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BackgroundPatients with autoimmune diseases require appropriate pregnancy counceling, as their disease can negatively impact both maternal and fetal outcomes. In regards to Behçet’s disease (BD), although commonly diagnosed during the childbearing age, only few studies focused on its impact on gestation, with contradictory results.ObjectivesWe aimed to collect new data on BD and its impact on pregnancy by designing a single-center cohort study, reporting the pregnancy and maternal outcomes in BD patients attending the Multidisciplinary Centre for Immunopathology and Rare Diseases at Giovanni Bosco Hospital (Turin, Italy).MethodsWe retrospectively included 26 women diagnosed with BD followed at our center. Out of 26 patients, data from 33 pregnancies in 16 women were collected. Ten patients were never pregnant. Table 1 shows the demographical and clinical characteristics and the pregnancy outcomes of the enrolled patients.Table 1.Clinical characteristics and pregnancy outcome of the Behçet’s disease patients enrolled in the study.Patients(tot=31)Nulliparous; n (%)10/26 (38,46%)Primigravida; n (%)16/26 (61,54%)DemographicsAge at BD diagnosis; mean± S.D.40,56±11,42Age at first pregnancy; mean ± S:D.24,86 ± 6,56Clinical characteristics of BDOral ulcers; n (%)26/26 (100 %)Genital ulcers; n (%)21/26 (80,76%)Folliculitis; n (%)15/26 (57,69%)Erythema nodosum; n (%)6/26 (23,08%)Ocular manifestations; n (%)11/26 (42,31%)HLA B-51+; n (%)13/26 (50%)Pregnancy outcomeRate of live births; n (%)27/33 (81,81%)Any complications; n (%)12/33(36,36%) Early miscarriage before 10 weeks of gestation; n (%)6/33 (18,18%) Gestational diabetes; n (%)3/33 (9,09%) Placental abruption; n (%)2/33 (6,06%) Vaginal bleeding before 20 weeks of gestation; n (%)1/33 (3,03%)Worsening of oral and genital ulcerations; n (%) *2/ 11 (18%)* This information was available only for 11/33 pregnancies.BD – Behçet’s diseaseS.D – Standard deviationResultsAll included patients fulfilled the international criteria for BD (1), with a median age at the time of diagnosis of BD and first pregnancy of 40,56±11,42 and 24,86±6,86 respectively. The frequency of clinical manifestations was oral ulcers (100%), genital ulcers (80,76%), erythema nodosum (23,08%), folliculitis (57,69%), ocular manifestations (42,31%) and HLA B-51+ (50%), respectively.The overall rate of live births was 81,81% (27/33). Twelve women experienced pregnancy complications (36,36%), in particular we report one case of vaginal bleeding before 20 weeks of gestation, six miscarriages before the 10th week of gestation, three cases of gestational diabetes and two placental abruptions. Among the analyzed pregnancies, a worsening of oral and genital ulcerations was observed in the 18% of them, while in the remaining cases a subjective amelioration of aphtosis and other concurrent symptoms was reported by the patients. No systemic flare, worsening of ocular complications and VTE was reported during pregnancy and in the post-partum period.ConclusionThe results of our study highlight an overall favorable rate of life births in BD women and a positive rate of disease remission in pregnancy. Currently, the studies focusing on pregnancy outcomes in BD are scarce, however it might be appropriate to give a specialized counseling to a BD woman planning for a pregnancy and follow these patients with a multidisciplinary approach.References[1]Chan T-M, Meng &, Chiou J, Kuo C-F. Adverse pregnancy outcomes in women with Behçet’s disease: population-based registry linkage study in Taiwan. Available from: https://doi.org/10.1007/s10067-021-05688-6[2]Davatchi F, Assaad-Khalil S, Calamia KT, Crook JE, Sadeghi-Abdollahi B, Schirmer M, et al. The International Criteria for Behçet’s Disease (ICBD): A collaborative study of 27 countries on the sensitivity and specificity of the new criteria. J Eur Acad Dermatology Venereol [Internet]. 2014 Mar [cited 2021 May 1];28(3):338–47. Available from: https://pubmed.ncbi.nlm.nih.gov/23441863Disclosure of InterestsNone declared

