One-year efficacy of a 3-week intensive multidisciplinary non-pharmacological treatment program for fibromyalgia patients.

OBJECTIVE: The aim of the present study was to investigate the long-term efficacy of a 3-week intensive residential multidisciplinary non-pharmacological treatment program (including individually prescribed and monitored aerobic exercise and cognitive behavioural therapy) on fibromyalgia symptoms and aerobic fitness. METHODS: Twenty-five women with fibromyalgia participated in six experimental sessions (pre-admission, immediately before and immediately after the treatment, and to 2, 5 and 12 months afterwards) in which they underwent clinical, psychophysical and psychological examinations: pain intensity (VAS), pain area (percentage of total body surface), deep pressure pain threshold at 18 tender point sites measured with a pressure algometer, an incremental step test with blood lactate determination and calculation of the individual intensity of exercise corresponding to 2 mM of lactate concentration (W2, index of aerobic fitness). Depression and coping were evaluated with the Center for Epidemiologic Studies Depression Scale (CES-D) and Brief Pain Coping Inventory (BPCI), respectively. RESULTS: Pain intensity, pain area and number of positive tender points were significantly reduced up to 12 months, while deep pressure pain threshold and W2 increased. CES-D score decreased until two months. Among the 18 items of the BCPI, only item 3 ("physical exercise/stretching") changed significantly, increasing until 12 months. CONCLUSION: In fibromyalgia patients, whose symptoms before treatment were constant, a 3-week intensive residential multidisciplinary treatment program showed one-year efficacy in improving pain and aerobic fitness. The acquisition of physical exercise as a coping strategy for chronic pain acceptance could explain the long-term effects of our brief treatment.

Familial aggregation of primary glomerulonephritis in an Italian population isolate: Valtrompia study.

Hereditary factors are suspected to contribute to the pathogenesis of sporadic primary glomerulonephritis, but their contribution is difficult to delineate in the general population. We studied the prevalence of primary glomerulonephritis in an isolated population from the extreme northern Valtrompia valley, Northern Italy. Investigation of medical records, community urinary screening program and molecular characterization of the population's ancestry were performed; genealogies of affected individuals were researched. Forty-three patients with primary glomerulonephritis were identified: 25 had biopsy-proven disease (11 immunoglobulin A (IgA) nephropathy; eight mesangial proliferative glomerulonephritis without IgA deposits; four focal segmental glomerular sclerosis; two membranous nephropathy), and 18 had clinical glomerulonephritis. All 43 patients originated from three mountain villages (Collio, San Colombano, and Bovegno). In contrast, we found only four cases of primary glomerulonephritis in two nearby villages (Pezzaze and Tavernole) that shared similar population histories and lifestyles, demonstrating heterogeneity of risk factors for glomerulonephritis (P=3 x 10(-5)). All 43 affected individuals could be traced back to common ancestors (XVI-XVII centuries), enabling the construction of three large pedigree including three parent-child affected pairs and five affected siblings pairs. Molecular data showed lower genetic diversity and increased inbreeding in the Valtrompia population compared to the control population. Molecular and genealogical evidence of limited set of founders and the absence of shared nephritogenic environmental factors suggest that our patients share a common genetic susceptibility to the development of primary glomerulonephritis. Further molecular study of our families will offer the possibility to shed light on the genetic background underlying these glomerular disorders.

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells.

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic acid:Galbeta1,3GalNAcalpha2, 3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1, 3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1, 3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.

Incidence of antiperinuclear factor in patients with psoriatic arthritis.

Antiperinuclear factor (APF) is considered a disease marker of rheumatoid arthritis (RA) and its diagnostic value is obvious in patients who are seronegative for rheumatoid factor (RF) activity. We have evaluated APF positivites in 76 patients with psoriatic arthritis, 38 uncomplicated psoriatic patients, 119 patients with non-inflammatory rheumatic diseases (NIRD), 36 RF- and 123 RF + RA patients and 204 healthy controls. APFs were investigated with an indirect immunofluorescence (IIF) test using epithelial cells from human buccal mucosa as a substrate. 6/76 (7.9%) PA patients were APF+. The incidence was greater than in healthy controls (2/204; p < 0.01) and similar to the incidences in patients with uncomplicated psoriasis (1/38; p = NS) and patients with non-inflammatory rheumatic disease NIRD (5/119; p = NS). However, the incidence was much lower than in RF- (19/36; p < 0.001) as well as RF+ (111/123; p < 0.001) RA patients. Finally, we emphasise that 3 out of 6 APF positivities shown by PA patients were found in our 3 patients with pustolotic arthroosteitis, a new specific entity in the spectrum of PA.

