis without MC syndrome [ 24 , 25 ]; similarly, hepatocellular carcinoma less frequently complicates MC syndrome compared to the whole population of HCV-positive individuals [ 24 , 25 ]. These differences are quite intriguing, but very difficult to fully explain. It is possible that the light consumption of alcohol and/or the relatively low prevalence of HCV genotype 1b [ 24 , 25 ] may explain, at least in part, the rather benign clinical course of liver involvement and its scarce prognostic relevance in MC series.

Membranoproliferative glomerulonephritis type 1 is another important organ involvement, which may severely affect the prognosis and survival of the disease [ 24 , 25 , 34 - 37 ]. MC-related nephropathy is a typical immune-complex-mediated glomerulonephritis, although other immunological mechanisms have also been hypothesized [ 24 , 25 , 38 , 39 ].

Widespread vasculitis involving medium-small sized arteries, capillaries and venules with multiple organ involvement may develop in a small proportion of patients [ 3 , 6 , 10 , 24 , 25 , 40 ]. This extremely severe complication may involve the skin, kidney, lungs, central nervous system, and gastrointestinal tract. In rare cases intestinal vasculitis may suddenly complicate the disease, often in patients with renal and/or liver involvement; pain simulating an acute abdomen is the presenting symptom of intestinal vasculitis. A timely diagnosis and aggressive steroid treatment are necessary for this life-threatening complication.

Interstitial lung involvement has been anecdotally observed in MC syndrome as well as in patients with isolated HCV infection [ 3 , 25 , 41 - 43 ]. Almost invariably, lung involvement in MC is characterized by subclinical alveolitis, as demonstrated by means of bronco-alveolar lavage in unselected patient series [ 44 ]; this condition may predispose to harmful infectious complications and, in rare cases, it may lead to clinically evident interstitial lung fibrosis. The hyperviscosity syndrome, due to high levels of serum cryoglobulins is another rare clinical manifestation of MC [ 27 ]. Generally, there was no relationship between the severity of clinical symptoms, such as glomerulonephritis, skin ulcers, or diffuse vasculitis and the serum levels of cryoglobulins and/or hemolytic complement [ 3 , 24 , 25 ]. Low complement activity is almost invariably detectable in MC, with the typical pattern of low or undetectable C4 and normal or relatively normal C3 serum levels, regardless the disease activity (Table 2 ). Moreover, in vitro consumption of complement can be also observed due to the anti-complement activity of some cryoimmunoglobulins [ 3 ]. Of interest, a sudden increase in C4, raised to abnormally high levels, can be observed in MC patients developing a B-cell lymphoma [ 45 ]. The lack of correlation between circulating cryoglobulin levels and the severity/activity of MC manifestations might be explained on the basis of different hypotheses: the pathogenic role of other non-cryoprecipitable immune-complexes, their intrinsic capacity to activate the complement, and/or the in situ formation of immune-complexes, with a relative concentration of HCV virions [ 3 , 14 , 24 , 25 ].

Some endocrinological disorders are significantly more frequent in MC patients compared to the general population [ 3 , 12 , 13 , 24 , 25 , 46 - 50 ]. The most common thyroid disorders are autoimmune thyroiditis, subclinical hypothyroidism, and thyroid cancer; while hyperthyroidism is less frequent, generally as reversible complication of interferon treatment [ 51 - 54 ]. Moreover, a statistically increased incidence of diabetes mellitus has been observed in HCV-positive patients with and without MC syndrome compared to the general population [ 55 - 57 ]. Finally, HCV-positive males with or without cryoglobulinemic vasculitis may develop erectile dysfunction, attributable to hormonal and/or neuro-vascular alterations [ 49 ].

B-cell lymphom a is the most frequent neoplastic manifestation complicating MC, often as late manifestation of the MC syndrome [ 58 - 65 ]. This complication may be related to peripheral B-lymphocyte expansion and to lymphoid infiltrates observed in the liver and bone marrow of MC patients [ 19 , 39 , 58 , 59 ]. In particular, these infiltrates have been regarded by some authors as "early lymphomas", since they are sustained by lymphoid components indistinguishable from those of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL) and immunocytoma (Ic) [ 3 , 12 , 25 , 59 ]. However, unlike frank malignant lymphomas, they tend to remain unmodified for years or even decades and are followed by overt lymphoid tumors in about 10% of cases [ 3 ]. These characteristics justify the proposed term of "monotypic lymphoproliferative disorder of undetermined significance (MLDUS)" [ 3 , 12 , 25 , 59 ]. Interestingly enough, type II MC-related MLDUS has its highest incidence in the same geographic areas where about 30% of patients with 'idiopathic' B-cell lymphomas also display HCV-positivity, and where an increased prevalence of HCV genotype 2a/c has been observed in both MC and lymphomas [ 3 , 12 , 25 , 59 ]. The type II MC-associated MLDUS presents two main pathological patterns; namely, the B-CLL-like and the Ic-like [ 3 , 12 , 25 , 59 ]. In the clinical practice, it is not rare to observe the appearance of malignant B-cell lymphomas in patients with mild MC clinical course, sometimes unexpectedly during a routine evaluation. It is possible to observe a sudden decrease or disappearance of serum cryoglobulins and RF, sometimes associated with abnormally high levels of C4, as presenting symptom of complicating B-cell malignancy [ 40 ].

Other neoplastic manifestations, i.e . hepatocellular carcinoma or papillary thyroid cancer, are less frequently observed [ 3 , 11 , 24 , 50 , 59 ]. In this light, the MC can be regarded as a pre-neoplastic disorder [ 61 ]; consequently, a careful clinical monitoring is recommendable, even in the presence of mild MC syndrome [ 3 , 11 ].

Etiopathogenesis

Since the first description of MC syndrome [ 5 ], chronic hepatitis has been reported as frequent manifestation appearing during the clinical course of the disease [ 3 - 6 , 11 , 24 ]; therefore, a possible role for hepatotropic viruses in the pathogenesis of the disease has long been suggested [ 3 , 12 ]. A role for hepatitis B virus (HBV) has been firstly investigated [ 66 ]; however, HBV viremia is rarely recorded, while anti-HBV antibodies largely varied among different MC patient populations [ 3 ]. It can be estimated that HBV can represent a causative factor of MC in less than 5% of individuals (Fig. 5 ).

Soon after the discovery of hepatitis C virus (HCV) as the major etiologic agent of non-A-non-B chronic hepatitis [ 67 ], a possible role of HCV infection in MC was proposed independently by two pioneering reports [ 68 , 69 ] showing a significantly higher prevalence of serum anti-HCV antibodies compared to the general population. This hypothesis was definitely demonstrated in 1991, when the presence of HCV RNA was detected by means of polymerase chain reaction (PCR) in 86% of Italian MC patients [ 70 ]. Successively, an increasingly number of studies including clinico-epidemiological observations, as well as both histopathological and virological investigations (HCV RNA detection by PCR and/or in situ hybridization) have been definitely established the preeminent role of HCV in the pathogenesis of MC [ 8 - 14 , 21 , 25 , 59 , 71 - 76 ]. The prevalence of serum anti-HCV antibodies and/or HCV RNA in MC patients ranged from 70% to almost 100% among different patient populations [ 3 , 11 - 13 , 25 ]. Given the striking association between MC and HCV infection, the term 'essential' is now referred to a minority of MC patients (in Italy <5%, Fig. 5 ) [ 3 , 11 - 13 , 25 ].

