Prognostic value of specific KRAS mutations in patients with colorectal peritoneal metastases.

BACKGROUND: There is little evidence on KRAS mutational profiles in colorectal cancer (CRC) peritoneal metastases (PM). This study aims to determine the prevalence of specific KRAS mutations and their prognostic value in a homogeneous cohort of patients with isolated CRC PM treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. MATERIALS AND METHODS: Data were collected from 13 Italian centers, gathered in a collaborative group of the Italian Society of Surgical Oncology. KRAS mutation subtypes have been correlated with clinical and pathological characteristics and survival [overall survival (OS), local (peritoneal) disease-free survival (LDFS) and disease-free survival (DFS)]. RESULTS: KRAS mutations occurred in 172 patients (47.5%) out of the 362 analyzed. Two different prognostic groups of KRAS mutation subtypes were identified: KRASMUT1 (G12R, G13A, G13C, G13V, Q61H, K117N, A146V), median OS > 120 months and KRASMUT2 (G12A, G12C, G12D, G12S, G12V, G13D, A59E, A59V, A146T), OS: 31.2 months. KRASMUT2 mutations mainly occurred in the P-loop region (P < 0.001) with decreased guanosine triphosphate (GTP) hydrolysis activity (P < 0.001) and were more frequently related to size (P < 0.001) and polarity change (P < 0.001) of the substituted amino acid (AA). When KRASMUT1 and KRASMUT2 were combined with other known prognostic factors (peritoneal cancer index, completeness of cytoreduction score, grading, signet ring cell, N status) in multivariate analysis, KRASMUT1 showed a similar survival rate to KRASWT patients, whereas KRASMUT2 was independently associated with poorer prognosis (hazard ratios: OS 2.1, P < 0.001; DFS 1.9, P < 0.001; LDFS 2.5, P < 0.0001). CONCLUSIONS: In patients with CRC PM, different KRAS mutation subgroups can be determined according to specific codon substitution, with some mutations (KRASMUT1) that could have a similar prognosis to wild-type patients. These findings should be further investigated in larger series.

Laparoscopic cytoreductive surgery and hyperthermic intraperitoneal chemotherapy: Long term oncologic outcomes from the international PSOGI registry.

The laparoscopic approach for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (L-CRS + HIPEC) in highly selected patients was previously reported from the PSOGI registry with a demonstrable reduction in length of stay and post-operative morbidity. This study aims to update this international PSOGI registry with a larger cohort of patients and a longer follow-up period. METHODS: An international registry was designed through a networking database (REDCAP®). All centers performing L-CRS + HIPEC were invited through PSOGI to submit data on their cases. Variables such as demographics, clinical outcomes, and survival were analyzed. RESULTS: A total of 315 L-CRS + HIPEC cases were provided by 14 worldwide centers. A total of 215 patients were included in the L-CRS + HIPEC group. The median peritoneal cancer index (PCI) was 3 (3-5). The median length of stay was 7 days (5-10) and the major morbidity (Clavien-Dindo ≥3) was 6.1% after 30 days. The 5-year disease-free survival (DFS) per tumor origin was: 94% for PMP-LG, 85% for PMP-HG, 100% for benign multicyst peritoneal mesothelioma (MPM), 37.4% for colonic origin, and 54%(at 3 years) for ovarian origin. The 5 years overall survival (OS) per tumor origin was: 100% for PMP-LG, PMP-HG and MPM; 61% for colonic origin, and 74% (at 3 years) for ovarian origin. In addition, a total of 85 patients were analyzed in the laparoscopic risk-reducing HIPEC (L-RR + HIPEC). The median length of stay was 5 days (4-6) and the major morbidity was 6% after 30 days. The 5-year DFS per tumor origin was: 96% for perforated low grade appendiceal mucinous neoplasm (LAMN II) and 68.1% for colon origin. The 5 years OS per tumor origin was: 98% for LAMN II and 83.5% for colonic origin. CONCLUSIONS: Minimally invasive CRS + HIPEC is a safe procedure for selected patients with peritoneal carcinomatosis in specialized centers. It improves perioperative results while providing satisfactory oncologic outcomes. L-RR + HIPEC represents a promising strategy that could be evaluated in patients with high risk of developing peritoneal carcinomatosis into prospective randomized trials.