Antiperinuclear factor in psoriatic arthropathy.

BACKGROUND: Antiperinuclear factor (APF) is an autoantibody directed against (pro)-filaggrin molecules. OBJECTIVE: We evaluated whether APF determination is useful for the diagnosis of psoriatic arthritis (PA). METHODS: We determined APF titers in sera from patients with PA (n = 76), psoriasis vulgaris (n = 38), noninflammatory rheumatic diseases (NIRDs, n = 119), rheumatoid arthritis (RA, n = 159) both with negative (n = 36) and positive (n = 123) rheumatoid factor (RF) tests, as well as from 204 healthy controls. We used an indirect immunofluorescence test on epithelial cells from human buccal mucosa as a substrate. RESULTS: In patients with PA, 7.9% of the serum samples were APF-positive. The incidence was greater than in healthy controls (1.0%; P < .01), similar to those with uncomplicated psoriasis (2.6%; P = NS) and NIRDs (4.0%; P = NS), and lower than in RF-negative (52.7%; P < .001) and RF-positive (90.2%; P < .001) patients with RA. Three APF-positive patients with PA had symmetric joint involvement and 3 had pustulotic arthroosteitis. CONCLUSION: The APF test may be useful in differentiating PA from RA, and APF may be specific for two PA subgroups.

Familial clustering of IgA nephropathy: further evidence in an Italian population.

Several lines of evidence suggest that genetic factors have an important role in the pathogenesis of immunoglobulin A (IgA) nephropathy. We report the prevalence of familial IgA nephropathy in a referral center in northern Italy and present the data on HLA genotypes in the families identified. Twenty-six of 185 patients (14%) with IgA nephropathy investigated in Brescia, Italy, were related to at least one other patient with the disease. Restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta and HLA-DQ alpha and beta genes, as well as polymerase chain reaction-based oligonucleotide typing, was performed in family members. The 26 patients with IgA nephropathy belonged to 10 families. Familial relationships between the patients varied greatly, ranging from parent-child to sib-pair to more distant familial relationships. No common nephrotoxic factor was identified in the families. The intervals separating the apparent onset of disease in relatives with IgA nephropathy varied from 8 months to 13 years. In patients with a family history of IgA nephropathy, there was an increased incidence of HLA-DRB1*08 compared with those with sporadic IgA nephropathy. The study shows that a significant number of the patients with IgA nephropathy followed up in Brescia had a family history of disease. The fact that the Italian population, an ethnic group not previously examined, also presents an increased familial susceptibility to IgA nephropathy suggests that familial predisposition is a very common finding for IgA nephropathy. Thus, clinicians should become aware that IgA nephropathy may aggregate within families in a substantial number of cases. In addition, this subgroup of patients with IgA nephropathy offers an ideal opportunity to elucidate the molecular genetics of this disease.

Autosomal dominant medullary cystic disease: a disorder with variable clinical pictures and exclusion of linkage with the NPH1 locus.

BACKGROUND: The nephronophthisis-medullary cystic disease (NPH/MCD) complex represents a heterogeneous group of hereditary tubulointerstitial nephritis. The most common variant is juvenile recessive NPH, for which a gene locus (NPH1) has been mapped on chromosome 2q13. MCD is a less common dominant condition usually recognized later in life, which resembles NPH in many aspects, still presenting remarkable clinical differences. Nothing is known about the chromosome locus of MCD. METHODS: Five MCD families were studied. Diagnosis was made by inference from family history, type of inheritance, clinical signs and histology. Multipoint linkage analysis was performed by markers D2S293, D2S340 and D2S160 spanning the entire NPH1 locus. RESULTS: Diagnosis of MCD was made in 28 affected members (16 males; 12 females), belonging to five families. Histological diagnosis was available in 10 patients; clinical diagnosis in 11; seven deceased relatives had diagnosis of chronic nephritis. The age at diagnosis ranged from 8 to 65 years. Renal medullary cysts were found in a minority of patients. In family 1, the disease was associated with hyperuricaemia and gouty arthritis. Progression of renal disease presented intra- and extra-family variability with members of the same family showing mild elevation of creatinine or terminal renal failure. The NPH1 locus associated to recessive NPH was excluded from linkage to the dominant MCD. CONCLUSIONS: MCD might be more common than previously assumed. Variability in clinical presentation and absence of histopathological hallmarks contribute to make the diagnosis uncommon. The most remarkable clinical difference with NPH is the age of onset in some kindreds and a delayed progression towards renal failure. The exclusion of linkage to the NPH1 locus suggests the existence of an MCD responsible locus, still to be mapped.

Synthesis of bisected glycoforms of recombinant IFN-beta by overexpression of beta-1,4-N-acetylglucosaminyltransferase III in Chinese hamster ovary cells.

