The anticytokine neuropeptide alpha-melanocyte-stimulating hormone in synovial fluid of patients with rheumatic diseases: comparisons with other anticytokine molecules.

The aim of this study was to determine if the anticytokine neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) occurs, along with interleukin 1 receptor antagonist (IL-1ra) and soluble tumor necrosis factor receptor (sTNFr), in synovial fluid of patients with rheumatoid arthritis (RA), juvenile chronic arthritis (JCA), or osteoarthritis. The data show that alpha-MSH does occur in the synovial fluid and its concentrations are greater in patients with RA than in those with osteoarthritis. Synovial fluid concentrations of IL-1ra and sTNFr were likewise greater in RA. Further, concentrations of alpha-MSH, IL-1-ra, and sTNFr were greater in patients with polyarticular/systemic-onset JCA than in those with pauciarticular disease, that is in patients with greater joint inflammation. Concentrations of alpha-MSH were greater in synovial fluid than in plasma in a substantial proportion of patients, suggesting local production of the peptide; this is the first indication that the anticytokine molecule alpha-MSH is produced within a site of inflammation. Further, it appears that local production of alpha-MSH is induced particularly in those arthritic joints that have more intense inflammatory reactions. This finding, combined with previous evidence of the marked anti-inflammatory activity of alpha-MSH, suggests that the peptide acts locally to modulate proinflammatory influences in rheumatic diseases.

Rheumatoid factors and circulating immune complexes in HIV-infected individuals.

We studied serological aspects of autoimmunity in patients with AIDS, AIDS-related complex (ARC) and in individuals at risk for AIDS. Immunoglobulin (Ig) M, IgG and IgA rheumatoid factors (RF) were quantified by enzyme-linked immunosorbent assay (ELISA), Ig by radial immunodiffusion, and circulating immune complexes (CIC) by the CIC-conglutinin and CIC-complement 1q (C1q) assays. Mean IgM RF levels were normal in AIDS patients, but those of ARC patients were higher and more frequent than the levels defined by agglutination methods. Similar observations were made for intravenous drug users (IVDU) and for both HIV-seropositive and HIV-seronegative homosexual men. Mean IgG RF levels were normal in AIDS and ARC patients but high in homosexual men and, to a lesser degree, in IVDU. IgA RF levels were high in many AIDS and ARC patients, in homosexual men, and in haemophiliac and control groups. The selective increase of the IgA isotype in AIDS was confirmed by the Ig results, which also showed an IgG increase in all groups. IgM were mainly high in people with ARC. CIC were detected in 68% of ARC patients by both methods, and in 55% of AIDS patients by CIC-Clq. A high incidence of positive samples in all at-risk populations, but particularly in seronegative individuals, was observed using CIC-conglutinin. CIC-C1q also revealed larger amounts of CIC in HIV-seronegative individuals, mainly in homosexual men. The study of these humoral aspects of autoimmunity provides useful information on the impairment of B-cells in patients with AIDS and ARC.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of rheumatoid factors of different isotypes and of circulating immune complexes in patients with inflammatory bowel disease.

The behaviour of some serological aspects of autoimmunity was studied in patients with inflammatory bowel disease (IBD) by determining the incidence and levels of serum IgM, IgG and IgA rheumatoid factors (RF) and of circulating immune-complexes (IC) in 67 patients suffering from ulcerative colitis (UC) and 53 from Crohn's disease (CD). Sera from 40 young healthy subjects were included as controls. RF were detected by an enzyme linked immuno-assay (ELISA) and IC by the CIC-conglutinin and the CIC-C1q assays. ELISA detected a higher incidence of IgM RF in both UC and CD groups than the routine agglutination tests, although the mean levels were non significantly higher than in controls. Mean IgG RF levels were significantly above normal in UC and CD patients, the latter showing a greater incidence of elevated values and higher mean levels. IgA RF were significantly elevated only in CD patients; mean values for the UC group were within the normal range. The CIC-conglutinin test did not detect a significant incidence or high mean levels of IC and the CIC-C1q assay turned up a sizeable number of positive samples only in the CD group. Although still only preliminary, these overall findings suggest that RF activity is somehow present in patients with IBD. The greater prevalence of IC in CD patients, together with the patterns of IgG and IgA RF support the concept that this disease possibly involves autoimmune phenomena more than ulcerative colitis.

Circulating immune complexes in serum and in cerebrospinal fluid of patients with multiple sclerosis. Characterization and correlation with the clinical course.

We studied circulating immune complexes (IC) in the serum and cerebrospinal fluid (CSF) of patients with clinically defined multiple sclerosis (MS), in order to establish a correlation with the clinical course of the disease and to investigate the molecular composition of the IC isolated from patients in active phase of the disease. Serum IC levels were found to be significantly increased in patients from the progressive and active relapsing-remittent subgroups with both the CIC-conglutinin and C1q-binding methods. High levels of IC in CSF were detected only in the subgroup consisting of the relapsing-remittent patients in disease exacerbation when IC were determined by the C1q-binding test. No significant increase in serum or in CSF were found using the mRF-I test. The preliminary results of a qualitative investigation on serum IC in MS indicated that they are heterogeneous in nature, their size is mainly of the intermediate type, and they contain IgG, IgM, complement components and beta 2-microglobulins, the latter presenting an observation both new and interesting for studies on serum IC in MS patients.