Because of the frequent association between MC and HCV, the behavior of MC is closely linked to the natural history of HCV chronic infection [ 3 , 11 - 13 , 24 , 25 ]. However, MC is also the result of concomitant genetic and/or environmental factors, which remain largely unknown (Fig. 6 ). HCV has been recognized to be both a hepato- and lymphotropic virus, as suggested by the presence of active or latent viral replication in the peripheral lymphocytes of patients with type C hepatitis or MC [ 3 , 73 , 77 ]. HCV is an RNA virus without reverse trascriptase activity; therefore viral genome cannot integrate in the host genome [ 63 ]. Probably, HCV may exert a chronic stimulus of the immune-system, through different viral proteins such as core protein [ 9 , 22 , 63 ]. Chronic stimulation of the lymphatic system exerted through viral epitopes, autoantigen production, and/or molecular mimicry mechanism has been suggested by the presence, in HCV-positive patients, of anti-GOR antibodies, which are cross-reactive autoantibodies directed to both HCV core and a nuclear antigen named GOR [ 3 , 59 , 63 ]. Other Authors suggested that HCV, in association with very low-density lipoprotein (VLDL), might induce a T-independent primordial B-cell population producing monoclonal immunoglobulin with WA idiotype [ 14 ]. In turn, HCV-VLDL complexes may trigger RF production as consequence of somatic mutations of WA clones; the possible evolution to B-cell lymphomas might be the consequence of the accumulation of stochastic genetic aberrations [ 59 ]. Chronic stimulation of the B-cell by HCV epitopes may produce the expansion of some B-cell subpopulations with favorable and/or dominant genetic characteristics. This hypothesis recalls the pathogenetic role of Helicobacter pylori in MALT lymphoma of the stomach, for which different evolutive phases are requested [ 59 ].

Another important pathogenetic factor is the interaction between HCV E2 protein and CD81 molecule, a quite ubiquitary tetraspannin present on B-cells surface [ 78 ]; the consequence may be the strong and sustained polyclonal stimulation of B-cell compartment (Fig. 6 ). A following pathogenetic step of HCV-related autoimmune-lymphoproliferative disorders may be the t [ 14 , 18 ] translocation observed in B-cells of HCV-infected individuals [ 63 , 79 ]. Even if not definitely confirmed [ 80 , 81 ], the t [ 14 , 18 ] translocation might lead to abnormally elevated expression of Bcl-2 protein with consequent inhibition of apoptosis and abnormal B-cell survival. Interestingly, the relevant prevalence of t [ 14 , 18 ] translocation in patients with only type C hepatitis (about 37–38%), become particularly high in patients with HCV-related cryoglobulinemic syndrome, ranging 85% in type II MC [ 63 ]. It is possible to hypothesize that during chronic HCV infection, several factors (including the interaction between HCV E2 protein and CD81 molecule, the high viral variability, and the persistent infection of both hepatic and lymphatic cells) may favor a sustained and strong B-cell activation (Fig. 6 ). This latter may in turn favors the apparition of t [ 14 , 18 ] translocation and Bcl-2 overexpression; the consequent B-lymphocyte expansion is responsible for autoantibody production, including the cryoglobulins [ 3 , 11 , 12 , 63 , 77 - 79 ]. In addition, the prolonged B cell survival may represent a predisposing condition for further genetic aberrations, which may lead to frank B-cell malignancy as late complication of MC syndrome [ 11 , 20 , 24 , 82 ].

Of interest, HCV-driven lymphoproliferation may explain the pathogenetic role of HCV infection also in 'idiopathic' B-cell lymphomas [ 11 , 59 , 64 , 65 , 80 ]. This association was firstly described in unselected Italian patients with 'idiopathic' B-cell lymphomas [ 64 ] and successively confirmed by different epidemiological and laboratory studies, mainly in the same geographical areas where HCV-associated MC is commonly found [ 65 ].

Given its biological characteristics, HCV may be involved in a wide number of autoimmune and lymphoproliferative disorders [ 3 , 11 , 12 , 63 , 77 - 79 ]. Fig. 6 summarizes the main causative factors -infectious, toxic, genetic, and/or environmental- that are potentially involved in the pathogenesis of MC [ 11 , 12 , 79 , 82 - 84 ]. These factors, alone or in combination, may trigger two multistep pathogenetic processes, not mutually exclusive, responsible for MC and other HCV-related disorders. The first one produces a 'benign' poly-olygoclonal B-lymphocyte proliferation responsible for organ- and non-organ-specific autoimmune disorders, including the immune-complex-mediated cryoglobulinemic vasculitis; the second one is characterized by different oncogenetic alterations, which ultimately may lead to malignant complications [ 11 , 82 ]. Comparable pathogenetic mechanisms could be also hypothesized for HCV-negative MC, this intriguing clinical subset might be correlated to other infectious agents or associated to some well-known autoimmune/rheumatic or lymphoproliferetive disorders (Fig. 5 , 6 ) [ 13 ]. Th1-mediated tissue alterations are probably responsible for some endocrine disorders, i.e. diabetes type 2 and thyroid dysfunction, in HCV-associated MC patients [ 57 ]. Our preliminary data suggest that HCV infection of thyrocytes, as well as of beta cells, may upregulate CXCL10 gene expression and secretion (as shown in human hepatocytes); the consequent recruitment of Th1 lymphocytes that secrete IFN-γ and TNF-α may in turn induce CXCL10 secretion by infected cells, thus perpetuating the immune cascade responsible for these endocrine disorders. All the above pathogenetic mechanisms could be also hypothesized for HCV-negative MC, this intriguing clinical subset might be correlated to other infectious agents or associated to some well-known autoimmune/rheumatic or lymphoproliferetive disorders (Fig. 5 , 6 ) [ 13 ].

MC with or without overt clinical syndrome has been reported in patients with a great number of infectious agents, usually as anecdotally observations [ 9 ]. A significant prevalence of MC has been observed in patients with human immunodeficiency virus (HIV) infection, with and without HCV co-infection [ 85 ]. HIV alone may exert a continuous antigenic stimulation of B-lymphocytes; these latter may be responsible for type III MC production earlier in the course of HIV infection. In some patients the B-cell disorder may evolve to monoclonal MC with typical clinical syndrome. As observed for HCV infection, the prevalence of other virus-related MC seems to show a variable prevalence among patient's series from different geographical areas [ 86 ]. Moreover, a number of clinico-epidemiological studies revealed a heterogeneous distribution of different HCV-related extra hepatic manifestations, including some autoimmune disorders such as 'primary' Sjogren's syndrome [ 87 - 98 ].

Cryoglobulinemic syndrome may share a number of etiopathogenetic events and clinical features with both autoimmune diseases such as autoimmune hepatitis, Sjögren's syndrome, and polyarthritis, and B-cell lymphomas [ 3 , 11 , 12 , 25 , 28 , 85 - 100 ]. Therefore, a differential diagnosis should be carefully done in all patients with MC syndrome (Fig. 7 , see also the paragraph 'differential diagnosis'); a correct disease classification may decisively affect the overall clinico-therapeutic approach and prognosis.

Diagnostic methods

There are no available diagnostic criteria for MC; in 1989 the Italian Group for the Study of Cryoglobulinemias has proposed preliminary criteria for MC classification [ 101 ]. A revised version of these criteria (Table 3 ), including clinico-pathological and virological findings, has been successively proposed [ 3 ]. Circulating mixed cryoglobulins, low C4, and orthostatic skin purpura are the hallmarks of the disease; moreover, leukocytoclastic vasculitis, involving medium- and, more often, small-sized blood vessels (arterioles, capillaries, and venules) is the typical pathological finding of involved tissues. It is easily detectable by means of skin biopsy of recent vasculitic lesions (within the first 24–48 hours) [ 3 , 11 , 12 , 25 ].

The detection of serum mixed (IgG-IgM) cryoglobulins is necessary for a correct classification of MC syndrome (Table 3 , Fig. 1 ). Unfortunately, there are no universally accepted methodologies for cryoglobulin measurements, but simple standardized indications are often sufficient for testing cryoglobulinemia [ 3 , 9 , 23 ]. Since cryoglobulins present a high thermal instability, the measurement of Ig cryoprecipitate should be performed immediately in the same place where the blood is sampled. For a correct evaluation of serum cryoglobulins, it is necessary to avoid false-negative results due to Ig cold precipitation also at room temperature. For this purpose, the first steps (blood sampling, clotting, and serum separation by centrifugation) should be always carried out at 37°C and the cryocrit determination and cryoglobulin characterization at 4°C (after 7 days). The serum with cryoglobulins should be tested for reversibility of the cryoprecipitate by rewarming an aliquot at 37°C for 24 hours. Moreover, cryocrit determinations (percentage of packed cryoglobulins referred to total serum after centrifugation at +4°C; Fig. 1 ) should be done on blood samples without anticoagulation to avoid false-positive results due to cryofibrinogen or heparin-precipitable proteins. Without the above relatively simple precautions, not only will the quantities of cryoglobulins measured be incorrect, but the test may completely fail to even detect cryoglobulins.