Genetic engineering of oligosaccharide biosynthesis pathways in mammalian cells makes possible generation of new recombinant glycoproteins of potential importance in the biopharmaceutical industry. Most prior investigations of glycosylation engineering of secreted heterologous glycoproteins involve terminal steps of oligosaccharide biosynthesis. In particular, increasing the frequency of bisected structures within the glycoform distribution has not before been considered. A Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III). Interferon beta (IFN-beta) was chosen as a model and potential therapeutic secreted heterologous protein to demonstrate the effect of recombinant GnT-III-expression on product glycosylation. IFN-beta with bisected oligosaccharides was produced by the GnT-III-engineered CHO cells but not by the unmodified parental cell line.

Familial membranous nephropathy.

Numerous HLA studies suggest that genetic factors play an important role in the development of membranous nephropathy (MN). We studied seven patients with idiopathic MN, from three unrelated families of Italian ancestry. Complement phenotype analysis and restriction fragment length polymorphism (RFLP) typing of HLA class II and of the switch region genes were done in family members. In the first family, the father, one son, and one daughter had MN; another daughter had clinical glomerulonephritis. The three members with MN shared one HLA haplotype carrying DR beta 11; in the two siblings with the disease, the second HLA haplotype carried the DR beta 3.2 allele. In families 2 and 3, two brothers had MN: in family 2, they differed in at least one haplotype; in family 3, they differed in both haplotypes. Only family 3 was informative with regard to the RFLP of the switch region genes: the two siblings were identical for both Ig heavy chain haplotypes. No clinical, laboratory or morphologic features consistent with a secondary form of the disease were found. Familial clustering of MN suggests a genetically transmitted mechanism.

Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells.

Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities.

Study of spontaneous apoptosis in HIV+ patients: correlation with clinical progression and T cell loss.

In vitro spontaneous apoptosis (SA) of lymphocytes was studied in HIV infection to evaluate possible clinical and prognostic correlations, in a transsectional study of 101 individuals in different clinical categories and in a prospective longitudinal study of 18 asymptomatic individuals (mean follow-up, 17.2 months). The rate of SA was higher in HIV+ patients than in healthy controls (p < 0.001) and was higher in patients with AIDS than in the other HIV+ individuals (p < 0.001). It was inversely correlated with the peripheral blood CD4+ (R -0.61; p < 0.001) and CD8+ (R -0.46; p < 0.001) cell numbers. In a group of long-term survivors (LTS), it was significantly lower than in a control group of asymptomatic HIV+ patients with a similar number of circulating CD4+ lymphocytes but a shorter follow-up (p < 0.02). In the five asymptomatic HIV-infected individuals who showed a clinical progression, peaks of SA rates above the normal range before the clinical event were much more frequent than in those who remained asymptomatic (p < 0.0001), even though they were fairly homogeneous as far as CD4+ cell count and viral load were concerned. The median levels of SA rates were moreover correlated with the rate of total T cell loss (R -0.46; p 0.053). This study suggests that evaluation of the SA levels may provide a predictive factor for clinical and immunological progression of HIV-related immunodeficiencies and strengthen the hypothesis for the role of this phenomenon in the pathogenesis of this progression.

The anti-beta2-glycoprotein I activity in human anti-phospholipid syndrome sera is due to monoreactive low-affinity autoantibodies directed to epitopes located on native beta2-glycoprotein I and preserved during species' evolution.

To characterize the reactivity pattern of Abs directed to beta2-glycoprotein I (anti-beta2GPI) in patients with anti-phospholipid syndrome, we have purified anti-beta2GPI Abs by affinity chromatography using the IgG fractions from sera of five different anti-phospholipid syndrome patients. Affinity-purified anti-beta2GPI were shown to be representative of Abs found in human sera because their activity could be virtually abolished from the IgG preparations after repeated absorptions on immobilized human beta2GPI column. Our results show that affinity-purified anti-beta2GPI: 1) do react with beta2GPI in the absence of any phospholipid, as demonstrated by the lack of phosphorus contaminant in the employed reagents, as well as by their comparable binding activity before and after extensive delipidation procedure; 2) can recognize beta2GPI regardless of its origin from different animal species; 3) are able to bind soluble beta2GPI with a mean Kd value of 4.65 x 10(-6) M (range 3, 4-7, 2 x 10(-6) M); 4) significantly enhance their binding avidity when beta2GPI is linked to a solid support; and 5) appear to be mainly monoreactive autoantibodies. In conclusion, we have shown that human polyclonal anti-beta2GPI are low affinity, mainly monoreactive autoantibodies directed to an epitope located on native beta2GPI, preserved along the species evolution.