ELISA determined IgM, IgG and IgA rheumatoid factors in rheumatoid arthritis and in other connective tissue diseases.

We used an adaptation of an enzyme-linked immunoadsorbent assay (ELISA) to determine serum levels of IgM, IgG and IgA rheumatoid factors (RF) in 50 patients with classic or definite rheumatoid arthritis (RA) according to the ARA criteria, balanced for positive or negative-routine Latex-RF reaction. A control group of 50 young normal subjects and a reference group of 44 patients with other connective tissue diseases (OCTD) were also studied. We confirmed the high sensibility of the method, together with its good specificity and reproducibility. For the IgM RF a very significant correlation was found between ELISA results and Latex-RF titration (p less than 0.001). Many Latex-RF negative RA patients had high ELISA levels of IgM RF, suggesting that this assay reveals, at least in part, hidden or non-agglutinating IgM RF. Among the OCTD group only some SLE cases, mainly Latex-RF positive, had enhanced IgM RF on ELISA. Considered quantitatively, IgG RF did not play a significant diagnostic role for RA (p greater than 0.05), because they were also found, with widely dispersed values, in normal subjects, and because the mean increase in RA patients was relatively small. Interestingly, IgA RF were above the normal range in many RA patients, both Latex-RF positive or negative. The mean values differed significantly from those of controls (p less than 0.005), and a correlation was observed between IgA RF levels and IgA containing immune-complexes. Normal IgA RF values were observed in SLE patients, even if Latex-RF positive, suggesting that their increase in RA patients is not the mere expression of a polyclonal B cell activation.

IgM, IgG and IgA rheumatoid factors and circulating immune complexes in patients with AIDS and AIDS-related complex with serological abnormalities.

To investigate some humoral aspects which may reflect the involvement of B lymphocytes in the acquired immunodeficiency syndrome (AIDS), we used an enzyme-linked immunoassay (ELISA) to determine the levels of IgM, IgG and IgA rheumatoid factors (RF) in 16 patients suffering from full-blown AIDS and 32 patients with AIDS-related complex (ARC), in the clinical form of lymphoadenopathy syndrome (LAS), compared with 40 healthy, young heterosexual subjects. Both AIDS and ARC patients showed a greater incidence of high IgM RF levels, with mean values significantly higher than controls, but with no differences between the two pathological groups. IgG RF behaviour was similar in the two patient populations and the healthy subjects. IgA RF were significantly raised in AIDS and ARC. Further information on RF was obtained by determination of the immunoglobulin levels of the respective isotypes in the same patients. Mean IgG levels were above normal in AIDS and ARC patients, but the latter group showed a higher incidence of increased values and higher mean levels. The IgA isotype was significantly increased mainly in AIDS patients. The behaviour of IgM was virtually the same in the three groups studied. A difference between AIDS and ARC patients was established by the detection of circulating immune-complexes (IC) by the C1q-binding and CIC-conglutinin assays. IC were significantly high, by both methods, only in the ARC group, but normal or very low in AIDS. These overall findings suggest once again the impairment of B cell function in AIDS, with prevalent hyperactivation in ARC and exhaustion in full-blown AIDS, and apparent preservation, in the latter group, of the antibody responses which are more closely related to the activity of subsets of T helper cells.

Detection, by an enzyme linked immunoadsorbant assay (ELISA), of IgM, IgG and IgA rheumatoid factors in patients with essential mixed cryoglobulinemia.

In order to investigate on some yet unclear aspects of the relationship between Rheumatoid Factor (RF) activity, cryoprecipitation and clinical features of Essential Mixed Cryoglobulinemia (EMC), we determined, by an ELISA technique, the IgM, IgG and IgA RF in 16 patients with EMC, 16 patients affected with seropositive Rheumatoid Arthritis (AR), taken as a reference, and 16 young, normal controls. For IgM RF a high rate of incidence of positive samples (11/16 = 69%) from the EMC group was found, with mean values significantly higher than controls (p less than 0.001) and quite similar to those of the RA group. The ELISA assay was performed at room temperature (RT) and at 37 degrees C, and only a slight increase of adsorbance values in the latter condition was observed. On the contrary, temperature revealed to be a critical experimental condition for the detection of IgG RF. In fact at 37 degrees C the mean values of EMC patients were similar to those of RA and control groups, while at RT a marked increase of adsorbance values was observed, in spite of the inactivation of IgMRF due to the preliminary pepsin digestion of the samples. More interesting are our results concerning IgA RF. In fact a high incidence of overnormal values (9/16 = 56.2%) were found in the EMC group, similar to those of RA patients, with mean values which significantly differ from the normal subjects (p less than 0.01). No relevant differences derived from the temperature at which the test was carried out. The relationship between IgA RF and clinical course of EMC seems to be a very stimulating aim for further investigations.

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.

Isolation and characterization of circulating immune complexes in patients with rheumatoid arthritis.