BackgroundMedication non-adherence has a significant impact on the health and well-being of individuals with chronic diseases. Indeed, with respect to important risk factors of Rheumatoid Arthritis (RA), such as cardiovascular risk factors, it is known that up to 50% of patients will stop taking medication for these conditions during the first year of prescription [1].ObjectivesTo support the management of RA patients treated with Tofacitinib, we designed the TuTOR (Tailoring Tofacitinib Oral therapy in Rheumatoid arthritis) Mobile App.MethodsA prospective-controlled study evaluated the impact of TuTOR App on medical adherence in 20 RA patients, that began treatment with Tofacitinib jointly with the App. We used a crossover design alternating Paper-Diary and TuTOR App, with monthly clinical assessments.ResultsSeventeen patients with RA (mean age at inclusion 59±13yrs; 88% females) the study. A statistically significant decrease of DAS28 was observed since the first month of therapy with Tofacitinib (mean DAS28 at baseline 3.9±1 vs. 1° month 3.1±1, p=0.0016). Similarly, Numerical Rating Scale(NRS) of perceived activity of disease (5.8±2.1 vs.3.7±2.5, p=0.02), and subjective fatigue (6.1±2.3 vs 4.3±2.6; p=0.01) progressively decreased. No differences were reported in DAS28 and in all the NRS between the use of the TuTOR App and the Paper-Diary. A significant decrease was observed also in HAQ during the follow-up (baseline 1.38±1.11 vs.six months 0.83±0.9; p=0.01).Most of the patients (82%) when filling out the self-reporting questionnaires preferred the TuTOR App in helping them to remember to take the pills. Further 82% of patients used the TuTOR App regularly (vs.53% Paper-Diary) and 76% of patients would use it in the future (vs.53% Paper-Diary). Three patients suspended the therapy with Tofacitinib due to gastrointestinal intolerance.ConclusionBoth digital- and paper-devices can help maximize the adherence to therapy, leading to an improvement in disease’s activity, highlighting the need of supports for medication adherence.References[1]BJ van den B, HE Z, CH van den E. Medication adherence in patients with rheumatoid arthritis: a critical appraisal of the existing literature. Expert Rev Clin Immunol 2012;8:337–351. Available at: https://pubmed.ncbi.nlm.nih.gov/22607180/. Accessed July 13, 2021.Table 1.Baseline demographic and clinical characteristics of the patients enrolled in the study, who completed follow-up. RA – Rheumatoid ArthritisPatients with RA(N=17)DEMOGRAPHICSAge (mean ± S.D.)59,4 ± 13,5Sex (n; %)M (2; 11,8), F (15; 88,2)Etnicity (caucasian; n; %)16; 94Etnicity (Hispanic; n; %)1; 6CLINICAL CHARACTERISTICSAge at diagnosis (mean ± S.D.)44,7±14,47Follow-up length (years; mean ± S.D.)6,33± 5,17Positive Rheumatoid Factor (n; %)17; 100Positive Anti-Cyclic citrullinated peptides (n;%)15; 88.2Structural articular damage at radiography (n; %)11; 7Figure 1.Decrease of disease activity assessed by DAS28 in the paper-diary and TuTOR App groups.Disclosure of InterestsMassimo Radin Grant/research support from: The study is supported by the Investigator-Initiated Research Studies Grant—CREARE (Pfizer)., Marta Arbrile: None declared, Irene Cecchi Grant/research support from: The study is supported by the Investigator-Initiated Research Studies Grant—CREARE (Pfizer)., Alice Barinotti: None declared, Simone Baldovino: None declared, Elisa Menegatti: None declared, Daniela Rossi Grant/research support from: The study is supported by the Investigator-Initiated Research Studies Grant—CREARE (Pfizer)., Savino Sciascia Grant/research support from: The study is supported by the Investigator-Initiated Research Studies Grant—CREARE (Pfizer)., Dario Roccatello Grant/research support from: The study is supported by the Investigator-Initiated Research Studies Grant—CREARE (Pfizer).

