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BackgroundAntiphospholipid Syndrome (APS) is a rare autoimmune disease characterized by thrombotic events and/or pregnancy morbidities in the presence of confirmed positivity for antiphospholipid antibodies (aPL). Complement was demonstrated to be involved in aPL-related pregnancy loss in animal models and several groups investigated the significance of complement levels in human disease. C3 and C4 serum levels were assessed in several cohorts of pregnant patients with APS and/or aPL positivity in order to relate complement consumption with adverse pregnancy outcome (APO).According to some authors, hydroxychloroquine (HCQ) can control the activation of the complement system, improve pregnancy outcome and reduce aPL title.ObjectivesThis study was designed to verify the effect of HCQ in addition to low dose aspirin (LDA) + low molecular weight heparin (LMWH) treatment in a multicenter cohort of primary APS (PAPS) and aPL carriers pregnant women and the possible correlation with preconception serum C3/C4 levels.MethodsMedical records of pregnant women with confirmed positivity for aPL antibodies attending twelve referral centers from January 2010 to December 2020 were retrospectively evaluated. We considered as aPL-related APO: spontaneous abortions (ResultsWe have analyzed 164 singleton PAPS/aPL carrier pregnancies (22 aPL carriers - 13%) in 128 patients: all were treated with combination therapy (LDA+LMWH), and in 30 HCQ was added. 58 pregnancies (43%) had low levels of preconception C3/C4. A triple aPL positivity was observed in 54 pregnancies, 14 of them were treated with combination therapy + HCQ. When considering the whole cohort, the addition of HCQ had not significantly improved the gestational outcome. Further stratification was performed on the basis of complement consumption. In the group of patients with preconception low C3/C4 levels the addition of HCQ had not significantly improved pregnancy outcome. We have lastly evaluated 40 pregnancies with a high-risk profile (triple aPL positivity and complement consumption), in which we have found that HCQ significantly improved gestational outcome (p=0.018, Table 1).Table 1.Relationship between APO, therapy during pregnancy and risk profile.All pregnancies (n=164)Reduced C3/C4 (n=58)Triple aPL+ and reduced C3/C4 (n=40)LDA+LMWH (n, %)LDA+LMWH+HCQ (n, %)pLDA+LMWH (n, %)LDA+LMWH+HCQpLDA+LMWHLDA+LMWH+HCQp(n, %)(n, %)(n, %)APO62 (46%)16 (53%)ns32 (68%)4 (36%)ns23 (77%)3 (30%)0.018No APO72 (54%)14 (47%)15 (32%)7 (64%)7 (23%)7 (70%)Total1343047113010This observation could not be confirmed in patients with single or double aPL positivity.ConclusionThe study shows that administering HCQ in addition to combination therapy can improve gestational outcome in aPL/PAPS high-risk patients. This observation confirms that HCQ exerts a beneficial effect on aPL pregnancies by complement inhibition as it was shown in animal models. In addition, our results provide the clinicians a useful tool to implement conventional treatment in patients at high risk of pregnancy complication or loss.References[1]De Carolis S, et al. Is there any role for the hydroxychloroquine (HCQ) in refractory obstetrical antiphospholipid syndrome (APS) treatment? Autoimmun Rev 2015;14:760-2.[2]Mekinian A, et al. The efficacy of hydroxychloroquine for obstetrical outcome in antiphospholipid syndrome: data from a European multicenter retrospective study. Autoimmun Rev 2015;40:498-502.[3]Mekinian A et al. Obstetrical APS: is there a place for hydroxychloroquine to improve the pregnancy outcome? Autoimmun Rev 2015;14:23-9.Disclosure of InterestsNone declared

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4(+)CCR6(+)IL-7R(+)T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6(+)B helper T cells were phenotypically distinct from follicular helper T (T(FH)) cells and lacked BCL6 expression. In peripheral blood, a CCR6(+)T cell population with similar characteristics was identified, which lacked Th17- and T(FH)-associated gene signatures and differentiation-associated surface markers. CD4(+)CCR6(+)T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4(+)CCR6(+)IL-7R(+)T cells were associated with the presence of pathogenic anti-dsDNA (double-stranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10–producing CCR6(+)T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE.

