Preliminary classification criteria for the cryoglobulinaemic vasculitis.

BACKGROUND: To develop preliminary classification criteria for the cryoglobulinaemic syndrome or cryoglobulinaemic vasculitis (CV). METHODS: Study part I developed a questionnaire for CV to be included in the formal, second part (study part II). Positivity of serum cryoglobulins was defined by experts as an essential condition for CV classification. In study part II, a core set of classification items (questionnaire, clinical and laboratory items, as agreed) was tested in three groups of patients and controls-that is, group A (new patients with the CV), group B (controls with serum cryoglobulins but lacking CV) and group C (controls without serum cryoglobulins but with features which can be observed in CV). RESULTS: In study part I (188 cases, 284 controls), a positive response to at least two of three selected questions showed a sensitivity of 81.9% and a specificity of 83.5% for CV. This questionnaire was employed and validated in study part II, which included 272 patients in group A and 228 controls in group B. The final classification criteria for CV, by pooling data from group A and group B, required the positivity of questionnaire plus clinical, questionnaire plus laboratory, or clinical plus laboratory items, or all the three, providing a sensitivity of 88.5% and a specificity of 93.6% for CV. By comparing data in group A versus group C (425 controls), the same classification criteria showed a sensitivity 88.5% and a specificity 97.0% for CV. CONCLUSION: Classification criteria for CV were developed, and now need validation.

Selective enhancement of the fluorescent pseudomonad population after amending the recirculating nutrient solution of hydroponically grown plants with a nitrogen stabilizer.

Fluorescent pseudomonads have been associated, via diverse mechanisms, with suppression of root disease caused by numerous fungal and fungal-like pathogens. However, inconsistent performance in disease abatement, after their employment, has been a problem. This has been attributed, in part, to the inability of the biocontrol bacterium to maintain a critical threshold population necessary for sustained biocontrol activity. Our results indicate that a nitrogen stabilizer (N-Serve, Dow Agrosciences) selectively and significantly enhanced, by two to three orders of magnitude, the resident population of fluorescent pseudomonads in the amended (i.e., 25 microg ml(-1) nitrapyrin, the active ingredient) and recycled nutrient solution used in the cultivation of hydroponically grown gerbera and pepper plants. Pseudomonas putida was confirmed as the predominant bacterium selectively enhanced. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA suggested that N-Serve selectively increased P. putida and reduced bacterial diversity 72 h after application. In vitro tests revealed that the observed population increases of fluorescent pseudomonads were preceded by an early growth suppression of indigenous aerobic heterotrophic bacteria (AHB) population. Interestingly, the fluorescent pseudomonad population did not undergo this decrease, as shown in competition assays. Xylene and 1,2,4-trimethylbenzene (i.e., the inert ingredients in N-Serve) were responsible for a significant percentage of the fluorescent pseudomonad population increase. Furthermore, those increases were significantly higher when the active ingredient (i.e., nitrapyrin) and the inert ingredients were combined, which suggests a synergistic response. P. putida strains were screened for the ability to produce antifungal compounds and for the antifungal activity against Pythium aphanidermatum and Phytophthora capsici. The results of this study suggest the presence of diverse mechanisms with disease-suppressing potential. This study demonstrates the possibility of using a specific substrate to selectively enhance and maintain desired populations of a natural-occurring bacterium such as P. putida, a trait considered to have great potential in biocontrol applications for plant protection.

Emergency in cryoglobulinemic syndrome: what to do?

The clinical course of cryoglobulinemic syndrome (CS) is usually slow; however, fast aggravations have been frequently reported in recent years. In these cases vasculitic ischemic tissue damage accounts for glomerular involvement, neuropathy, cutaneous ulcers, ischemic heart disease, lung or jejunal impairment and stroke. Other critical events in CS may be represented by sepsis, liver insufficiency, hepatocellular carcinoma and non-Hodgkin's lymphomas. Sometimes emergency can not be controlled and the evolution is fatal. Long-term follow up, emergency outcome and cause of death have not been considered in controlled studies, in large series. Here we report a 53-year old woman affected by IgG-IgMk type II HCV-related mixed cryoglobulinemia, who presented several critical events over the course of the disease, which required therapeutical emergency interventions. The latter consisted of plasma exchange, cytotoxic agents, corticosteroids, intravenous immunoglobulin, antihypertensive drugs, antibiotics, and rituximab. Eventually no therapy was effective and the patient died from a catastrophic-like syndrome. This case is relevant because it enables us to consider some important steps in the treatment of emergency in CS.