After isolating and washing the cryoprecipitate, the identity of cryoglobulin components can be determined by immunoelectrophoresis or immunofixation. These analysis must be performed at 37°C to avoid precipitation and hence loss of the cryoglobulin during the procedures. More sophisticated methodologies, such immunoblotting or two-dimensional polyacrylamide gel electrophoresis, can be proposed for laboratory investigations [ 102 - 104 ]. While the detection of serum cryoglobulins is fundamental for the diagnosis of MC, the levels of serum cryoglobulins usually do not correlate with the severity and prognosis of the disease [ 3 , 11 , 12 , 24 , 25 ]. Very low levels of cryocrit, often difficult to quantify, can be associated with severe and/or active cryoglobulinemic syndrome; on the contrary, high cryocrit values may characterize a mild or asymptomatic disease course. In rare cases, very high cryocrit levels, possibly associated to cryogel phenomenon, can be associated to classical hyperviscosity syndrome [ 24 , 25 , 27 ]. On the contrary, a sudden decrease or disappearance of serum mixed cryoglobulins, with or without abnormally high levels of C4, can be regarded as alarming signal of complicating B-cell malignancy [ 45 ].

Table 4 summarizes the clinico-serological work-up at patient's first evaluation in order to correctly classify the MC syndrome and to identify possible overlapping disorders (see next paragraph), and/or comorbidities. The prevalence of these latter, in particular atherosclerosis, may be correlated with the disease duration, as well as with cumulative side effects of prolonged treatments. Moreover, the diagnosis and monitoring of the major MC manifestations is fundamental for timely treatments, especially for life-threatening liver, renal, and/or neoplastic complications [ 3 , 11 , 12 , 24 , 25 ].

Differential diagnosis

The term 'essential' MC was originally referred to autonomous disease when other well known systemic, infectious or neoplastic disorders have been ruled out by means of a wide clinico-serological work-up [ 5 ]. However, in some patients a definite diagnosis may be difficult because of the clinical polymorphism of the disease. Moreover, the association of MC with HCV infection may further complicate the differential diagnosis with other immunological HCV-related disorders: there is a frequent clinico-pathological overlapping among different HCV-related disorders. Cryoglobulinemic syndrome can represent a crossroads between some autoimmune diseases (autoimmune hepatitis, Sjögren's syndrome, polyarthritis, glomerulonephritis, thyroiditis, type 2 diabetes, etc .) and malignancies (B-cell lymphomas, hepatocellular carcinoma) [ 3 , 11 , 12 , 25 , 28 , 87 - 98 ]. It is possible to observe in the same patient a slow progression from mild HCV-associated hepatitis to various extrahepatic manifestations (arthralgias, sicca syndrome, Raynaud's phenomenon, RF positivity, etc .), and ultimately to overt MC syndrome with typical clinico-serological manifestations. In only a minority of MC patients a malignancy may develop, generally after a long lasting follow-up period [ 3 , 11 , 12 , 24 , 25 ]. Therefore, a careful patient evaluation is necessary for a correct diagnosis of MC syndrome, particularly with regards to other RF-positive, systemic rheumatic disorders such as rheumatoid arthritis (RA) and primary Sjögren's syndrome (pSS) (Fig. 7 ).

While arthralgias are one of the most frequent symptoms, clear signs of sinovitis are quite rare. Generally, patients develop mild, non-erosive oligoarthritis [ 28 , 90 ], often sensitive to low doses of corticosteroids with or without hydroxychloroquine. In contrast, a rheumatoid-like polyarthritis is more frequent in patients with HCV-related hepatitis without MC syndrome [ 28 ]. In patients with HCV-associated MC and symmetrical, erosive polyarthritis the diagnosis of overlapping MC/RA syndrome can be suspected. In these cases, the detection of serum anti- cyclic citrullinated peptide antibodies, markers of classical RA, may represent a useful diagnostic tool [ 91 , 92 ].

Almost half MC patients complain of sicca syndrome; however, only a few cases meet the current criteria for the classification of pSS. MC and pSS may share various symptoms: xerostomia and/or xerophthalmia, arthralgias, purpura, RF and serum cryoglobulins, and the possible complication with B-cell lymphoma [ 3 , 11 , 12 , 25 , 87 , 93 - 98 ]. However, a careful patient clinical assessment is usually sufficient for a correct diagnosis in the large majority of cases by considering some important findings: histopathological alterations of salivary glands and specific autoantibody pattern (anti-RoSSA/LaSSB) of pSS are rarely found in MC patients; conversely, HCV infection, cutaneous leukocytoclastic vasculitis, and visceral organ involvement (renal, liver) are seldom recorded in primary Sjögren's syndrome (Fig. 7 ). Given the above considerations, it has been recently proposed that the presence of HCV infection per se should be considered exclusion criteria for the classification of pSS [ 99 , 100 ]. In rare cases in which the differential diagnosis may result very difficult, particularly in HCV-negative individuals, it might be correct to classify the disorder as overlap syndrome. However, patients with MC/pSS overlap syndrome are often characterized by more severe clinico-prognostic evolution; they show a significant low rate of anti-RoSSA/LaSSB along with a high prevalence of mixed cryoglobulinemia, hypocomplementemia, systemic autoimmune manifestations, and complicating lymphomas (94, 97, 98). In the clinical practice, this particular condition could be better regarded as vasculitic syndrome with relevant implications on the patient monitoring and treatment [ 11 , 24 , 25 ].

Finally, autoimmune hepatitis, the old 'lupoid' hepatitis, may be associated to HCV infection, mainly in the same countries where HCV-associated MC is more frequently found [ 88 ]. Moreover, it may share with MC syndrome a number of extrahepatic symptoms, including serum mixed cryoglobulins [ 89 ]. The differential diagnosis between these two conditions may be problematic: some typical features of MC (leukocytoclastic vasculitis, hypocomplementemia, glomerulonephritis), as well as the presence of serum autoantibodies commonly found in autoimmune hepatitis (anti-smooth muscle antibodies) can be taken into account.

Management

Considering its complex etiopathogenesis and clinical polymorphism, the treatment of MC syndrome is particularly challenging. For a correct therapeutic approach, three important factors (Fig. 6 ), namely, the HCV infection, the presence of autoimmune disorder, and the possible neoplastic complications should be considered [ 3 , 25 ]. Following the main steps of the etiopathogenetic process, from HCV infection to B-lymphocyte proliferation, and lastly to cryoglobulinemic vasculitis (Fig. 8 ), the disease can be treated at different levels by means of etiologic, pathogenetic, and/or symptomatic therapies. Since HCV represents the main causative factor of the disease by exerting a chronic stimulus on the immune-system [ 3 , 8 - 12 , 14 , 24 ], an attempt at HCV eradication by alpha-interferon treatment should be done in all cases of HCV-associated MC [ 3 , 11 , 12 , 24 , 105 - 109 ]. However, the beneficial effect observed with this drug is often transient and possibly associated with important immune-mediated side effects such as peripheral sensory-motor neuropathy, thyroiditis, and rheumatoid-like polyarthritis [ 51 - 54 ]. Probably, in predisposed subjects alpha-interferon (which is both antiviral and immunomodulating agent) can trigger or exacerbate some pre-existing, often subclinical, symptoms. Unfortunately, there are no available parameters for predicting this harmful complication; thus, alpha-interferon therapy should be avoided at least in those patients with clinically evident peripheral neuropathy. On the whole, the usefulness of alpha-interferon treatment in MC patients is limited by the low rate of responders and frequent side effects. The association of alpha-interferon and ribavirin might achieve the eradication of HCV infection in a higher number of treated subjects, even if the long-term effects are inconstant and often unpredictable [ 3 , 11 , 12 , 24 , 105 - 114 ]. After careful patient's evaluation of contraindications and/or possible side effects, a standard treatment with alpha-interferon and ribavirin [ 111 ] should be attempted in MC patients with prevalent clinico-pathological findings of chronic hepatitis (Fig. 9 , 10 ). Controlled clinical trials might establish the actual usefulness of antiviral therapy in HCV-associated MC and better evaluate the predictive markers of response to treatment (HCV genotypes, host HLA alleles?). Hopefully, with the rapid growth of molecular biology a vaccine against HCV might be available in the near future. A vaccine-based therapy [ 115 ] with recombinant HCV proteins in HCV-infected individuals could be able to prevent the progression of viral infection and possibly to interrupt the self-perpetuating autoimmune mechanism underlying the MC.