The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling.

ObjectivesTo assess the prevalence of the main causes of morbi-mortality in the antiphospholipid syndrome (APS) during a 10-year-follow-up period and to compare the frequency of early manifestations with those that appeared later.MethodsIn 1999, we started an observational study of 1000 APS patients from 13 European countries. All had medical histories documented when entered into the study and were followed prospectively during the ensuing 10 years.Results53.1% of the patients had primary APS, 36.2% had APS associated with systemic lupus erythematosus and 10.7% APS associated with other diseases. Thrombotic events appeared in 166 (16.6%) patients during the first 5-year period and in 115 (14.4%) during the second 5-year period. The most common events were strokes, transient ischaemic attacks, deep vein thromboses and pulmonary embolism. 127 (15.5%) women became pregnant (188 pregnancies) and 72.9% of pregnancies succeeded in having one or more live births. The most common obstetric complication was early pregnancy loss (16.5% of the pregnancies). Intrauterine growth restriction (26.3% of the total live births) and prematurity (48.2%) were the most frequent fetal morbidities. 93 (9.3%) patients died and the most frequent causes of death were severe thrombosis (36.5%) and infections (26.9%). Nine (0.9%) cases of catastrophic APS occurred and 5 (55.6%) of them died. The survival probability at 10 years was 90.7%.ConclusionsPatients with APS still develop significant morbidity and mortality despite current treatment. It is imperative to increase the efforts in determining optimal prognostic markers and therapeutic measures to prevent these complications.

Summary: Purpose: To measure anti-cardiolipin (aCL), anti-β2 glycoprotein I (anti-β2GPI), and anti-prothrombin (aPT) antibodies in young patients with epilepsy, and to correlate their presence with demographic data, clinical diagnoses, laboratory and neuroradiologic findings, and antiepileptic drugs (AEDs). Methods: Sera from one hundred forty-two consecutive patients with epilepsy with a median age of 10 years were tested for aCL and anti-β2GPI autoantibodies by solid-phase assays. aPT antibodies also were assayed in sera from 90 patients. Positive results were confirmed after a minimum of 6 weeks. Antinuclear antibodies (ANAs) and antibodies against extractable nuclear antigens (ENAs) also were tested. Results: An overall positivity of 41 (28.8%) of 142 sera was found. Fifteen patients were positive for aCL, 25 for anti-β2GPI, and 18 for aPT antibodies. Several patients (12%) displayed more than one specificity in their serum. Only one of these patients had a concurrent positivity for ANAs and ENAs. A predominance of younger patients was found in the antibody-positive group. All types of epilepsy were represented in the positive group. No relation between antibody positivity and AEDs was found. Diffuse ischemic lesions at computed tomography (CT)/magnetic resonance imaging (MRI) scans were present in higher percentages in patients who were antibody positive. No positive patient had a history of previous thrombosis or other features related to systemic lupus erythematosus (SLE), and no patient was born of a mother with SLE. Conclusions: Our study suggests a relation between epilepsy and aPL in young patients. A pathogenetic role for these autoantibodies cannot be excluded, and their determination might prove useful even from a therapeutic point of view.

To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl-coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in vitro.Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E-selectin, and intercellular adhesion molecule I (ICAM-1) expression by cell enzyme-linked immunosorbent assay and as interleukin-6 (IL-6) messenger RNA (mRNA) expression by RNA protection assay. E-selectin-specific nuclear factor kappaB (NF-kappaB) DNA-binding activity was evaluated by the gel-shift assay. HUVECs were activated by polyclonal affinity-purified IgG, human monoclonal IgM anti-beta2GPI antibodies, human recombinant IL-1beta, tumor necrosis factor alpha, or lipopolysaccharide (LPS).Fluvastatin reduced, in a concentration-dependent manner (1-10 microM), the adhesion of U937 to HUVECs and the expression of E-selectin and ICAM-1 induced by anti-beta2GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, displayed similar effects but to a lesser extent than fluvastatin. The inhibition of E-selectin expression exerted by fluvastatin was related to the impairment of NF-kappaB binding to DNA. Moreover, the drug attenuated the expression of IL-6 mRNA in HUVEC exposed to anti-beta2GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 microM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin.Endothelial activation mediated by anti-beta2GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.

