Effect of an oral preparation containing hyaluronic acid, chondroitin sulfate, hydrolyzed collagen type II and hydrolyzed keratin on synovial fluid features and clinical indices in knee osteoarthritis. A pilot study.

The aim of this study was to evaluate the effect of an oral preparation containing a naturally occurring matrix of hydrolyzed collagen type II, chondroitin sulfate (CS), and hyaluronic acid (HA), and bioactive oligopeptides of natural hydrolyzed keratin (K) in patients affected by knee OA through the evaluation of synovial fluid (SF) and clinical changes before and after treatment. Thirty patients with knee OA and swollen joint were included in the study and submitted to arthrocentesis. Patients were randomized in two groups: 1) the treatment group (N.15) took a dietary supplement containing 120 mg HA, 240 mg CS and 300 mg K once a day for 4 weeks; 2) the control group (N.15) was only submitted to arthrocentesis. Patient symptoms were evaluated at the beginning and at the end of the study by the WOMAC self-assessment questionnaire, the Lequesne algofunctional index, and the VAS forms. SF changes were evaluated by measuring local inflammatory indices, cytokines IL-1ß, IL-8, IL-6, IL-10 and GM-CSF. The group of patients treated with the oral supplement showed an improvement in the clinical indices WOMAC (p<0.01), Lequesne (p=0.014) and VAS pain (p<0.01). On the contrary, no significant changes were found in the control group. The SF collected from the treated group showed a reduction of IL-8 (p=0.015), IL-6 and IL-10 levels, while no changes in cytokines were observed in the control group. This pilot study suggests that an oral administration of a preparation containing a combination of HA, CS and K can improve some clinical parameters and affect cytokine concentrations in SF in patients with knee OA.

Clinical value of anti-domain I-ß2Glycoprotein 1 antibodies in antiphospholipid antibody carriers. A single centre, prospective observational follow-up study.

BACKGROUND: There seems to be a clear correlation between antibodies against domain I (anti-DI) of ß2Glycoprotein I and severe clinical profiles in antiphospholipid syndrome (APS) patients. We investigated the clinical significance of anti-DI antibodies in a cohort of aPL carriers. METHODS: One hundred and five carriers persistently positive for IgG anti-ß2Glycoprotein 1 antibodies (a-ß2GPI) and/or IgG anticardiolipin (aCL) and/or lupus anticoagulants (LAC) were tested for the presence of anti-DI antibodies using the QUANTA Flash® Beta2GPI-Domain I chemiluminescence immunoassay. RESULTS: Anti-DI antibodies were detected in 44 aPL carriers (41.9%) and they were significantly associated to triple aPL positivity (LAC plus IgG a-ß2GPI plus IgG aCL antibodies). Isolated LAC and a-ß2GPI antibodies were significantly associated to anti-DI negative aPL carriers. During a 82.2 month mean follow-up, ten aPL carriers (9.5%) developed a first thrombotic event so becoming APS patients. Anti-DI antibodies, triple aPL positivity, thromboembolic risk factors and autoimmune disorders significantly prevailed in carriers becoming APS. Logistic regression analysis showed that anti-DI positivity was an independent risk factor for thrombosis. CONCLUSIONS: Anti-DI antibody positivity can be considered a new risk factor predictive of the first thrombotic event in aPL carriers, instead, negative anti-DI may be useful to identify low-risk aPL carriers.

Identification of discrete epitopes of Ro52p200 and association with fetal cardiac conduction system manifestations in a rodent model.