We studied soluble immune complexes in sera and synovial fluids of ten patients suffering from Rheumatoid Arthritis (RA), six seropositive and four seronegative. Isolation of the immune complexes was obtained by a combination of gel filtration and affinity chromatography from biological fluids. The first step involved preparation of a globulin-enriched fraction by precipitation of either serum or synovial fluid with 5% polyethylene glycol 6000. The precipitate was solubilized in Borate NaCl buffer and fractioned by gel filtration in Ultrogel AcA 34. Eluted peaks were submitted to affinity chromatography on a Protein A-Sepharose CL 4B column. The recovered material was analyzed by double immunodiffusion and electrophoresis in 6-20% polyacrylamide gel containing sodium dodecylsulphate (SDS-PAGE) and was shown to contain IgG, IgM, IgA with small amounts of C1q, C3 and C4. Observation occurred that there is a different distribution of macromolecular and intermediate complexes in seropositive and seronegative samples respectively, total C1q binding activity being equal.

IgG rheumatoid factors behaviour in young normal subjects following vaccination.

In order to contribute to explain the finding of Rheumatoid Factors of the IgG class in all normal subjects, even at low levels, a study on their behaviour in a group of young subjects serving in the army, at the time of anti-typhoid and antitetanic vaccination, and in the following two months, was conducted. Parallely some aspecific serological parameters of autoimmunity were determined. The results demonstrate, first of all, a good antibody response to the vaccine in all subjects observed. Immunoglobulins of the IgG, IgA, and IgM class were normal in all samples. Complement factors C3 and C4 were slightly decreased after the second dose of vaccine, suggesting a modest consumption probably due to immune complexes formation. Factor C3A showed a slight increase, may be to be considered as its property of an acute phase reactant. Autoantibodies determined by immunofluorescence were constantly negative. IgM rheumatoid factors became transitory positive in a significant proportion. IgG rheumatoid factors demonstrated a two-phase increase, more evident after the second dose of vaccine. We can conclude, mainly for the latest parameter, that these results reveal an autoimmune serological response, even quantitatively modest, concomitant to the specific sensitization resulting from vaccination.

Peripheral neuropathy and solitary myeloma: analysis of serum and CSF IgG in two cases.

Clinical and laboratory findings of two patients affected by solitary myeloma, IgG3 lambda type, with peripheral neuropathy are reported. In both cases the same IgG isoelectrofocusing pattern was found in cerebrospinal fluid and serum samples. Data are consistent with damage of the blood-brain barrier.

CSF immunoglobulins G in multiple sclerosis.

Three different methods were compared to detect an intrathecal IgG synthesis in cerebrospinal fluid of patients with Multiple Sclerosis. A preliminary investigation was carried out on serum and cerebrospinal fluid proteins fractionated by means of analytical isotachophoresis. This technique shows many advantages but isoelectrofocusing is still the most available technique in diagnostic trials.

Multiple tests in the diagnosis of multiple sclerosis.

Visual evoked potentials (VEPs), somatosensory evoked potentials (SEPs), E-UFA test and CSF isoelectrofocusing (IEF) were applied to 42 multiple sclerosis patients. VEPs were abnormal in 29 cases. SEPs in 31 cases, E-UFA in 30 cases, CSF IEF in 38 cases. At least two tests were abnormal in 41 out of 42 patients; 16 cases revealed pathological responses in three tests (38,1%), 15 in four of them (35,7%). The authors discuss the value of using several tests in the diagnosis of multiple sclerosis and emphasize their importance for the purpose of diagnostic classification.

Measles antibodies in multiple sclerosis patients.

The Authors have assayed in this work the antimeasles antibodies in serum and CSF in subject affected by M.S. and controls. The M.S. patients have been subdivided in two groups with and without immunosuppressive therapy. In 27% out of 48 patients production intrathecal antimeasles antibodies was demonstrated.

[Evaluation of some inflammatory and immune parameters in juvenile rheumatoid arthritis during the acute phase and in remission]. / Valutazione di alcuni parametri flogistici ed immunitari nell'artrite reumatoide infantile in fase di acuzie e di remissione.

A serologic study was carried out in 27 children.-- 12 females and 15 males -- affected with juvenile rheumatoid arthritis (JRA), systemic and polyarticular type, in active phase and in remission. Beside the routine assays (ESR and haemometry), a dosage of serum immunoglobulins (IgA, M, G) and complement components (C3 and C4) was carried out; antinuclear antibodies with immunofluorescence, rheumatoid factors with classical method (latex test and Waaler-Rose reaction) and antigamma factors (AGGF) according to modified Schur's method were looked for. Polymorphonuclear function was assayed employing NBT test, phagocytosis and killing with Klebsiella. The results confirmed that the most reliable activity index is the ESR, while the WBC count is move variable and that the rheumatoid factors according to the usual techniques are almost always absent. As a whole, in JRA the levels of IgM, IgG, IgM-AGGF, C4 assayed higher than in controls. In the different subgroups, the systemic disease is characterized by higher serum IgA and IgG-AGFF. The PMN function was globally normal.