A biological response modifier (BRM) has been defined as an agent able to modulate effector mechanisms or mediators of host defence. Some antibiotic molecules have been shown to display a BRM like activity, being able to enhance immune responses (certain cephalosporins), to synergize with the immune effectors (macrolides, quinolones) or alternatively, to depress immune functions (tetracyclines or antimycotic drugs). The BRM-like activity of different antibiotic molecules has been widely reported in in vitro studies as well as ex vivo in experimental animal models. Only recently some Authors have approached the problem by investigating whether the in vivo administration of antibiotic was able to affect different immune effector functions, either in healthy subjects or in patients. The main question in the field is the possible clinical impact of the connections between antibiotics and the immune system, particularly in subjects with acquired immunodeficiency in whom the impairment of the immune responses leads to increased susceptibility to infectious processes. Ex vivo data seem to suggest that cefodizime, one of the newest third-generation cephalosporins, is able to enhance phagocyte and mononuclear cell functions in healthy volunteers, thus confirming the possibility of combining an antibacterial efficacy with the ability to restore or enhance immune responses. Comparable data in studies investigating the effect of cefodizime on immune functions in immunocompromised patients such as elderly subjects, hemodialyzed or diabetic patients, BPCO subjects, patients undergoing surgical stress and patients with multiple myeloma are more important from a practical clinical point of view.(ABSTRACT TRUNCATED AT 250 WORDS)

From a clinical point of view Brouet’s classification of cryoglobulinemias, so widely accepted, has several limitations particularly because it cannot have a nosological meaning and also because of some technical aspects; in effect some cryoglobulins cannot be easily immunochemically classified: for example there are cryoglobulins consisting exclusively of polyclonal IgM or IgG [7]. But in order to discuss the problem of the classification of cryoglobulinemias we will present our case series; this collects also patients seen by Dr. M. Galli in the Clinic of Infectious Diseases of the University of Milan and by Dr. G. Monti in the Hospital of Saronno.

The effect of hydrocortisone (HC) on in vitro human polymorphonuclear leukocyte cytotoxicity was studied. HC was able to inhibit reversibly and in a dose-dependent way the antibody-dependent cellular cytotoxicity (ADCC) and the phytohemagglutinin-dependent cellular cytotoxicity (PDCC). The killing defect was partially overcome by increasing the antibody or the phytohemagglutinin (PHA) concentrations on the target cells. HC inhibited the PDCC more efficiently than ADCC; in fact the inhibition was present even at 10-5 M HC levels. Higher concentrations caused a progressive reduction in both tests. Inhibition of these PMN-mediated cytotoxicities may explain, in part, bot the anti-inflammatory actions of steroids and their deleterious effects on host defenses.

May anti-phospholipid or other autoantibodies interfere with trophoblast-endothelial cells interaction in women with unexplained pregnancy losses?The sera of 72 women with recurrent spontaneous abortions (RSA) containing antibodies to endothelial cells (28), trophoblast (14), and cardiolipin (10) or lacking antibodies (25), and 26 controls were examined in an inhibition assay of trophoblast adhesion to endothelial cells using an ELISA based on the recognition of trophoblast by antibodies to cytokeratin.Adhesion of trophoblast to endothelial cells was time- and dose-dependent. Patients and control sera inhibited trophoblast adhesion with mean values of 37\% and 7\%, respectively. Inhibition above 2SD of the mean control value was still observed in 58\% of the patients sera and 8\% of the control sera. Sera containing antibodies to endothelial cells had higher inhibitory effect (38\%) than those with antibodies to trophoblast (23\%) and cardiolipin (28\%) or lacking antibodies (26\%).Antibodies and other undefined factors in the sera of women with RSA inhibit adhesion of trophoblast to endothelial cells.