Congenital heart block (CHB) is a potentially lethal condition characterized by a third-degree atrioventricular block (AVB). Despite anti-Ro52 antibodies being detected in nearly 90% of mothers of affected children, CHB occurs in only 1-2% of anti-Ro/Sjögren's-syndrome-related antigen A (SSA) autoantibody-positive pregnancies. Maternal antibodies have been suggested to bind molecules crucial to fetal cardiac function; however, it remains unknown whether a single antibody profile associates with CHB or whether several specificities and cross-reactive targets exist. Here, we aimed to define further the reactivity profile of CHB-associated antibodies towards Ro52p200 (amino acid 200-239). We first analysed reactivity of a monoclonal anti-Ro52 antibody shown to induce AVB in rats (7.8C7) and of sera from anti-Ro52p200 antibody-positive mothers of children with CHB towards a panel of modified Ro52p200 peptides, and subsequently evaluated their potential to induce AVB in rats upon transfer during gestation. We observed that CHB maternal sera displayed a homogeneous reactivity profile targeting preferentially the C-terminal part of Ro52p200, in contrast to 7.8C7 that specifically bound the p200 N-terminal end. In particular, amino acid D233 appeared crucial to maternal antibody reactivity towards p200. Despite low to absent reactivity towards rat p200 and different binding profiles towards mutated rat peptides indicating recognition of different epitopes within Ro52p200, immunoglobulin (Ig)G purified from two mothers of children with CHB could induce AVB in rats. Our findings support the hypothesis that several fine antibody specificities and cross-targets may exist and contribute to CHB development in anti-Ro52 antibody-positive pregnancies.

APS - Diagnostics and challenges for the future.

Diagnosis of antiphospholipid syndrome (APS) is essentially based on the detection of circulating antiphospholipid (aPL) antibodies. Progress have been made on the standardization of tests exploring the presence of aPL as guidelines on coagulation and immunological tests were recently published in the literature. Clinical relevance of aPL profile has come from prospective cohort studies in populations with a homogeneous antibody profile supporting the view that triple positivity is a high risk pattern in patients and carriers. In addition to the classic ones, several other tests have been proposed for the diagnosis of APS. The detection of antibodies directed to domain 1 and 4/5 of ß2-Glycoprotein I (ß2GP1) were found to be particularly sound. Several issues remain to be addressed. We do not yet know what is the physiological function of ß2GP1 and the pathophysiology of thrombosis and pregnancy loss in these patients. Moreover, treatment is poorly defined especially in the case of feared catastrophic APS.

Laboratory testing for antiphospholipid syndrome.

This is a practical report on laboratory tests for the diagnosis of antiphospholipid syndrome (APS). After a general definition of APS, this study deals with appropriateness and timing in requesting the determination of antiphospholipid (aPL) antibodies. Lupus anticoagulant (LAC), anticardiolipin (aCL), and anti ß2-glycoprotein I (aßGPI) are the mandatory tests to be performed, while other tests are not yet validated for clinical use. Interpretation of results is an important discussed issue that implies a close liaison between clinical pathologists and clinicians. Finally, a personal definition of APS according to aPL antibody profile closes the manuscript.

Attributable mortality of carbapenem-resistant Klebsiella pneumoniae infections in a prospective matched cohort study in Italy, 2012-2013.

BACKGROUND: In Italy, infections with carbapenem-resistant Klebsiella pneumoniae (CRKP) have increased markedly since 2009, creating unprecedented problems in healthcare settings and limiting treatment options for infected patients. AIM: To assess the attributable mortality due to CRKP in ten Italian hospitals and to describe the clinical characteristics of patients with an invasive CRKP and carbapenem-susceptible K. pneumoniae (CSKP) infection. METHODS: We conducted a matched cohort study, and calculated crude and attributable mortality for CRKP. The attributable mortality was calculated by subtracting the crude mortality rate of the patients with CSKP from the crude mortality rate of the patients with CRKP. We also described the clinical characteristics of CRKP and CSKP patients and analysed the determinants of mortality by using conditional Poisson regression. FINDINGS: The study included 98 patients, 49 with CRKP and 49 with CSKP. CRKP patients had undergone more invasive procedures and also tended to have more serious conditions, measured by higher Simplified Acute Physiology Score II. The attributable mortality of CRKP at 30 days was 41%. CRKP patients were three times more likely to die within 30 days [matched incidence rate ratio (mIRR): 3.0; 95% confidence interval (CI): 1.5-6.1]. Adjusting for potential confounders, the risk remained the same (adjusted mIRR: 3.0; 95% CI: 1.3-7.1). CONCLUSION: CRKP infection had a marked effect on patient mortality, even after adjusting for other patient characteristics. To control the spread of CRKP we recommend prioritization of control measures in hospitals where CRKP is found.