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.

Risk-based secondary prevention of obstetric antiphospholipid syndrome.

Treatment of pregnant women with antiphospholipid syndrome (APS) should be set apart from that from thrombotic APS patients. Patients with a history of pregnancy morbidity but no vascular thrombosis are usually treated with a prophylactic dose of heparin plus low-dose aspirin; whereas, those with previous vascular thrombosis alone or associated with previous pregnancy morbidity, are commonly treated with a therapeutic dose of heparin generally combined with low-dose aspirin. However, in about 20% of pregnant APS women these regimens fail. In this context, we conducted a case-control study on a large multicentre cohort of conventionally treated pregnancies to verify whether specific laboratory profiles and/or clinical characteristics are predictive of unsuccessful pregnancy outcome during conventional treatments. Multivariate analysis showed that pregnancy failure during conventional therapies was independently associated with a history of both thrombosis and pregnancy morbidity, the presence of systemic lupus erythematosus (SLE) or other systemic autoimmune diseases and triple antiphospholipid antibody positivity. With the aim to discover the most effective and safe treatments in high-risk pregnant APS women a large-scale multicentre study focusing on the effect of treatments on pregnancy outcome in women with APS and further risk factors for pregnancy failure has been designed.

Hypertension negatively affects the pregnancy outcome in patients with antiphospholipid syndrome.

The impact of hypertension in the pregnancies from autoimmune patients is not unequivocally defined. We have prospectively followed 168 pregnancies from 135 patients from four Italian centres to verify the potential impact of hypertension in the antiphospholipid syndrome (APS). The rate of preeclampsia, mean neonatal weight and gestational age at delivery were significantly lower in patients with both APS and hypertension than in patients with hypertension or APS alone. This information may be relevant for counselling and care of these patients.

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.

[Confirmation of antiphospholipid antibody positivity: a year's results in a cohort of 113 patients]. / Conferma della positività nell'autoimmunità antifosfolipidica: risultati acquisiti in un anno in una coorte di 113 pazienti.

OBJECTIVE: To evaluate the confirmation rate of antiphospholipid antibodies (aPL), to analyze their behaviour at confirmation time, and to study the clinical value of their confirmation. METHODS: Blood samples from 380 subjects, enrolled in this study from June 1, 2007 to May 31, 2008, were tested for anti-cardiolipin (aCL) and anti-beta2glycoprotein (abeta2GPI) antibodies using an ELISA method and for Lupus anticoagulant (LA) using a series of clotting tests. The samples of the 113 subjects resulting positive at the first testing time were assayed again to confirm antiphospholipid positivity. RESULTS: aPL positivity was confirmed in 67 out of the 113 subjects (59.3%). Medium-high antibody levels of all, except IgM aCL, aPL/ELISA had a significantly higher confirmation rate with respect to that in subjects with low levels. The confirmation rate in the category I antibody patients (multiple positivity) was higher than that in the category II antibody subjects (single positivity). LA positivity was confirmed only when it was associated to other aPL. The cut-off of 40 GPL produced a confirmation rate equal to that resulting from a 99th percentile cut-off. Confirmation of aPL positivity made it possible for us to confirm the diagnosis of antiphospholipid syndrome (APS) in 8 out of the 113 subjects originally resulting positive (7.1%). APS clinical features were vascular thrombosis in 4 of these and pregnancy morbidity in the other 4. CONCLUSIONS: Our data emphasize aPL positivity confirmation selectivity, and medium-high antibody levels and category I antibodies (multiple positivity) had the best confirmation rates.

Laboratory classification categories and pregnancy outcome in patients with primary antiphospholipid syndrome prescribed antithrombotic therapy.