Isolation and characterisation of wheat cDNA clones encoding PR4 proteins.

Two cDNA clones encoding the previously characterised PR4 proteins wheatwin 1 and wheatwin2 from wheat (Triticum aestivum cv. S. Pastore) have been identified and named wPR4a and wPR4b, respectively. The clones have been isolated by screening a cDNA library with a specific cDNA probe obtained by RT-PCR. The wPR4a and wPR4b cDNAs contain open reading frames of 441 and 447 bp that encode for wheatwin1 and wheatwin2, respectively.

HCV genotypes in bone marrow and peripheral blood mononuclear cells of patients with mixed cryoglobulinemia.

OBJECTIVE: The association of the hepatitis C virus (HCV) with the cryoglobulinemic syndrome is well known, but its pathogenetic mechanism still remains to be clarified. HCV-RNA has been found in the peripheral blood mononuclear cells (PBMC) of infected subjects. We investigated the presence of the HCV genome in bone marrow cells (BMC), and the distribution of different HCV genotypes in individuals with mixed cryoglobulinemia (MC) and in noncryoglobulinemic controls. METHODS: 15 anti-HCV positive subjects with MC, 7 non-cryoglobulinemic patients with type C chronic active hepatitis (CAH) and 2 anti-HCV negative controls were studied. HCV-RNA was detected by nested PCR of the highly conserved 5'-NCR sequence. HCV typing was carried out by means of the hybridization of the same amplified region with specific probes. RESULTS: HCV-RNA was present in the PBMC of a large proportion of the MC patients and controls without any significant differences. On the contrary, HCV-RNA was present in the bone marrow cells of all the patients with MC and in 43% of the CAH controls. The HCV 1b and 2a genotypes seem to be the most prevalent among MC patients. Nevertheless, the patients with type II MC had a very high prevalence of the 2a genotype (77%). CONCLUSION: The results suggest that the presence of HCV-RNA in bone marrow cells may be correlated to the pathogenetic mechanism of MC. Other studies are needed to confirm the frequent association of HCV genotype 2 with MC.

The natural history of cryoglobulinemia: symptoms at onset and during follow-up. A report by the Italian Group for the Study of Cryoglobulinemias (GISC)

OBJECTIVE: The cryoglobulinemic syndrome (CS) may be associated with other diseases; when it is not, it is termed essential. Recently the natural history of this disease has been re-evaluated on the basis of its close association with HCV markers. In this context, the GISC has studied a large series of patients from several Italian centres. METHODS: In a multicentric retrospective study of 913 cases, we evaluated the clinical and laboratory signs at onset in patients with essential mixed cryoglobulinemias (EMC) (654 cases) or secondary cryoglobulinemias (SC) (259 cases) and sought to define possible diagnostic criteria typical of the different CS. We also carried out a retrospective 5-year cohort study on 192 patients selected randomly from the 913 cases described above. In particular, we examined the correlation between the presence/absence of concomitant diseases and the presence of HCV markers on the clinical evolution and survival of the patients. RESULTS: Purpura, rheumatoid factors (RF), significant C4 consumption, and Brouet's classification type II were more frequent in EMC. Purpura, Meltzer's triad, and Raynaud's phenomenon improved, while renal involvement tended to worsen over time. During the follow-up we did not note a significant change in clinical staging in the patients with Brouet's type II cryoglobulins, in the patients with HCV-related markers, or in the overall series. The most frequent causes of death in 34 patients were liver, cardiovascular, renal, and lymphoproliferative diseases. Lymphomas were diagnosed in 11 patients during follow-up, with particular frequency in the HCV-marker positive patients. CONCLUSION: Specific clinical and laboratory features typical of the different CS subgroups could not be identified on the basis of these data. Our follow-up data seem, however, to confirm a role for HCV in the mixed cryoglobulinemias.