Antibodies to Domain 4/5 (Dm4/5) of ß2-Glycoprotein 1 (ß2GP1) in different antiphospholipid (aPL) antibody profiles.

BACKGROUND: Determination of the three recommended tests for the diagnosis of antiphospholipid syndrome [Lupus Anticoagulant (LA), anticardiolipin (aCL) and anti ß2-Glycoprotein 1 (aß2GP1) antibodies] allow physicians to allocate patients into classification (risk) categories. OBJECTIVES: To measure antibodies of IgG isotype directed towards Domain 4/5 (Dm4/5) of ß2GP1. PATIENTS/METHODS: In this cross-sectional study we measured IgG aß2GP1-Dm4/5 in a group of individuals positive for IgG aß2GP1 and classified as triple (LAC+, IgG aCL+, IgG aß2GP1+, n=32), double (LAC-, IgG aCL+, IgG aß2GP1+, n=23) or single positive (LA-, IgG aCL-, IgG aß2GP1+, n=10). RESULTS: Geometric mean and standard deviation of IgG aß2GP1 values expressed as Chemiluminescent Units (CU) in triple, double, single positive groups and in 40 healthy individuals were 1795±783, 321±181, 29±8 and 5.0±1.0, respectively (ANOVA p<0.0001). Geometric mean and standard deviation of IgG aß2GP1-Dm4/5 expressed as Optical Density (OD) in triple, double and single positive groups and in 40 healthy individuals were 0.16±0.13, 0.16±0.15 and 0.26±0.15, 0.13±0,11, respectively (ANOVA p<0.002). Individuals in the single positive group, expressed significantly higher values with respect to triple (p=0.04) and double (p=0.03) positive groups. Approximate OD cut-off value (99° percentile) calculated in 40 normal control subjects is 0.404. Positivity to IgG aß2GP1-Dm4/5 according to this cutoff was found in only 5 individuals, 3 in triple positive and 2 in single positive groups and was not associated with thromboembolism. CONCLUSION: Mean level of IgG aß2GP1-Dm4/5 is higher in single positive group. There is no association between positivity to IgG aß2GP1-Dm4/5 and thromboembolic events.

Are biological drugs safe in pregnancy?

The introduction of biological therapies has significantly improved the outcome of inflammatory rheumatic diseases. As most of these diseases affect women and men in childbearing age, some concerns have been voiced as to the safety of these drugs in relation to reproduction and pregnancy. Data from many hundreds of pregnancies in patients affected by inflammatory bowel disease and inflammatory arthritis have suggested that exposure to anti-TNF therapies at conception and/or during pregnancy is not associated with adverse pregnancy outcomes or any increase in congenital abnormalities. However, the exposure to anti-TNFα agents, particularly to monoclonal antibodies, in late pregnancy is associated with high drug levels in the newborn and their long-term effects on children remain unknown. Therefore, limiting the use of anti-TNFα to the first 30 weeks of pregnancy is recommended to reduce fetal exposure. Live-virus vaccines should be given only when levels of anti-TNFα drugs are undetectable in the serum of infants. Studies suggest that many of these drugs do enter breast milk in small amounts, but the extent to which the infant absorbs them is less clear. Limited reports have not suggested adverse pregnancy outcomes in women whose partners were exposed to anti-TNF therapies at the time of conception. Pregnancy data for rituximab, abatacept, anakinra, tocilizumab and belimumab are limited and their use in pregnancy cannot currently be recommended.

Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile.