In patients with 'essential' MC the immunosuppressive treatment, i.e . cyclophosphamide or rituximab, is still the first-line intervention (Fig. 9 , 10 ). For HCV-associated MC, immunomodulating/immunosuppressive treatments should be considered, especially in patients with more severe complications. These treatments include steroids, low-antigen-content (LAC) diet, plasma exchange, and immunosuppressors [ 3 , 11 , 12 , 24 , 116 - 122 ]. In particular, both traditional plasma exchange and double-filtration plasma exchange are able to markedly reduce the levels of circulating immune-complex, especially the cryoglobulins [ 24 , 121 , 122 ]. Oral cyclophosphamide (50–100 mg/day for 2–6 weeks) during the tapering of apheretic sessions can reinforce the beneficial effect of plasma exchange; moreover, it can prevent the rebound phenomenon that may be observed after the aphaeresis discontinuation [ 24 ]. Plasma exchange is particularly useful in severe MC complications such as active membranoproliferative glomerulonephritis (Fig. 8 , 9 , 10 ).

LAC-diet is a particular dietetic treatment that can improve the clearance circulating immune-complexes by restoring the activity of the reticulo-endothelial system, overloaded by large amounts of circulating cryoglobulins [ 122 , 123 ]. LAC-diet has been designed to reduce the input of alimentary macromolecules crossing the mucosal barrier of the gut; some foods, particularly dairy products and eggs, present a potential antigenic activity, and consequently might be involved in the pathogenesis of some diseases in humans. The reduction of the alimentary input of macromolecules directed to mononuclear phagocytic system may improve its function in those conditions characterized by abnormal endogenous production of immune-complexes responsible for organ damage, i.e . mixed cryoglobulinemia and other immune-complex mediated diseases. An impaired function of mononuclear phagocytic system has been also demonstrated in patients with IgA nephropathy [ 123 ]. Given the above pathogenetic considerations, LAC-diet has been usefully employed in patients with clinically mild MC symptoms and IgA nephropathy (reduction of 24-hour proteinuria). Usually, LAC-diet and/or low dosage of steroids (6-methyl-prednisolone 2–4 mg/day) may be sufficient to improve mild manifestations (arthralgias, sporadic purpura, etc .) of the MC (Fig. 8 , 9 , 10 ); patients with mild-moderate symptoms, such as palpable purpura, are particularly sensitive to the smallest variations of daily steroid dosage (1–2 mg).

In clinical practice, MC treatment should be tailored for the single patient according to the severity of clinical symptoms (Fig. 9 , 10 ). Therefore, patients with severe vasculitic manifestations must be promptly treated with high doses of steroids and/or plasma exchange and/or cyclophosphamide or rituximab. Some recent clinical studies suggested that sequential or combined antiviral/immunosuppressive treatment could represent a rather useful therapeutic strategy [ 124 , 125 ]. The rationale of such aggressive therapies could be particularly indicated in MC patients with major clinical manifestations and partial/transitory remission after standard treatments [ 25 , 124 , 125 ]. On the contrary, clinically asymptomatic patients usually do not need any treatment, even in the presence of high levels of cryocrit. In all cases, a careful clinical monitoring of the disease is mandatory, with particular attention to neoplastic complications. Preliminary studies suggested the synergic affect of some therapies in association with antiviral treatment, namely the cyclosporine A or tumor necrosis factor inhibitors, in the setting of chronic hepatitis type C [ 126 - 128 ]. These agents may contribute to viral clearance and/or to improve the immune-mediated hepatic and extrahepatic inflammatory manifestations; future clinical trials might evaluate their potential usefulness even in MC, possibly in selected patient subsets.

Prognosis

The natural history of MC is not predictable and strongly depends on concomitant diseases and complications and response to treatment. Morbidity due specifically to cryoglobulinemia may also be significant (infections, cardiovascular diseases, progressive renal failure, advanced neuropathy). The overall prognosis is worse in patients with renal disease, liver failure, lymphoproliferative disease, and malignancies. Mean survival is estimated to be approximately 50–60% at 10 years after diagnosis [ 24 ]. Careful monitoring of life-threatening MC complications (mainly nephropathy, widespread vasculitis, and B-cell lymphoma or other malignancies) should be carried out in all MC patients.

Unresolved questions

MC was first described as distinct disorder in 1966 [ 5 ]; 25 years later the discovery of the strong association between MC and HCV infection [ 3 , 11 , 12 , 25 , 70 ] represented a decisive contribute for a better understanding of the etiopathogenetic mechanisms responsible for the disease, and consequently for an adequate therapeutic strategy.

However, the following points remain still to be clarified:

1- Since the etiopathogenesis of HCV-associated MC syndrome is a multifactorial and multistep process, two important aspects need further investigations: a) HCV may represent the simple triggering factor or it could also contribute to self-perpetuating mechanism of the disease: b) nature and role of other etiopathogenetic co-factors;

2- The etiopathogenesis of 'essential' MC syndrome;

3- The actual role of HCV eradication on the natural history of HCV-associated MC syndrome;

4- The usefulness of sequential or combined antiviral/immunesuppressive treatments compared to traditional therapeutic approach.

Abbreviations

Ig: immunoglobulin; RF: rheumatoid factor; MC: mixed cryoglobulinemia; HCV: hepatitis C virus; MPGN: membranoproliferative glomerulonephritis; B-CLL: B-cell chronic lymphocytic leukemia; Ic: immunocytoma; MLDUS: monotypic lymphoproliferative disorder of undetermined significance; HBV: hepatitis B virus; PCR: polymerase chain reaction; VLDL: very low-density lipoprotein; MALT: mucosa-associated lymphoid tissue; HIV: human immunodeficiency virus; RA: rheumatoid arthritis; pSS: primary Sjögren's syndrome; LAC-diet: low-antigen-content diet.

Competing interests

The author declares that they have no competing interests.

Wintrobe M Buell M Hyperproteinemia associated with multiple myeloma. With report of a case in which an extraordinary hyperproteinemia was associated with trombosis of the retinal veins and symptoms suggesting Raynaud's disease Bull Johns Hopkins Hosp 1933 52 156 65

Lospalluto J Dorward B Miller W Jr Ziff M Cryoglobulinemia based on interaction between a gamma macroglobulin and 7S gamma globulin Am J Med 1962 32 142 145 14466769 10.1016/0002-9343(62)90191-2

Ferri C Zignego AL Pileri SA Cryoglobulins (review) J Clin Pathol 2002 55 4 13 11825916 10.1136/jcp.55.1.4 PMC1769573

Brouet JC Clouvel JP Danon F Klein M Seligmann M Biologic and clinical significance of cryoglobulins Am J Med 1974 57 775 88 4216269 10.1016/0002-9343(74)90852-3

Meltzer M Franklin EC Elias K McCluskey RT Cooper N Cryoglobulinemia. A clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity Am J Med 1966 40 837 56 4956871 10.1016/0002-9343(66)90200-2

Gorevic PD Kassab HJ Levo Y Kohn R Meltzer M Prose P Franklin E Mixed Cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients Am J Med 1980 69 287 308 6996482 10.1016/0002-9343(80)90390-3

Invernizzi F Galli M Serino G Monti G Meroni PL Granatieri C Zanussi C Secondary and essential cryoglobulinemias. Frequency, nosological classification, and long-term follow-up Acta Haematol 1983 70 73 82 6408882 10.1159/000206699

Monti G Galli M Invernizzi F Pioltelli P Saccardo F Monteverde A Pietrogrande M Renoldi P Bombardieri S Bordin G Candela M Ferri C Gabrielli A Mazzaro C Migliaresi S Mussini C Ossi E Quintiliani L Tirri G Vacca A GISC. Italian Group for the Study of Cryoglobulinaemias Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease QJM 1995 88 115 26 7704562

Dammacco F Sansonno D Mixed cryoglobulinemia as a model of systemic vasculitis Clin Rev Allergy Immunol 1997 15 97 119 9209804 10.1007/BF02828280