BACKGROUND: A relationship between antibody profile and pregnancy outcome in patients with a previous diagnosis of primary antiphospholipid syndrome (APS) has not been clearly documented. METHODS: Women attending our Center with primary APS characterized by the presence in the blood of one or more of the following: Lupus Anticoagulant (LA), IgG/IgM anticardiolipin (aCL), IgG/IgM anti-human beta2-Glycoprotein I (abeta2GPI) antibodies (confirmed after a minimum of 3 months) were considered eligible for this study. Women who became pregnant during the study period with the exception of those with congenital thrombophilia or other congenital abnormalities were included in our analysis. Primary outcome events, defined as early abortion or fetal death, were evaluated in relation to the laboratory classification category assigned to each patient at the time they were diagnosed with APS. RESULTS: A total of 97 pregnancies occurring in 79 primary APS patients during the study period were analyzed. Twelve out of 97 pregnancies were unsuccessful, 11 out of 65 (16.9%) in category I patients (more than one positive laboratory test) and 1 out of 32 (3.1%) in category II patients (single positive test; adjusted hazard ratio 1.9; 95% CI, 0.2 to 18.9, p=0.6). Pregnancy loss took place in 10 out of 19 pregnancies (52.6%) in women belonging to category I with triple positivity and in 1 out of 46 pregnancies (2.2%) in patients with double positivity. The rate of pregnancy loss was more frequent in the 19 pregnancies of patients with triple positivity than in the 46 pregnancies of double positive patients (adjusted hazard ratio 23, 95% CI, 1.3 to 408, p=0.03). CONCLUSION: Poor pregnancy outcomes occur more frequently in category I than in category II primary APS patients. However, it has been seen that a greater predictability is achieved when category I patients are grouped into triple and double positivity states.

Peritoneal carcinomatosis arising from rectal or colonic adenocarcinoma treated with cytoreductive surgery (CRS) hyperthermic intraperitoneal chemotherapy (HIPEC): two different diseases.

PURPOSE: Peritoneal carcinomatosis (PC) from colorectal cancer (CRC) has poor survival. Multi-modal treatment including systemic chemotherapy, cytoreductive surgery (CRS), and hyperthermic intraperitoneal chemotherapy (HIPEC) can be used in selected patients with curative intent. The majority published works consider PC of CRC origin as a homogenous disease. Aim of this study is to stress the different biological behaviors and survival of PC according to colonic or rectal origin. METHODS: Data of CRS and HIPEC procedures for PC of CRC origin performed at MD Anderson Cancer Center-Madrid (Spain) have been collected, dividing patients into two groups according to colonic or rectal PC. Clinical, operatory, and postoperatory variables of the two groups have been analyzed to compare survival-related rates and PC origin. RESULTS: In the years 2004-2015, 114 procedures of CRS followed by HIPEC for peritoneal metastasis of different origin have been performed; of these, 36 procedures were for colorectal PC (31 patients in colonic and 5 in rectal group). Two groups are homogenous after analysis of clinical, operatory, and follow-up data. Median survival (OS) is significantly higher in colonic compared to rectal group (47.83 vs. 22.0 months, p 0.008). 3- and 5-year survival rate is 74 and 50% in colonic group vs. 20 and 0% in rectal group. CONCLUSION: Rectal origin PC has a more aggressive behavior compared to colonic origin, reflecting in a worst prognosis of patients affected by rectal origin PC. According to our data and literature, indications of multi-modal treatment including CRS and HIPEC should be more restrictive for rectal cancer PC. Authors should differentiate colonic and rectal origin of PC when reporting cases in the literature.

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.

[Digital ulcers in a cohort of 333 scleroderma patients]. / Ulcere digitali in una coorte di 333 pazienti sclerodermici.

Digital ulcers (DU) at the hands are one of the more frequent and severe complications in systemic sclerosis. Data on their prevalence and distribution in the different subsets of disease are variable in the literature. We studied the frequency of DU in a cohort of 333 scleroderma patients followed in the last 10 years in our Unit. DU have been recorded in 133 patients (39,9%), more frequently in males, in patients with cutaneous diffuse form of disease and in patients with anti-Scl70 ANA specificity. Complications of DU have been observed in 12,3% of cases. Surgery of the hands has been required in 8,7% of patients. The more effective treatment of DU are i.v. prostanoids, performed usually in day hospital, with high costs for the National Health Service. Recently the efficacy of bosentan, an oral receptor antagonist of endothelin, has been demonstrated, thus opening new perspectives in the treatment of DU in systemic sclerosis.