Anti-beta 2-glycoprotein I antibodies: a marker of antiphospholipid syndrome?

Anticardiolipin (aCL) and anti-beta 2-glycoprotein I(anti beta 2GPI) antibodies have been shown in animal models as not cross-reacting antibody populations. This observation prompted us to prove if anti-beta 2GPI exist in human sera by using a reliable method and then to investigate if these are independent from aCl antibodies. We have developed a new ELISA for the detection of anti-beta 2GPI antibodies employing the coating of the protein in carbonate buffer to irradiated microtitre plates and the filtration of serum samples, that makes irrelevant the binding to the uncoated wells. IgG F(ab)2 fragments from IgG positive sera were shown bind beta 2GPI, providing that the binding was a specific antibody binding, mediated by the antigen binding site of the antibody molecule: moreover the antibodies were not able to differentiate native and delipidated beta 2GPI coated plates, making a possible role of a phospholipid contaminant unlikely. On the other hand, the phosphorus content of native as well as delipitated beta 2GPI was undetectable. IgG, but not IgM, anti-beta 2GPI antibodies were classically inhibited by the addition of soluble beta 2GPI, while cardiolipin liposomes appear to modify the reaction in a completely different way, possibly by the described interaction between cardiolipin and beta 2GPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal expansion of large granular lymphocytes with a CD4+ CD8dim+/- phenotype associated with hairy cell leukemia.

Peripheral blood lymphoid cell expansions with an unusual CD3+, CD4+, CD8dim+/-, CD11b+, CD57+ immunophenotype have recently been reported. They frequently have the morphology of large granular lymphocytes (LGL) and can be either monoclonal or polyclonal. Their significance is still unclear and no association with hematological neoplasms has been described. We report the case of a patient with a monoclonal expansion of LGL associated with a B-cell-derived hairy cell leukemia. The two lymphoid clones were not physically associated since T-LGL were found in the peripheral blood and hairy cells were detected in the bone marrow and kidney.

The ability of CD4+ cells from HIV+ individuals to express CD40 ligand after in vitro stimulation is not impaired.

Interaction between the B cell glycoprotein CD40 and its ligand (CD40L), expressed by activated T cells, is of crucial importance in the generation of specific antibody response, which is impaired in HIV+ individuals. We studied the expression of CD40L by lymphocytes activated with PMA plus ionomycin in 17 HIV+ individuals and 12 healthy donors. In HIV+ individuals, the percentage of cells expressing CD40L was lower than that in the controls (22.6 +/- 14.7 vs 40.1 +/- 12.0; P < 0.002) and clearly correlated with that of CD4+ peripheral blood lymphocytes (r = 0.83; P < 0.001); therefore, the reduced CD40L expression might be explained by the decrease of the CD4+ cells. In fact, the expression of CD40L by purified CD4+ cells was comparable in individuals with and without HIV infection. These data indicate that the ability of CD4+ cells from HIV individuals to express CD40L is not impaired, at least after optimal stimulation in vitro.

Insertion of a short human immunodeficiency virus (HIV)-2 gp36 sequence into an HIV-1 p24 recombinant protein results in a polypeptide with potent and TCRBV-restricted T cell triggering activity.

In the present work we investigate whether artificial alterations of the structure of an inactive retrovirus-encoded protein could transform it in a superantigen. As a model system we used a recombinant human immunodeficiency virus (HIV)-1 p24 protein and two of its variants in which a short peptide corresponding to sequences of gp41 of HIV-1 (HIV-1 p24*) or gp36 of HIV-2 (HIV-1-2 p24*) has been inserted nearby the carboxy-terminal end of HIV-1 p24. As expected both HIV-1 p24 and HIV-1 p24* were inactive, while HIV-1-2 p24* was a potent inducer of human, but not murine, T cell proliferation. The possibility that the observed activity was due to contaminants was ruled out since the proliferative response could be specifically inhibited by a monoclonal anti-p24 antibody and by a peptide encompassing the area of HIV-1 p24/HIV-2 gp36 junction. Furthermore, the data exclude the possibility that the gp36 insertion is per se responsible for the observed proliferative activity. The analysis of the functional, phenotypic and molecular properties of the responding cells demonstrated that the response was class II dependent and that the activated cells were predominantly CD4+CD8- expressing a strongly biased repertoire of TCRBV segments. Collectively, these data strongly suggest that the HIV-1-2 p24* fusion protein shares common functional properties typical of superantigen molecules. Thus, our demonstration that a viral protein can be transformed into a superantigen simply by the insertion of a short peptide at the carboxy-terminal end has important implications for understanding the mode of action of retrovirus-encoded superantigens.