Lamprecht P Gause A Gross WL Cryoglobulinemic vasculitis (review) Arthritis Rheum 1999 42 2507 16 10615995 10.1002/1529-0131(199912)42:12<2507::AID-ANR2>3.0.CO;2-#

Ferri C Antonelli A Mascia MT Sebastiani M Fallahi P Ferrari D Pileri SA Zignego AL HCV-related autoimmune and neoplastic disorders: the HCV syndrome Dig Liver Dis 2007 39 S13 21 17936215 10.1016/S1590-8658(07)80005-3

Ferri C Antonelli A Mascia MT Sebastiani S Fallhai P Ferrari D Giunti M Pileri SA Zignego AL B-cells and mixed cryoglobulinemia Autoimmun Rev 2007 7 114 20 18035320 10.1016/j.autrev.2007.02.019

Mascia MT Ferrari D Campioli D Sandri G Mussini C Ferri C Non HCV-related mixed cryoglobulinemia Dig Liver Dis 2007 39 S61 64 17936226 10.1016/S1590-8658(07)80013-2

Abel G Zhang QX Agnello V Hepatitis C virus infection in type II mixed cryoglobulinemia (review) Arthritis Rheum 1993 36 1341 9 8216392 10.1002/art.1780361003

Pawlotsky J Mustapha B Andre C Voisin M-C Intrator L Roudot-Thoraval F Deforges L Duvoux C Zairani E-S Duval J Immunological disorders in C virus chronic active hepatitis: a prospective case-control study Hepatology 1994 19 841 8 8138255

Lunel F Musset L Cacoub P Frangeul L Cresta P Perrin M Grippon P Hoang C Valla D Piette JC Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage Gastroenterology 1994 106 1291 300 7513667 10.1016/0016-5085(94)90022-1

Herrine SK Approach to the patient with chronic hepatitis C virus infection Ann Intern Med 2002 136 747 57 12020143 10.7326/0003-4819-136-10-200205210-00010

Ferri C Bertozzi MA Zignego AL Erectile dysfunction and hepatitis C virus infection JAMA 2002 288 698 9 12169072 10.1001/jama.288.6.698

Monteverde A Ballarè M Pileri S Hepatic lymphoid aggregates in chronic hepatitis C and mixed cryoglobulinemia Springer Semin Imunopathol 1997 19 99 110 10.1007/BF00945028 9266634

Pozzato G Mazzaro C Crovatto M Modolo ML Ceselli S Mazzi G Sulfaro S Franzin F Tulissi P Moretti M Santini GF Low-grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia Blood 1994 84 3047 53 7949176

Sansonno D De Vita S Cornacchiulo V Carbone A Baiocchi M Dammacco F Detection and distribution of hepatitis C-related proteins in lymph nodes of patients with type II cryoglobulinemia and neoplastic or non-neoplastic lymphoproliferation Blood 1996 88 4638 45 8977256

Dammacco F Sansonno D Piccoli C Racanelli V D'Amore FP Lauletta G The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinemia, and Overt B-cell malignancy Semin Liver Dis 2000 20 143 57 10946420 10.1055/s-2000-9613

Kallemuchikkal U Gorevic PD Evaluation of cryoglobulins Arch Pathol Lab Med 1999 123 119 25 10050784 10.5858/1999-123-0119-EOC

Ferri C Sebastiani M Giuggioli D Cazzato M Longombardo G Antonelli A Puccini R Michelassi C Zignego AL Mixed cryoglobulinemia: demographic, clinical, and serological features, and survival in 231 patients Sem Arthritis Rheum 2004 33 355 74 10.1016/j.semarthrit.2003.10.001 15190522

Ferri C Mascia MT Cryoglobulinemic vasculitis: Review Curr Opin Rheumatol 2006 18 54 63 16344620 10.1097/01.bor.0000198002.42826.c2

Bombardieri S Ferri C Migliorini P Pontrandolfo A Puccetti A Vitali C Pasero G Cryoglobulins and immune complexes in essential mixed cryoglobulinemia Ric Clin Lab 1986 16 281 288 2947311 10.1007/BF02909351

Ferri C Mannini L Bartoli V Gremignai G Genovesi-Ebert F Cristofani R Albanese B Pasero G Bombardieri S Blood viscosity and filtration abnormalities in mixed cryoglobulinemia patients Clin Exp Rheumatol 1990 8 271 81 2379342

Fadda P La Civita L Zignego AL Ferri C Hepatitis C virus infection and arthritis. A clinico-serological investigation of arthritis in patients with or without cryoglobulinemic syndrome Reumatismo 2002 54 316 23 12563365 10.4081/reumatismo.2002.316

Ferri C La Civita L Cirafisi C Siciliano G Longombardo G Bombardieri S Rossi B Peripheral neuropathy in mixed cryoglobulinemia: clinical and electrophysiological investigations J Rheumatol 1992 19 889 95 1328632

Ammendola A Sampaolo S Ambrosone L Ammendola E Ciccone G Migliaresi S Di Iorio G Peripheral neuropathy in hepatitis-related mixed cryoglobulinemia: electrophysiologic follow-up study Muscle Nerve 2005 31 382 5 15515001 10.1002/mus.20184

Di Lullo L De Rosa FG Coviello R Sorgi ML Coen G Zorzin LR Casato M Interferon toxicity in hepatitis C virus-associated type II cryoglobulinemia Clin Exp Rheumatol 1998 16 506 9706438

La Civita L Zignego AL Lombardini F Monti M Longombardo G Pasero G Ferri C Exacerbation of peripheral neuropathy during alpha-interferon therapy in a patient with mixed cryoglobulinemia and hepatitis B virus infection J Rheumatol 1996 23 1641 3 8877939

Lidove O Cacoub P Hausfater P Wechsler B Frances C Leger JM Piette JC Cryoglobulinemia and hepatitis C: worsening of peripheral neuropathy after interferon alpha treatment Gastroenterol Clin Biol 1999 23 403 6 10384347

Casato M Saadoun D Marchetti A Limal N Picq C Pantano P Galanaud D Cianci R Duhaut P Piette JC Fiorilli M Cacoub P Central nervous system involvement in hepatitis C virus cryoglobulinemia vasculitis: a multicenter case-control study using magnetic resonance imaging and neuropsychological tests J Rheumatol 2005 32 484 8 15742440

Druet P Letonturier P Contet A Mandet C Cryoglobulinaemia in human renal diseases. A study of seventy-six cases Clin Exp Immunol 1973 15 483 96 4778718 PMC1553960

Longombardo G Ferri C Marchi S Costa F Lombardini F Vacri L Bombardieri S Migliorini P Immune response to an epitope of the NS4 protein of hepatitis C virus in HCV-related disorders Clin Immunol Immunopathol 1998 87 124 9 9614926 10.1006/clin.1997.4498

Uchiyama-Tanaka Y Mori Y Kishimoto N Nose A Kijima Y Nagata T Umeda Y Masaki H Matsubara H Iwasaka T Membranous glomerulonephritis associated with hepatitis C virus infection: case report and literature review Clin Nephrol 2004 61 144 50 14989635 10.5414/cnp61144

Sabbatini A Dolcher MP Marchini B Chimenti D Moscato S Pratesi F Bombardieri S Migliorini P Alpha-enolase is a renal-specific antigen associated with kidney involvement in mixed cryoglobulinemia Clin Exp Rheumatol 1997 15 655 8 9444422

Quartuccio L Fabris M Salvin S Isola M Soldano F Falleti E Beltrami CA De Re V De Vita S Bone marrow B-cell clonal expansion in type II mixed cryoglobulinaemia: association with nephritis Rheumatology (Oxford) 2007 46 1657 61 17893101 10.1093/rheumatology/kem209

Kaplanski G Maisonobe T Marin V Grès S Robitail S Farnarier C Harlé JR Piette JC Cacoub P Vascular cell adhesion molecule-1 (VCAM-1) plays a central role in the pathogenesis of severe forms of vasculitis due to hepatitis C-associated mixed cryoglobulinemia J Hepatol 2005 42 334 340 15710215 10.1016/j.jhep.2004.11.034