Hepatitis C antibody and cryoglobulins in patients with cirrhosis and hepatocellular carcinoma.

OBJECTIVE: To assess: (1) the role of hepatitis C virus (HCV), hepatitis B virus (HBV) and alcoholism as risk factors for hepatocarcinoma (HCC) in patients with liver cirrhosis, and (2) the presence of cryoglobulins in HCV + patients with and without HCC. PATIENTS: 82 cirrhotic patients, 41 with and 41 without HCC, who were admitted consecutively to our General Medicine Division from January 1992 to June 1994, were studied. RESULTS: The prevalence of HBV markers, anti-HCV and alcoholism in patients with liver cirrhosis and HCC was 39%, 63.4% and 24%, and in cirrhotics without HCC it was 31.7%, 51.2% and 63.4%, respectively. Cryoglobulins were present in 87.5% of the patients with HCC (78.5% anti-HCV +) and in 57.8% of the patients without HCC (81.8% anti-HCV +). The cryoglobulins, as characterized in 11 cases, were type III in nine cases and type II in two. CONCLUSIONS: Patients with cirrhosis, especially when associated with HCV and HBV infection, are at high risk for HCC and therefore require careful follow-up. Moreover, a strong association between HCV and the cryoglobulins in cirrhotics with and without HCC was evident, thus supporting the possible role of this virus in stimulating lymphocytes to produce cryoglobulins.

Anti-GOR antibodies, HCV, liver disease and cryoglobulinemia.

OBJECTIVE: We investigated the presence of anti-GOR antibodies in patients with essential mixed cryoglobulinemia, since both autoimmune pathogenetic processes and a high prevalence of HCV infection are present in this syndrome. METHODS: We compared these cases to patients with HCV-related chronic active hepatitis or alcoholism, and to ex-blood donors. A total of 60 patients with biopsy-proven chronic liver disease were studied. RESULTS: HCV related markers, cryoglobulins, anti-GOR antibodies and ANA were detected in all of the groups. CONCLUSION: Our data would appear to indicate that anti-GOR are related to the presence of HCV chronic hepatitis and not to cryoglobulinemia or chronic liver damage.

Colchicine in the treatment of mixed cryoglobulinemia.

OBJECTIVE: The best treatment for cryoglobulinemic syndrome (CS) is still an unsolved problem. Recently colchicine has been successfully used to treat vasculitides and other immune-mediated diseases. Therefore, we undertook to treat 17 CS patients with colchicine (1 mg/day for 6-48 months), 8 of them with essential mixed cryoglobulinemia (EMC) and 9 with CS secondary to liver disease. METHODS: In all patients the clinical and laboratory features were evaluated at the beginning of the study and during the first 6-12 months; 10 cases were followed for a longer period (18-48 months). RESULTS: During the first period symptoms improved as follows: purpura in 15 of 17 patients, weakness in 9 of 14 and leg ulcers in 3 of 5. Hepatic and renal function tests, hypocomplementemia, rheumatoid factor (RF) titres and the cryocrit also improved. Prolonged follow-up showed a relapse in the different variables, although they remained at better levels than at the beginning. Only the cryocrit showed a further reduction. CONCLUSION: Though this was a preliminary open study it shows that colchicine is an efficient treatment in CS and suggests that a controlled clinical study should be performed to assess its real value.

Hepatitis C virus RNA in the bone marrow of patients with mixed cryoglobulinemia and in subjects with noncryoglobulinemic chronic hepatitis type C.

Hepatitis C virus (HCV) infection is associated with most mixed cryoglobulinemia (MC) syndromes. In this study, HCV RNA was detected in the peripheral blood mononuclear cells of 11 (73.3%) of 15 patients with MC and in 5 (71.4%) of 7 noncryoglobulinemic patients with chronic hepatitis type C. All patients with cryoglobulinemia and 3 (42.8%) of the 7 without cryoglobulinemia (P < .05) had HCV RNA in bone marrow cells. Subjects in both groups with HCV-positive bone marrow also had HCV RNA in serum. The majority of patients with MC syndromes were infected with HCV subtypes 1b and 2a. Two patients with MC had different genotypes in serum and cells. Further studies are needed to determine which bone marrow cell population is preferentially infected by HCV and to determine if this phenomenon is involved in inducing the production of cryoglobulins.

Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC. Italian Group for the Study of Cryoglobulinaemias.

In a multi-centre retrospective study, we compared clinical and laboratory data in 913 patients with cryoglobulinaemias, divided as: (i) essential cryoglobulinaemias; (ii) cryoglobulinaemias secondary to connective tissue diseases (CTD), lymphoproliferative or other haematological diseases (LPD), chronic liver diseases (CLD), and 'other diseases'. Purpura was the commonest presenting feature in all groups and was more common in essential cryoglobulinaemias (p < 0.0001). Meltzer's triad (purpura, arthralgia, weakness) was less frequent, but similarly distributed. Renal involvement was randomly distributed. Neurological impairment was less frequent in cryoglobulinaemias secondary to CLD (p < 0.002). Raynaud's phenomenon, arthritis and sicca syndrome were more frequent in cryoglobulinaemias secondary to CTD. Essential cryoglobulinaemias had a significantly higher percentage of serum complement C4 < 8 mg/dl (p < 0.004), of detectable rheumatoid factor activity (p < 0.0002), and of type II cryoglobulins (p < 0.0001). Liver involvement was evident at presentation in 32.6% of essential cryoglobulinaemias, 27.1% of cryoglobulinaemias secondary to LPD and 12.2% of cryoglobulinaemias secondary to CTD. Antibodies to hepatitis B surface (HBsAg) and core (HBc) antigens were more frequent in cryoglobulinaemias secondary to CLD; anti-HBs antibodies were randomly distributed. Antibodies to hepatitis C (HCV) were tested for in 224 patients, and prevalence was high in all the groups, but lower in cryoglobulinaemias secondary to CTD (p < 0.0001). Type II and type III essential cryoglobulinaemias differed significantly in renal involvement (p < 0.0001), cryocrit > 3% (p < 0.0001), C4 < 15 mg/dl (p < 0.001), HBsAg prevalence (p < 0.01) and purpura (p < 0.05). Despite the high prevalence of HCV markers in all groups, the role of HCV in essential cryoglobulinaemia is not well defined; HBV seems to play only a marginal role.

[Chronic eosinophilic pulmonitis with eosinophilic pleurisy. A report on 2 clinical cases seen by the authors]. / Polmonite cronica eosinofila con pleurite eosinofila. Descrizione di due casi clinici di nostra osservazione.

We discuss the cases of two patients affected with chronic eosinophilic pneumonia (CEP) pleurisy and eosinophilia in pleural effusion, not previously mentioned in the literature, to point out their peculiarity, to consider differential diagnosis and the effect of steroid therapy. Both patients, a 57-year-old man and a 55-year-old woman, were atopic: they had been suffering from allergic rhinitis and asthma for several years when they suffered sudden onset of cough, dyspnea and thoracic pain. This symptomatology persisted for more than 6 weeks. Chest radiography highlighted pulmonary infiltrates, not fixed in the first case, fixed in the second. The laboratory features revealed eosinophilia in peripheral blood and in pleural effusion. These data conformed to the criteria suggested by Jederlinic et al. for the diagnosis of chronic eosinophilic pneumonia. Tuberculosis had been present in the remote history of the second case; the repeated research for mycobacteria was negative, and no improvement was seen after antitubercular chemotherapy for one month. We excluded the diagnosis of allergic bronchopulmonary aspergillosis because of the absence of both precipitating antibodies against Aspergillus fumigatus and bronchiectasis. Neither vasculitis nor autoantibodies were found; possible drug-related correlations were excluded; culture data and serological researches for infections were negative in both cases; no involvement of other districts correlated to hypereosinophilia was evidenced. Clinical and radiological remission was obtained in both cases after steroid therapy for a month at the dosage of 1-2 mg/kg daily. No clinical recurrence was seen during a follow-up period of 6 months. Pleural effusion has already been reported in patients with CEP, while we have not found any references to pleural fluid eosinophilia in this disease; this finding has instead been already reported in patients affected with acute eosinophilic pneumonia or hypereosinophilic syndrome.

Analysis of serum immune complexes isolated from patients with essential mixed cryoglobulinemia.