Anti-dermatophyte activity of Leguminosae plants from Southern Brazil with emphasis on Mimosa pigra (Leguminosae).

BACKGROUND: Intensive prophylactic use of antifungals leads to the increase of drug resistance and the need for new and more effective treatments are real. Plants from Leguminosae family are rich in flavonoids, for which numerous biological activities have been described, including antifungal effects. PURPOSE: To screen methanolic extracts from Leguminosae species looking for alternative sources for antifungal agents (anti-dermatophyte and anti-Candida) and their innocuity. METHODS: Antifungal activity was evaluated using the strains Candida albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, Epidermophyton floccosum, Trichophyton mentagrophytes, T. rubrum and, Microsporum gypseum in the broth microdilution method. Later, the minimum inhibitory concentration (MIC) for Mimosa pigra, Eriosema heterophyllum, and Chamaecrista nictitans was determined. The most promising extract was fractionated and cytotoxicity and genotoxicity of the most active fraction were also assayed. RESULTS: Fungicide and/or fungistatic activity against dermatophyte strains were presented by 60% of the methanolic extracts assayed. M. pigra, E. heterophyllum, and C. nictitans methanolic extracts could inhibit dermatophyte strains at concentrations ranging from 1.9 to 1000µg/mL. M. pigra showed the lowest MIC values for a dichloromethane fraction (1.9µg/mL) without DNA damage at 10 and 50µg/mL and 100% of cell viability of human leukocytes. CONCLUSION: Our results indicate that methanolic extracts from Leguminosae plants are potential sources of antifungal compounds, mainly the extract and fractions from M. pigra. The dichloromethane fraction from M. pigra did not showed in vitro toxicity according to the applied assays.

[Primary antiphospholipid syndrome and hyperhomocysteinemia: a study of a group of 29 patients]. / Sindrome da anticorpi antifosfolipidi primaria e iperomocisteinemia: studio di un gruppo di 29 pazienti.

OBJECTIVE: In order to investigate the potential role of hyperhomocysteinemia as an additional risk factor for thrombotic events, we studied its prevalence in patients with primary antiphospholipid syndrome (APS) and evaluated its association with different clinical features. METHODS: We enrolled 29 patients without any current evidence of underlying connective tissue disorder and fulfilling the Sapporo preliminary classification criteria for APS. RESULTS: Ten (34,4%) patients showed mild hyperhomocysteinemia (18,34 micromol/L +/- 2,04 DS). Nine had history of cerebrovascular disease, isolated (3 cases) or more often (6 cases) in association with other APS features. All patients, but one, showed multiple ischemic cerebral lesions. Seven of the 10 patients with hyperhomocysteinemia had multiple antiphospholipid antibody positivity and presented more frequently (6 cases) multi-site vascular involvement. CONCLUSIONS: The frequency of hyperhomocysteinemia in patients with primary APS is not negligible and appears to be associated with cerebral microangiopathic disease, multiple antiphospholipid antibody positivity and the simultaneous involvement of different vascular districts. For this reason and because hyperhomocysteinemia can be easily corrected with safe and relatively inexpensive therapeutic interventions, we advocate the measurement of homocysteinemia in every patient affected by APS and possibly in subjects with positive antiphospholipid antibody without a history of thrombosis.

[Isolated congenital heart block in undifferentiated connective tissue disease and in primary Sjögren's syndrome: a clinical study of 81 pregnancies in 41 patients]. / Il blocco cardiaco congenito nella connettivite indifferenziata e nella sindrome di Sjögren primitiva. Studio di 81 gravidanze in 41 pazienti.