Bombardieri S Paoletti P Ferri C Di Munno O Fornai E Giuntini C Lung involvement in essential mixed cryoglobulinemia Am J Med 1979 66 748 56 443251 10.1016/0002-9343(79)91112-4

Salaffi F Manganelli P Carotti M Subiaco S Cervini C Mixed cryoglobulinemia: effect of alpha-interferon on subclinical lymphocytes alveolitis Clin Exp Rheumatol 1996 14 219 20 8737733

Ferri C La Civita L Fazzi P Solfanelli S Lombardini F Begliomini N Monti M Longombardo G Pasero G Zignego AL Interstitial lung fibrosis and rheumatic disorders in patients with hepatitis C virus infection Br J Rheumatol 1997 36 360 5 9133969 10.1093/rheumatology/36.3.360

Manganelli P Salaffi F Subiaco S Carotti M Cervini C Consigli G Majori M Pesci A Bronchoalveolar lavage in mixed cryoglobulinaemia associated with hepatitis C virus Br J Rheumatol 1996 35 978 82 8883436 10.1093/rheumatology/35.10.978

Vitali C Ferri C Nasti P La Civita L Mazzantini M Longombardo G Bombardieri S Hypercomplementaemia as a marker of the evolution from benign to malignant B-cell proliferation in patients with type II mixed cryoglobulinaemia Br J Rheumatol 1994 33 791 2 8055216 10.1093/rheumatology/33.8.791

Antonelli A Ferri C Fallahi P Giuggioli D Nesti C Longombardo G Fadda P Pampana A Maccheroni M Ferrannini E Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia QJM 2004 97 499 506 15256607 10.1093/qjmed/hch088

Antonelli A Ferri C Fallahi P Sebastiani M Nesti C Barani L Barale R Ferrannini E Type 2 diabetes in hepatitis C-related mixed cryoglobulinaemia patients Rheumatology (Oxford) 2004 43 238 40 13130149 10.1093/rheumatology/keh011

Antonelli A Ferri C Fallahi P Nesti C Zignego AL Maccheroni M Thyroid cancer in HCV-related mixed cryoglobulinemia patients Clin Exp Rheumatol 2002 20 693 6 12412202

Ferri C Bertozzi MA Zignego AL Erectile dysfunction and hepatitis C virus infection JAMA 2002 288 698 9 12169072 10.1001/jama.288.6.698

Antonelli A Ferri C Fallahi P Thyroid cancer in patients with hepatitis C infection JAMA 1999 281 1588 10235149 10.1001/jama.281.17.1588

Ferri C Marzo E Longombardo G Lombardini F La Civita L Vanacore R Liberati AM Gerli R Greco F Moretti A Monti M Gentilini P Bombardieri S Zignego AL Alpha-Interferon in Mixed Cryoglobulinemia patients: a randomized crossover controlled trial Blood 1993 81 1132 6 8382969

Di Lullo L De Rosa FG Coviello R Sorgi ML Coen G Zorzin LR Casato M Interferon toxicity in hepatitis C virus-associated type II cryoglobulinemia Clin Exp Rheumatol 1998 16 506 9706438

La Civita L Zignego AL Lombardini F Monti M Longombardo G Pasero G Ferri C Exacerbation of peripheral neuropathy during alpha-interferon therapy in a patient with mixed cryoglobulinemia and hepatitis B virus infection J Rheumatol 1996 23 1641 3 8877939

Lidove O Cacoub P Hausfater P Wechsler B Frances C Leger JM Piette JC Cryoglobulinemia and hepatitis C: worsening of peripheral neuropathy after interferon alpha treatment Gastroenterol Clin Biol 1999 23 403 6 10384347

Antonelli A Ferri C Fallahi P Sebastiani M Nesti C Barani L Barale R Ferrannini E Type 2 diabetes in hepatitis C-related mixed cryoglobulinaemia patients Rheumatology (Oxford) 2004 43 238 40 13130149 10.1093/rheumatology/keh011

Mehta SH Brancati FL Sulkowski MS Strathdee SA Szklo M Thomas DL Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the United States Ann Intern Med 2000 133 592 9 11033586 10.7326/0003-4819-133-8-200010170-00009

Antonelli A Ferri C Galeazzi M Giannitti C Manno D Mieli-Vergani G Menegatti E Olivieri I Puoti M Palazzi C Roccatello D Vergani D Sarzi-Puttini P Atzeni F HCV infection: pathogenesis, clinical manifestations and therapy Clin Exp Rheumatol 2008 26 S39 47 18570753

Monteverde A Rivano MT Allegra GC Monteverde AI Zigrossi P Baglioni P Gobbi M Falini B Bordin G Pileri S mixed cryoglobulinemia, type II: a manifestation of low malignant lymphoma? Clinical-morphological study of 12 cases with special reference to immunohistochemical findings in liver frozen sections Acta Haematol 1988 79 20 25 3124457 10.1159/000205684

Ferri C Pileri S Zignego AL Goedert JJ Hepatitis C virus, B-cell disorders, and non-Hodgkin's lymphoma Infectious causes of cancer: targets for intervention National Cancer Institute (NIH) 2000 Totowa, NJ: The Humana Press Inc 349 368

Pozzato G Mazzaro C Crovatto Modolo ML Ceselli S Mazzi G Sulfaro S Franzin F Tulissi P Moretti M Santini GF Low-grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia Blood 1994 84 3047 53 7949176

La Civita L Zignego AL Monti M Longombardo G Pasero G Ferri C Mixed cryoglobulinemia as a possible preneoplastic disorder Arthritis Rheum 1995 38 1859 60 8849361 10.1002/art.1780381222

Monti G Pioltelli P Saccardo F Campanini M Candela M Cavallero G De Vita S Ferri C Mazzaro C Migliaresi S Ossi E Pietrogrande M Gabrielli A Galli M Invernizzi F Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias Arch Intern Med 2005 165 101 5 15642884 10.1001/archinte.165.1.101

Zignego AL Ferri C Giannelli F Giannini C Caini P Monti M Marrocchi EM Di Pietro E La Villa G Laffi G Gentilini P Prevalence of BCL-2 rearrangement in hepatitis C virus-related mixed cryoglobulinemia with or without complicating B-cell lymphoma Ann Intern Med 2002 137 571 80 12353944 10.7326/0003-4819-137-7-200210010-00008

Ferri C Caracciolo F Zignego AL La Civita L Monti M Longombardo G Lombardini F Greco F Capochiani E Mazzoni A Mazzaro C Pasero G Hepatitis C virus infection in patients with non-Hodgkin's lymphoma Br J Haematol 1994 88 392 4 7803287 10.1111/j.1365-2141.1994.tb05036.x

Matsuo K Kusano A Sugumar A Nakamura S Tajima K Mueller NE Effect of hepatitis C virus infection on the risk of non-Hodgkin's lymphoma: a meta-analysis of epidemiological studies Cancer Sci 2004 95 745 52 15471561 10.1111/j.1349-7006.2004.tb03256.x PMC11159764

Levo Y Gorevic PD Kassab HJ Zucker-Franklin D Franklin EC Association between hepatitis B virus and essential mixed cryoglobulinemia N Engl J Med 1977 296 1501 4 865530 10.1056/NEJM197706302962605

Choo GL Kuo G Weiner AJ Overby LR Bradley DW Houghton M Isolation of a cDNA clone derived from a blood-borne non- A non-B viral hepatitis genome Science 1989 244 359 61 2523562 10.1126/science.2523562

Pascual M Perrin L Giostra E Schifer JA Hepatitis C virus in patients with Cryoglobulinemia type II J Infect Dis 1990 162 569 70 2115556 10.1093/infdis/162.2.569

Ferri C Marzo E Longombardo G Lombardini F Greco F Bombardieri S Alpha-interferon in the treatment of mixed cryoglobulinemia patients. International Cancer Update. Focus on Interferon Alfa 2b. Cannes, France. November 1–4, 1990 Proceedings, Eur J Cancer 1991 27 81 2 10.1016/0277-5379(91)90583-y 1799489

Ferri C Greco F Longombardo G Palla P Marzo E Moretti A Mazzoni A Pasero G Bombardieri S Highfield P Corbishley T Association between hepatitis C virus and mixed cryoglobulinemia Clin Exp Rheumatol 1991 9 621 4 1662567