BACKGROUND: The revised classification criteria for the antiphospholipid syndrome state that antiphospholipid (aPL) antibodies (lupus anticoagulant [LAC] and/or anticardiolipin [aCL] and/or anti-ß2 -glycoprotein I [aß2 GPI] antibodies) should be detected on two or more occasions at least 12 weeks apart. Consequently, classification of patient risk and adequacy of treatment may be deferred by 3 months. OBJECTIVES: In order to early classify patient risk, we evaluated whether aPL positivity confirmation is related to aPL antibody profiles. PATIENTS AND METHODS: Consecutive patients referred to our center who were initially positive in one or more tests exploring the presence of aPL were tested after 3 months. During a 4-year period, 225 patients were initially positive in one or more tests, and 161 were available for confirmation after 3 months. Patients were classified as triple-positive (n = 54: LAC(+) , aCL(+) , aß2 GPI(+) , same isotype), double-positive (n = 50: LAC(-) , aCL(+) , aß2 GPI(+) , same isotype) and single-positive (n = 53: LAC or aCL or aß2 GPI antibodies as the sole positive test). RESULTS: Among subjects with triple positivity at initial testing, 98% (53 of 54) had their aPL profile confirmed after 12 weeks. The double-positive and single-positive groups had data confirmed in 42 of 50 (84%) and 23 of 57 (40%) subjects, respectively. CONCLUSIONS: Our results show that high-risk subjects with triple-positive aPL profiles are identified early, at the time of the initial screening tests.

Platelet and endothelial activation in catastrophic and quiescent antiphospholipid syndrome.

Antiphospholipid antibodies (aPL) seem to induce a prothrombotic state by activating endothelium and platelets, but no studies have evaluated systematically the effects of aPL from patients with the antiphospholipid syndrome (APS) in quiescent versus catastrophic phase. Our aims were to evaluate the in vitro effects on platelet activation of anti-ß2 glycoprotein I (anti-ß2GPI) antibodiesisolated from APS patientin either quiescent or catastrophic phase and to investigate ex vivo platelet and endothelial activation in patients with quiescent or catastrophic APS. Anti-ß2GPI antibodies were isolated from plasma of a pregnant woman in two different stages of APS (quiescent and catastrophic, respectively). They were co-incubated with washed platelets from healthy controls that were then challenged with TRAP-6 (thrombin receptor activating peptide 6) and the expression of P- selectin (P-sel) on platelets was assessed by flow cytometry. Moreover, plasma samples from six patients with quiescent, four with catastrophic APS and 10 controls were assessed for several markers of platelet and endothelial activation. The results showed that purified anti-ß2GPI antibodies co-incubated with platelets enhanced TRAP-6- induced platelet P-sel expression. Notably, anti-ß2GPI antibodies isolated during the catastrophic phase enhanced platelet P-sel expression more than antibodies isolated from the same patient in the quiescent stage of disease. Moreover, APS patients had significantly higher plasma levels of soluble (s) Psel, sCD40 ligand, soluble vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1 than control subjects. In addition, sP-sel and von Willebrand factor activity were significantly higher during catastrophic than in quiescent phase.

Correct laboratory approach to APS diagnosis and monitoring.

Triple positivity (positive Lupus Anticoagulant, anticardiolipin and anti ß2-glycoptrotein I antibodies) identifies the pathogenic autoantibody (anti Domain I of ß2-glycoptroteinI) that is present in patients with definite Antiphospholipid Syndrome (APS). This is supported by the fact that aß2GPI antibodies obtained by affinity purification in these patients possess LA activity. Moreover, patients and carriers of this profile carry a much higher risk of thrombosis and pregnancy loss than APS patients with positivity for only one of the tests. Thus, very different risk categories exist among patients with APS as well as among carriers of aPL. Clinical studies and interventional trials should first take these high risk subjects into consideration.

Interpretation of laboratory data and need for reference laboratories.

A single positive laboratory test among those exploring the presence of antiphospholipid antibodies is not associated with thromboembolic events and does not identify patients with antiphospholipid syndrome. On the other hand, more than one laboratory test positive, and in particular all three tests positive, is strongly associated to thromboembolic events and identifies high risk patients. Triple positivity is in fact related to the presence of a specific anti-ß2-glycoprotein I (anti-Domain I) antibody, also able to prolong coagulation tests. Monoclonal antibodies against Domain I with Lupus Anticoagulant activity might be candidate material for standardization of antiphospholipid assays. Much work remains to be done in this field.