OBJECTIVE: To study the incidence and the features of congenital heart block (CHB) in patients with undifferentiated connective tissue disease (UCTD) and primary Sjögren's syndrome (pSS). METHODS: We studied 81 pregnancies of 41 women attending the Outpatients' Clinic of the Rheumatology Unit of University Hospital of Padova from July 1989 to March 2004. Twenty five of these (61%) were affected with UCTD and 16 (39%) with pSS. Serologic inclusion criteria was anti-Ro/La positivity, assessed by counterimmunoelectrophoresis and ELISA. RESULTS: CHB was found in 2 out of the 46 (4.3%) pregnancies followed by our Staff and in 2 out of the 35 (5.7%) included in the retrospective part of the study. In 3 cases CHB was a 3rd degree block, causing pregnancy termination in 2. The only 2nd degree block was identified in one patient at the 22nd week of gestation and treated with dexamethasone and plasma-exchange. All of the women were positive to 52 kd and 60 kd Ro autoantibodies. CHB mothers had higher titer antibodies to 52 kd Ro protein than did the mothers with healthy infants (P = 0.026). Electrocardiographic abnormalities at birth were found in 3 out of 29 asymptomatic infants. One presented sinus bradycardia, the second abnormalities of ventricular repolarization, both regressed spontaneously, while the third ventricular extrasystoles which continue even now at 5 months. CONCLUSIONS: These results showed that in UCTD and pSS there is a higher incidence of CHB than that reported in Systemic Lupus Erythematosus. Electrocardiographic screening in all infants born to mothers with anti-Ro/La antibodies would seem an important measure to identify those with irreversible heart conduction abnormalities.

[Myositis specific and myositis associated autoantibodies in idiopathic inflammatory myopathies: a serologic study of 46 patients]. / Gli anticorpi miosite specifici e miosite associati nelle miopatie infiammatorie idiopatiche: studio sierologico di 46 pazienti.

OBJECTIVE: To characterize serum autoantibody profiles of patients with idiopathic inflammatory myopathies (IIM) by searching for myositis-specific (MSA) and myositis-associated (MAA) antibodies with sensitive and specific laboratory tests. METHODS: We tested the sera from 46 Caucasian patients diagnosed as affected with IIM at the Division of Rheumatology of Padova University (21 polimyositis, PM; 22 dermatomyositis, DM; 3 myositis overlap syndrome). All patients had definite IIM according to the criteria of Bohan-Peter. MSA including anti-tRNA synthetase (anti-Jo-1 and others) and anti-Mi-2 were determined by RNA immunoprecipitation and a modified immunoblot test, respectively. MAA (-U1RNP, -U2RNP, RoRNP, PM/Scl, Ku) were detected by counterimmunoelectrophoresis and immunoblot. RESULTS: Serum MSA and/or MAA were found in 30/46 (65%) patients with IIM. Twenty-three patients (50%) were positive for at least one MSA: anti-Jo-1 in 15 (33%), anti-Mi-2 in 6 (13%), and other anti-tRNA synthetase in 3 (6%). One patient was anti-Jo-1/Mi-2 positive. Moreover, 18 patients (39%) were positive for at least one MAA: anti-Ro/SSA in 13 (28%), anti-U1RNP in 4 (9%), anti-PM/Scl in 1 (2%) and anti-Ku in 1 (2%). Coexisting MSA and MAA were observed in 8 patients (17%), anti-Jo-1/SSA positive in most cases. Anti-Jo-1 was predominantly associated with PM (57% in PM vs 14% in DM), whereas anti-Mi-2 was exclusively found in DM patients (27%). Anti-synthetase antibodies were closely associated with interstitial lung disease and polyarthritis; anti-Mi-2 positive DM patients did not have lung involvement. Notably, anti-Ro/SSA antibody was frequently observed and almost equally detected in either PM or DM (about 30%): in more than 50% of cases the antibody was associated with one MSA. CONCLUSIONS: By means of analytically reliable methods, MSA was detected in 50% of our IIM patients. Searching for MSA in patients with IIM is recommended because of its diagnostic and prognostic value.

[Sensibility and specificity for pregnancy morbidity of anti-ß2-glycoprotein I antibodies in antiphospholipid syndrome]. / Sensibilità e specificità degli anticorpi anti-β2-glicoproteina I per l'impegno ostetrico della sindrome da anticorpi antifosfolipidi.