Misiani R Bellavita P Fenili D Borelli G Marchesi D Massazza M Vendramin G Comotti B Tanzi E Scudeller G Hepatitis C virus infection in patients with essential mixed cryoglobulinemia Ann Intern Med 1992 117 573 7 1326246 10.7326/0003-4819-117-7-573

Agnello V Chung RT Kaplan LM A role for hepatitis C virus infection in type II cryoglobulinemia N Engl J Med 1992 327 1490 5 1383822 10.1056/NEJM199211193272104

Ferri C Monti M La Civita L Longombardo G Greco F Pasero G Gentilini P Bombardieri S Zignego AL Infection of peripheral blood mononuclear cells by hepatitis C virus in mixed cryoglobulinemia Blood 1993 82 3701 4 8260706

Gumber S Chopra S Hepatitis C: a multifaceted disease. Review of extrahepatic manifestations (review) Ann Intern Med 1995 123 615 20 7677303 10.7326/0003-4819-123-8-199510150-00008

De Vita S Sansonno D Dolcetti R Ferraccioli G Carbone A Cornacchiulo V Santini G Crovatto M Gloghini A Dammacco F Boiocchi M Hepatitis C virus within a malignant lymphoma lesion in the course of type II mixed cryoglobulinemia Blood 1995 86 1887 92 7655017

Agnello V Abel G Localization of hepatitis C virus in cutaneous vasculitic lesions in patients with type II cryoglobulinemia Arthritis Rheum 1997 40 2007 15 9365090 10.1002/art.1780401113

Zignego AL Macchia D Monti M Thiers V Mazzetti M Foschi M Maggi E Romagnani S Gentilini P Bréchot C Infection of peripheral mononuclear blood cells by hepatitis C virus J Hepatol 1992 15 382 6 1332999 10.1016/0168-8278(92)90073-X

Pileri P Uematsu Y Campagnoli S Galli G Falugi F Petracca R Weiner AJ Houghton M Rosa D Grandi G Abrignani S Binding of hepatitis C virus to CD81 Science 1998 282 938 41 9794763 10.1126/science.282.5390.938

Zignego AL Ferri C Pileri SA Caini P Bianchi FB for the Italian Association of the Study of Liver Commission on Extrahepatic Manifestations of HCV infection Extrahepatic manifestations of Hepatitis C Virus infection: a general overview and guidelines for a clinical approach: Review Dig Liver Dis 2007 39 2 17 16884964 10.1016/j.dld.2006.06.008

Sansonno D Tucci FA De Re V Lauletta G Montrone M Libra M Dammacco F HCV-associated B cell clonalities in the liver do not carry the t(14;18) chromosomal translocation Hepatology 2005 42 1019 27 16231354 10.1002/hep.20887

Giannini C Giannelli F Zignego AL Association between mixed cryoglobulinemia, translocation (14;18), and persistence of occult HCV lymphoid infection after treatment Hepatology 2005 42 1019 27 16628675 10.1002/hep.20887

Machida K Cheng KT Sung VM Shimodaira S Lindsay KL Levine AM Lai MY Lai MM Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes Proc Natl Acad Sci USA 2004 101 4262 7 14999097 10.1073/pnas.0303971101 PMC384729

Migliaresi S Bresciani A Ambrosone L Spera M Barbarulo D Lombari V Pirozzi G Borgia G Lombardi ML Tirri G Manzo C Increased serum concentrations of soluble HLA-class I antigens in hepatitis C virus related mixed cryoglobulinaemia Ann Rheum Dis 2000 59 20 5 10627422 10.1136/ard.59.1.20 PMC1752989

De Re V Caggiari L Simula MP De Vita S Mazzaro C Lenzi M Massimo GM Monti G Ferri C Zignego AL Gabrielli A Sansonno D Dammacco F Libra M Sacchi N Talamini R Spina M Tirelli U Cannizzaro R Dolcetti R Role of the HLA class II: HCV-related disorders Ann N Y Acad Sci 2007 1107 308 18 17804559 10.1196/annals.1381.033

Dimitrakopoulos AN Kordossis T Hatzakis A Moutsopoulos HM Mixed cryoglobulinemia in HIV-1 infection: the role of HIV-1 Ann Intern Med 1999 130 226 30 10049201 10.7326/0003-4819-130-3-199902020-00027

Scotto G Cibelli DC Saracino A Prato R Palumbo E Fazio V Scarabaggio T Monno L Angarano G Cryoglobulinemia in subjects with HCV infection alone, HIV infection and HCV/HIV coinfection J Infect 2006 52 294 9 16026843 10.1016/j.jinf.2005.05.025

Tzioufas AG Boumba DS Skopouli FN Moutsopoulos HM Mixed monoclonal cryoglobulinemia and monoclonal rheumatoid factor cross-reactive idiotypes as predictive factors for the development of lymphoma in primary Sjögren's syndrome Arthritis Rheum 1996 39 767 72 8639173 10.1002/art.1780390508

Lenzi M Johnson PJ McFarlane IG Ballardini G Smith HM McFarlane BM Bridger C Vergani D Bianchi FB Williams R Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity Lancet 1991 338 277 80 1677111 10.1016/0140-6736(91)90418-O

Ferri C Longombardo G La Civita L Greco F Lombardini F Cecchetti R Cagianelli MA Marchi S Monti M Zignego AL Manns MP Hepatitis C virus as common cause of mixed cryoglobulinemia and chronic liver disease J Intern Med 1994 236 31 6 8021570 10.1111/j.1365-2796.1994.tb01116.x

Buskila D Hepatitis C-associated arthritis Curr Opin Rheumatol 2000 12 295 9 10910182 10.1097/00002281-200007000-00011

Wener MH Hutchinson K Morishima C Gretch DR Absence of antibodies to cyclic citrullinated peptide in sera of patients with hepatitis C virus infection and cryoglobulinemia Arthritis-Rheum 2004 50 2305 8 15248231 10.1002/art.20355

Bombardieri M Alessandri C Labbadia G Iannuccelli C Carlucci F Riccieri V Paoletti V Valesini G Role of anti-cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid arthritis from patients with chronic hepatitis C infection-associated polyarticular involvement Arthritis Res Ther 2004 6 137 41 10.1186/ar1041 PMC400432 15059277

Ramos-Casals M García-Carrasco M Cervera R Rosas J Trejo O de la Red G Sánchez-Tapias JM Font J Ingelmo M Hepatitis c virus infection mimicking primary Sjogren's syndrome: a clinical and immunologic description of 35 cases Medicine (Baltimore) 2001 80 1 8 11204499 10.1097/00005792-200101000-00001

Vasil'ev VI Probatova NA Varlamova EIu Tupitsin NN Simonova MV Safonova TN Bozh'eva LA Shornikova NS Logvinenko OA Belenin GIu Prognostic implications of mixed monoclonal cryoglobulinemia in Sjogren's disease Ter Arkh 2004 76 61 8 15471400

Ramos-Casals M De Vita S Tzioufas A Hepatitis C virus, Sjogren's syndrome and B-cell lymphoma: linking infection, autoimmunity and cancer Autoimmun Rev 2005 4 8 15 15652773 10.1016/j.autrev.2004.04.004

Ramos-Casals M Loustaud-Ratti V De Vita S Zeher M Bosch JA Toussirot E Medina F Rosas J Anaya JM Font J SS-HCV Study Group Sjogren syndrome associated with hepatitis C virus: a multicenter analysis of 137 cases Medicine 2005 84 81 9 15758837 10.1097/01.md.0000157397.30055.c9

Ramos-Casals M Brito-Zerón P Yagüe J Akasbi M Bautista R Ruano M Claver G Gil V Font J Hypocomplementaemia as an immunological marker of morbidity and mortality in patients with primary Sjogren's syndrome Rheumatology (Oxford) 2005 44 89 94 15381790 10.1093/rheumatology/keh407

Ioannidis JPA Vassiliou VA Moutsopoulos HM Long term risk of mortality and lymphoproliferative disease and predictive classification of primary Sjögren's syndrome Arth Rheum 2002 46 741 7 11920410 10.1002/art.10221