Secondary prevention in thrombotic antiphospholipid syndrome.

Secondary prevention of venous thromboembolism in antiphospholipid syndrome (APS) is usually made using vitamin K antagonists (VKAs) to maintain an international normalized ratio (INR) between 2.0 and 3.0. The optimal intensity of anticoagulation was determined in two prospective randomized controlled trials, both excluding the benefit of more intense anticoagulation. The same regimen is also recommended in patients with APS and arterial thromboembolism as aspirin does not appear to protect against recurrences. The duration of treatment is usually indefinite because of a substantial risk of recurrence.

Circulating ß2 glycoprotein I-IgG anti-ß2 glycoprotein I immunocomplexes in patients with definite antiphospholipid syndrome.

Beta2-glycoprotein I (ß(2)GPI), a relevant antigen in Antiphospholipid Syndrome (APS), binds anionic macromolecules including heparin (Hep). A possible formation of ternary complexes between ß(2)GPI, antibodies and Hep in APS is thus possible. The aim of this study was to evaluate Hep-ß(2)GPI interaction in patients with APS. The affinity of Heps of different length, including unfractionated Hep (UFH), low-molecular weight Hep (enoxaparin) and pentasaccharide (fondaparinux), to human ß(2)GPI was estimated by fluorescence spectroscopy, yielding dissociation constant (K(d)) values of 1.1, 24.0 and 89.4 µM, demonstrating that the longer UFH binds to ß(2)GPI far more tightly than the shorter ones. Plasma and protein G-purified IgGs from eight patients with APS (i.e. five with thromboembolic disease and three with catastrophic APS), were fractionated by affinity chromatography using a Hep (UFH)-bound column, eluted with a linear NaCl gradient. For each chromatographic analysis, fractions were collected in the whole NaCl gradient and tested by ELISA for the presence of ß(2)GPI and anti-ß(2)GPI IgG. The results of Hep-affinity chromatography and ELISAs concurrently indicate that either ß(2)GPI and anti-ß(2)GPI IgG elute from the Hep column in the same chromatographic peak, at a retention time identical to that of the purified, isolated ß(2)GPI, thus suggesting that circulating immunocomplexes containing ß(2)GPI are present in patients with APS.

Antiphosphatidylserine/prothrombin antibodies in primary antiphospholipid syndrome.

Antiprothrombin (aPT) antibodies may be detected by an enzyme-linked immunosorbent assay (ELISA) using a purified antigen or a phosphatidylserine/prothrombin complex (aPS/PT). IgG/IgM antibodies directed against aPS/PT were assessed in 158 patients with primary antiphospholipid syndrome (PAPS). They were detected in 80/158 (50.6%) PAPS patients; IgG alone was positive in 12 (7.6%), IgM alone in 36 (22.8%), and both IgG and IgM isotypes in 32 (20.2%) PAPS patients. IgG and IgM aPS/PT were significantly associated with both vascular thrombosis and pregnancy morbidity. IgG aPS/PT was significantly associated with venous thrombosis (p = 0.023), whilst IgG and IgM aPS/PT were associated with arterial thrombosis (p < 0.001 and p < 0.001, respectively). Logistic regression analysis showed that IgM and IgG aPS/PT were independent risk factors for thrombosis (odds ratio (OR) 3.5 [95% confidence interval (CI) 1.6-7.9] and OR 4.1 [95% CI 1.4-11.7], respectively) and IgM aPS/PT was an independent risk factor for arterial thrombosis (OR 2.7 [95% CI 1.1-6.7]). In conclusion, these findings indicate that aPS/PT are clinically relevant in PAPS.

In vitro effect of anti-ß2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation.