The analysis of the molecular composition of serum immune complexes (IC) in autoimmune diseases has assumed growing importance in the evaluation of their clinical significance and possible pathogenetic role. Essential Mixed Cryoglobulinemia (EMC) is typically a disorder characterized by the presence of high levels of IC with pathogenic action, by reduced serum complement levels and by an antiglobulin activity of the cryoprecipitable IgM. In this report we refer on the preliminary results of a study on the molecular composition of the cryoprecipitable IC isolated from the serum of five patients with EMC. The methodology we adopted involves the solubilization at 37 degrees C of the washed cryoprecipitates and the purification of the IgG-containing IC by affinity chromatography through a Protein A-Sepharose CL4B column. The analysis of the molecular component was carried out by electrophoresis in polyacrylamide gel containing sodium-dodecyl-sulphate (SDS-PAGE) followed by Immun-Blot. Our results, although very preliminary, suggest that this kind of study appears really suitable for the detection of specific antibody or antigenic components in the IC molecules involved in the etiopathogenesis of essential mixed cryoglobulinemia.

The cryoglobulinemic syndrome.

The clinical and immunochemical classification of 376 patients with cryoglobulinemia is presented. In 141 cases (37.5%) the syndrome was considered idiopathic. Among the secondary forms, chronic liver diseases and connective tissue diseases shared most frequently mixed cryoglobulins (18.8 and 15.4% of cases, respectively). Another common association was that with Waldenström's macroglobulinemia: cryoglobulins were found in 20% of these patients. In 5 patients (2 with the essential form and 3 with cryoglobulinemia secondary to Waldenström's disease) a Cl-inhibitor deficiency was discovered; the complement profile was characteristic of the acquired type and episodes of angioedema occurred. Studying the clinical course of 71 cases of essential mixed cryoglobulinemia followed-up for 4-20 years we concluded that the evolution of type II and type III cryoglobulinemias is different because that of type II seems to have a more severe course and may differentiate in a lymphoproliferative disorder; the immunochemical type of cryoglobulins seems therefore to have a prognostic significance. Because of the considerable overlap in the distribution of immunochemical types among the clinical subsets, a mixed classification (both biochemical and clinical) is proposed.

T lymphocyte subpopulations defined by monoclonal antibodies in essential mixed cryoglobulinemia and in secondary cryoglobulinemias.

T lymphocyte subpopulations defined by monoclonal antibodies were determined in patients with essential mixed cryoglobulinemia (EMC) and secondary cryoglobulinemias (SC). A decrease of circulating lymphocytes and a reduction in the absolute number of T3+, T4+ and T8+ (p less than 0.01) as well as in the percentage of T4+ lymphocytes (p less than 0.05) were found in EMC. A significant decrease of T8+ cells, both in percentage (p less than 0.01) and absolute number (p less than 0.001), was evidenced in SC, while T3+ and T4+ cell counts were not significantly different from those of healthy controls.

Absence of antinuclear antibodies in cryoprecipitates of patients with essential mixed or secondary non-lupoid cryoglobulinemia.

A panel of autoantibodies has been tested in serum samples and cryoprecipitates of 14 patients affected by essential mixed cryoglobulinemia (EMC) as well as in 12 subjects with secondary non-lupoid cryoglobulinemia (SC). Three out of 14 patients affected by EMC were ANA-positive (2 at 1:40 dilution and one at 1:80 dilution). Two out of 12 patients with SC were ANA-positive (both at 1:40 dilution). No autoantibodies were found in the cryoprecipitates. These data seem to confirm some previous observations and do not indicate a role of autoantibodies in the cryoprecipitate formation.

Causes of death in essential mixed cryoglobulinemia.

Ninety-three patients with essential mixed cryoglobulinemia have been followed for more than 5 years in 75% of cases; 21 patients died during this period and the mean age at death was 54 years. The most common cause of death was renal failure. The percentages of patients who survived after 5 and 10 years from the diagnosis were 87 and 74%, respectively. The cryoglobulin type seems to be an important prognostic factor: in our population the type II/III ratio is 6/7 at diagnosis, while it becomes 2/1 when the patients who died are considered.