Vitali C Bombardieri S Jonsson R Moutsopoulos HM Alexander EL Carsons SE Daniels TE Fox PC Fox RI Kassan SS Pillemer SR Talal N Weisman MH European Study Group on Classification Criteria for Sjögren's Syndrome Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group Ann Rheum Dis 2002 61 554 8 12006334 10.1136/ard.61.6.554 PMC1754137

Pillemer SR Smith J Fox P Bowman SJ Outcome measures for Sjogren's syndrome, april 10–11, 2003, Bethesda, Maryland, USA J Rheumatol 2005 31 143 149 15630740

Italian Group for the Study of Cryoglobulinemias (GISC) Ferri C Ed Metodologie di studio e protocolli diagnostici S Margherita di Pula, Cagliari 1989

Tissot JD Schifferli JA Hochstrasser DF Pasquali C Spertini F Clement F Frutiger S Paquet N Hughes GJ Schneider P Two-dimensional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide J Immunol Methods 1994 173 63 75 8034987 10.1016/0022-1759(94)90284-4

André M Mahammedi H Aumaitre O Tridon A Tissot JD Piette JC A "missed" cryoglobulin: the importance of in vitro calcium concentration Ann Rheum Dis 2000 59 490 2 10.1136/ard.59.6.490a PMC1753150 10885976

Mondelli MU Zorzoli I Cerino A Cividini A Bissolati M Segagni L Perfetti V Anesi E Garini P Merlini G Clonality and specificity of cryoglobulins associated with HCV: pathophysiological implications J Hepatol 1998 29 879 86 9875633 10.1016/S0168-8278(98)80114-1

Landau D Saadoun D Halfon F Martinot-Peignoux M Marcellin P Fois E Cacoub P Relapse of Hepatitis C Virus-Associated Mixed Cr yoglobulinemia Vasculitis in Patients With Sustained Viral Response Arthritis Rheum 2008 58 604 11 18240235 10.1002/art.23305

Casato M Lagana B Antonelli G Dianzani F Bonomo L Long-term results of therapy with interferon-alpha for type II essential mixed cryoglobulinemia Blood 1991 78 3142 785 1742481

Misiani R Bellavita P Fenili D Vicari O Marchesi D Sironi PL Zilio P Vernocchi A Massazza M Vendramin G Interferon alfa-2a therapy in cryoglobulinemia associated with hepatitis C virus N Engl J Med 1994 330 751 6 8107741 10.1056/NEJM199403173301104

Vassilopoulos D Calabrese LH Hepatitis C virus infection and vasculitis: implications of antiviral and immunosuppressive therapies Arthritis Rheum 2002 46 585 97 11920393 10.1002/art.10107

Mazzaro C Franzin F Tulissi P Pussini E Crovatto M Carniello GS Regression of monoclonal B-cell expansion in patients affected by mixed cryoglobulinemia responsive to a-interferon therapy Cancer 1996 77 2604 13 8640712 10.1002/(SICI)1097-0142(19960615)77:12<2604::AID-CNCR26>3.0.CO;2-V

Misiani R Bellavita P Baio P Caldara R Ferruzzi S Rossi P Tengattini F Successful treatment of HCV-associated cryoglobulinaemic glomerulonephritis with a combination of interferon-alpha and ribavirin Nephrol Dial Transplant 1999 14 1558 60 10383026 10.1093/ndt/14.6.1558

Zuckerman E Keren D Slobodin G Rosner I Rozenbaum M Toubi E Sabo E Tsykounov I Naschitz JE Yeshurun D Treatment of refractory, symptomatic, hepatitis C virus related mixed cryoglobulinemia with ribavirin and interferon-alpha J Rheumatol 2000 27 2172 8 10990230

McHutchison JG Gordon SC Schiff ER Shiffman ML Lee WM Rustgi VK Interferon alpha-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group N Engl J Med 1998 339 1485 92 9819446 10.1056/NEJM199811193392101

Mazzaro C Zora F Caizzi M Donada C Di Gennaro G Dal Maso K Carniello G Virgolini L Tirelli U Pozzato G Treatment with peg-interferon alfa-2b and ribavirin of hepatitis C virus-associated mixed cryoglobulinemia: a pilot study J Hepatol 2005 42 632 8 15826710 10.1016/j.jhep.2004.10.031

Saadoun D Resche-Rigon M Thibault V Piette JC Cacoub P Antiviral therapy for hepatitis C virus – associated mixed cryoglobulinemia vasculitis: a long-term followup study Arthritis Rheum 2006 54 3696 706 17075881 10.1002/art.22168

Barth H Schnober EK Neumann-Haefelin C Thumann C Zeisel MB Diepolder HM Hu Z Liang TJ Blum HE Thimme R Lambotin M Baumert TF Scavenger Receptor Class B is Required for Hepatitis C Virus Uptake and Cross-presentation by Human Dendritic Cells J Virol 2008 82 3466 79 18216094 10.1128/JVI.02478-07 PMC2268490

Zaja F De Vita S Mazzaro C Sacco S Damiani D De Marchi G Michelutti A Baccarani M Fanin R Ferraccioli G Efficacy and safety of rituximab in type II mixed cryoglobulinemia Blood 2003 101 3827 34 12560225 10.1182/blood-2002-09-2856

Sansonno D De Re V Lauletta G Tucci FA Boiocchi M Dammacco F Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20 Blood 2003 101 3818 26 12506023 10.1182/blood-2002-10-3162

Roccatello D Baldovino S Rossi D Mansouri M Naretto C Gennaro M Cavallo R Alpa M Costanzo P Giachino O Mazzucco G Sena LM Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis Nephrol Dial Transplant 2004 19 3054 61 15494358 10.1093/ndt/gfh469

Cacoub P Delluc A Saadoun D Landau DA Sene D Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemia vasculitis: where do we stand? Ann Rheum Dis 2008 67 283 7 17644544 10.1136/ard.2006.065565

Ferri C Moriconi L Gremignai G Migliorini P Paleologo G Fosella PV Bombardieri S Treatment of renal involvement in essential mixed cryoglobulinemia with prolonged plasma exchance Nephron 1986 43 246 51 3736735 10.1159/000183849

Moriconi L Ferri C Puccini R Casto G Baronti R Cecchetti R Gremignai G Cioni L Bombardieri S Double filtration plasmapheresis in the treatment of cryoglobulinemic glomerulonephritis Int J Art Org 1989 12 83 6

Ferri C Pietrogrande M Cecchetti C Tavoni A Cefalo A Buzzetti G Vitali C Bombardieri S Low-antigen-content diet in the treatment of mixed cryoglobulinemia patients Am J Med 1989 87 519 24 2816967 10.1016/s0002-9343(89)80607-2

Ferri C Puccini R Longombardo G Paleologo G Migliorini P Moriconi L Pasero G Cioni L Low-antigen-content diet in the treatment of patients with IgA nephropathy Nephrol Dial Transplant 1993 8 1193 1198 8302454

Lamprecht P Lerin-Lozano C Merz H Dennin RH Gause A Voswinkel J Peters SO Gutzeit O Arlt AC Solbach W Gross WL Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis Ann Rheum Dis 2003 62 1230 3 14644867 10.1136/ard.2002.004929 PMC1754408

Saadoun D Resche-Rigon M Sene D Perard L Piette JC Cacoub P Rituximab combined with Peg-Interferon-Ribavirin in refractory HCV-associated cryoglobulinemia vasculitis Ann Rheum Dis 2008 Jan 4 10.1136/ard.2007.081653 18178690

Galeazzi M Bellisai F Manganelli S Morozzi G Sebastiani GD Cyclosporine A for the treatment of autoimmune disorders in HCV infected patients Autoimmun Rev 2006 5 493 8 16920576 10.1016/j.autrev.2006.01.002

Zein NN for the Etanercept Study Group Etanercept as an adjuvant to interferon and ribavirin in treatment-naive patients with chronic hepatitis C virus infection: a phase 2 randomized, double-blind, placebo-controlled study J Hepatol 2005 42 315 22 15791697 10.1016/j.jhep.2004.11.025

Vassilopoulos D Calabrese LH Risks of immunosuppressive therapies including biologic agents in patients with rheumatic diseases and co-existing chronic viral infections. Review Curr Opin Rheumatol 2007 19 619 25 17917544 10.1097/BOR.0b013e3282f05b63