OBJECTIVE: Antiphospholipid antibodies (aPL) associated with thrombembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (ß2GPI) represents the major target antigen for aPL, but the pathogenic role of anti-ß2GPI antibodies (aß2GPI) is still unclear. Some authors assume they play a role in activating platelets. The effects of aß2GPI antibodies on platelet P-selectin expression were evaluated in this study. METHODS: Aß2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography during two different stages (catastrophic and quiescent) of the disease. Gel filtered platelets (100,000/µl) from healthy volunteers were incubated with ß2-GPI (20 µg/ml) and with different concentrations (5, 25 e 50 µg/ml) of aß2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. RESULTS: Aß2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6), a platelet agonist, at a subthreshold concentration. Aß2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47% versus 15%). CONCLUSIONS: TRAP-6 dependent platelet activation by aß2GPI antibodies is consistent with the "two hit" pathogenetic hypothesis for thrombosis. Aß2GPI antibodies induce higher platelet P-selectin expression during the active rather than in the acute phases.

The clinical significance of autoantibodies directed against prothrombin in primary antiphospholipid syndrome.

BACKGROUND: To evaluate the clinical significance of IgG/IgM antibodies directed against prothrombin (PT) in a homogeneous cohort of patients with primary APS (PAPS). METHODS: IgG/IgM anti-prothrombin (aPT) antibodies were measured using a commercial ELISA kit in 158 PAPS patients and in 214 control subjects (100 healthy blood donors and 114 patients with autoimmune diseases). RESULTS: IgG/IgM aPT antibodies were significantly associated with PAPS (OR, 95% CI: 52.0, 7.0-385.5; 9.8, 1.2-80.8, respectively). They were found to have a high specificity (IgG 99.50%, IgM 99.54%) but a low sensitivity (IgG 19.60%, IgM 3.80%) for PAPS. IgG aPT antibodies were significantly higher in the PAPS patients with thrombosis (OR, 95% CI: 69.2, 9.2-519.1) as well as in those with pregnancy morbidity alone (OR, 95% CI: 20.5, 2.4-174.5). The prevalence of IgG aPT was not significantly different in the thrombotic and obstetric patients, and the presence of IgM aPT antibodies was significant only in patients with thrombosis (OR, 95% CI: 2.6, 1.6-110.8). CONCLUSIONS: The study's findings confirm that IgG/IgM aPT antibodies are significantly associated with PAPS and indicate that IgG aPT antibodies are associated with clinical subsets of the disease. For the time being, however, the lower sensitivity of IgG/IgM antibodies with respect to conventional aPL antibodies precludes their inclusion in the recommendations for the diagnosis of PAPS.

Adjusted prophylactic doses of nadroparin plus low dose aspirin therapy in obstetric antiphospholipid syndrome. A prospective cohort management study.

OBJECTIVES: Current guidelines for the treatment of patients with obstetric antiphospholipid syndrome (APS) recommend low dose aspirin (LDA) and prophylactic doses of low molecular weight heparin (LMWH). Most clinicians use a fixed dosage of LMWH in pregnant APS women despite the fact that there are no clinical trials establishing that fixed doses are more efficacious than adjusted ones in preventing pregnancy complications. The efficacy and safety of adjusted single daily doses of LMWH (nadroparin) combined with LDA have thus been evaluated in 33 consecutive pregnancies in women with diagnosed obstetric APS. METHODS: LMWH doses were augmented as the pregnancies progressed and maternal/foetal weight increased. 70-80-90 U/Kg doses ranging between 3800 and 6650 U were administered daily during the first, second and third trimesters, respectively. LDA (100 mg/day) was also prescribed. RESULTS: Pregnancy outcome was successful in 97% of the patients studied, who delivered, between the 29th and 41st weeks of gestation (mean 37.4 ±2.1 SD), 32 infants with a mean birth weight of 3084 g ± 514 SD. One woman (3%) experienced a spontaneous abortion at the 8th week of gestation. CONCLUSIONS: The high live birth rate, the satisfactory mean gestational age and weight at birth and the absence of major pregnancy/neonatal-associated complications indicate that adjusted, once daily doses of LMWH together with LDA could be an efficacious treatment option for pregnant APS patients with no history of thrombosis.