OBJECTIVE: This study aimed to evaluate the sensitivity and specificity of the anti-β2-glycoprotein I (GPI) antibodies for pregnancy morbidity in the antiphosoplipid syndrome (APS). METHODS: 335 women were recruited and on the basis of their clinical features were subdivided into 2 groups homogenous for number and age. The first (study) group contained the women whose pregnancy complications satisfied the classification criteria for APS. The second (control) group was made up of women with pregnancy complications not included in the classification criteria for APS. Anti-β2-GPI, anticardiolipin antibodies (aCL) and lupus anticoagulants (LA) were determined in all of these women. RESULTS: The only antiphospholipid antibodies occurring with a significant frequency (p=0,00) in the women with pregnancy criteria for APS were the IgG anti-β2-GPI and the IgG aCL present respectively in 23,92% and in 27,60% of the women. Its association was found to be significant (p=0,000). The distribution of the different levels of positivity of the IgG and IgM anti-β2-GPI in the patients of the study and control groups was not significantly different. The highest sensitivity for pregnancy complications was that of the IgG aCL and of the IgG anti-β2-GPI whose difference was not statistically significant. The comparison of the specificity of the IgG and IgM anti-β2-GPI with that of the IgG and IGM aCL was not statistically significant. CONCLUSIONS: The importance of determining the IgG anti-β2-GPI as part of routine laboratory testing of women with pregnancy complications typical of APS was confirmed. Together with IgG aCL these antibodies have proved to be the most sensitive and specific markers of pregnancy complications in APS.

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.

Antiphospholipid syndrome: antibodies to Domain 1 of ß2-glycoprotein 1 correctly classify patients at risk.

BACKGROUND: Determination of lupus anticoagulant (LA), anticardiolipin (aCL) and ß2-Glycoprotein 1 (aß2GP1) antibodies is mandatory to classify patients with antiphospholipid syndrome (APS) into risk categories. OBJECTIVES: To measure relevant antibodies, considered to be those of the IgG isotype directed towards ß2GP1 and particularly those directed to Domain 1 (Dm1) of the molecule. PATIENTS/METHODS: In this cross-sectional study we measured IgG aß2GP1-Dm1 by a chemiluminescent immunoassay in a group of individuals initially 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 AND CONCLUSION: Geometric mean and standard deviation expressed as chemiluminescent units (CU) in triple, double and single positive groups were 273.0 ± 6.2, 18.2 ± 9.6 and 4.4 ± 2.2, respectively. The geometric mean obtained in 40 healthy subjects was 2.0 ± 2.0. Mean CU values were significantly different among groups and with respect to values found in 40 healthy subjects (P < 0.0001). Positive values of IgG aß2GP1-Dm1 (above 14.2 CU) were found in 45 individuals while 20 individuals (20/65 = 30.8%) positive for IgG aß2GP1 were negative for IgG aß2GPI-Dm1. There was a significant association between positive IgG aß2GP1-Dm1 and thromboembolic events (P = 0.001). Positive and negative values of IgG aß2GP1-Dm1 were consistently confirmed after 12 weeks, with only three low positive values being negative after 12 weeks. In conclusion, IgG aß2GP1-Dm1 seems a robust and reproducible test that in association with the classic tests may be useful in clinical practice in identifying individuals at high risk of developing thromboembolic events.

The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases.

Autoantibodies directed against intracellular antigens can be detected by immunoblotting (IB). Due to its high sensitivity this technique has many advantages, but it can give misleading results when the specific bands are weak or blurred against the background staining. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Moreover, these agents appear to have a renaturating action towards proteins and antigens. Tween 20 has a more pronounced renaturating effect on proteins than other detergents and thereby improves antigen-antibody binding. To evaluate the effect of Tween 20 on specific autoantibody detection by IB, we tested the sera of 162 patients with connective tissue diseases (CTDs) by adding this detergent at certain steps of the IB assay. We found that the use of Tween 20 in the IB procedure significantly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa and C, Sm B/B' and D. Moreover, it increased the sensitivity for the detection of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantly decreased the binding of autoantibodies specific for ribosomal P proteins, La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the method. We conclude that the addition of Tween 20 to standard IB is advantageous for anti-nuclear antigen antibody detection and improves the sensitivity of the method in revealing anti-Sm-positive sera in SLE. However, Tween 20 is not recommended for the detection of anti-cytoplasmic antibodies.