BackgroundCardiovascular diseases (CVDs) represent one of the most life-threatening conditions that can affect SLE patients. Assessing the potential CVD risk of these patients is still a challenge and an important aspect in the clinical practice. Recently the QRISK3 score has attempted to encompass for SLE augmented thrombotic risk by adding items (such as corticosteroid use) that are missing in traditional CVD risk scores.ObjectivesTo apply and compare the QRISK3 and the adjusted Global AntiPhospholipid Syndrome Score (aGAPSS), a validated score to assess CVD and overall thrombotic risk in aPL positive patients, in a cohort of SLE patients with and without a concomitant diagnosis of APS.Methods25-85 years old patients attending San Giovanni Bosco Hospital (Turin) during a period of 6 months (Sep 2019 – Feb 2020) with a confirmed diagnosis of SLE (2019 ACR/EULAR classification criteria) and/or a diagnosis of SAPS (Sidney criteria) were included in the study. QRISK3 has been calculated using the official online calculator (https://qrisk.org/). aGAPSS has been calculated using the validated point values based on aPL profile and independent risk factors: aCL=5, aβ2GPI=4, LA=4, aPS/PT=3, hyperlipidemia=3, hypertension=1.ResultsThe analysis included a cohort of 142 SLE patients: 34 SAPS (23.9%) and 108 SLE patients without APS (76.1%), with a mean age of 48±12.9 (SAPS=51.6±12.8/SLE without APS=46.9±12.8). Table 1 summarizes patients characteristics. When focusing on cerebrovascular/coronary events, we found a statistical significance with respect to aGAPSS (pt with event =10.1±6.2 vs pt without event=5.8±6.1; p=0.007), but not QRISK3. Also, a significant association was observed between the occurrence of these events and high risk aGAPSS: p=0.03 for aGAPSS≥8, p=0.01 for aGAPSS ≥9, p=0.008 for aGAPSS ≥10. Moreover, the aGAPSS but not the QRISK3 resulted to strongly correlate with the occurrence of any thrombotic event, both at the uni- and multivariate analysis (univariate: pt with event =8.17±7.1 vs pt without event= 5.41±5.6; p=0.012 / multivariate: p=0.009). The same was observed for gender: male gender resulted to correlate with the occurrence of any thrombotic event at both uni- and multivariate analysis (p=0.017 and p=0.03, respectively). Finally, when focusing on aPL profile, regardless the diagnosis, we found a statistical significance only with respect to aGAPSS (aPL+ =9.6±6.3 vs aPL- = 4.1±5.1; pTable 1.Demographic and clinical features of the patients included in the study.ConclusionBy encompassing factors that can contribute to CVD development in complex/autoimmune diseases, QRISK3 has been shown to be more accurate than traditional risk score in predicting SLE patients CVD risk. Nonetheless, the results of this analysis showed how the aGAPSS still seems to be the most valuable tool for this purpose when facing SLE patients. Moreover, the significance observed when focusing on patients’ aPL profile, suggests that adding this item to the QRISK3 could be a useful strategy to improve the management.References[1]Hippisley-Cox J, Coupland C, Brindle P. Development and validation of QRISK3 risk prediction algorithms to estimate future risk of cardiovascular disease: prospective cohort study. BMJ. 2017 May 23;357:j2099. doi: 10.1136/bmj.j2099.[2]Radin M, Sciascia S, Erkan D, et al. The adjusted global antiphospholipid syndrome score (aGAPSS) and the risk of recurrent thrombosis: Results from the APS ACTION cohort. Semin Arthritis Rheum. 2019 Dec;49(3):464-468. doi: 10.1016/j.semarthrit.2019.04.009.Disclosure of InterestsNone declared

The COVID-19 pandemic is forcing a rethink in robotics. In the form it is known today, robotics has been the prerogative of a broad community of insiders. But now, in the wreckage left behind by COVID-19, a new era is beginning. What does it hold? During the pandemic, increasing numbers of people had manifested the hope that robotics might bring novel solutions. And this interest has emerged beyond the usual boundaries of the experts or technology enthusiasts. This provides an opportunity to reinforce the community of people involved in the process of innovation. By involving citizens, the community becomes more comprehensive (that is, plural and diverse). This broadening will involve more practical knowledge and therefore produce better robots of many shapes and functions. If progress is possible in the industry, why not in the hospitals, shopping malls, restaurants, hotels, and schools? What is more, the approach endorsed by the Italian robotics community during the lockdown has established a new cooperation among those who labor with robots, and the professionals who work in hospitals, which is bound to last a long time. As a major impact, this experience will enable an improvement on science's relationship with and for society. This may entail a further shift: to value more scientific knowledge and scientific literacy.

Chronic cerebrospinal venous insufficiency (CCSVI) is a syndrome characterized by stenoses or obstructions of the internal jugular and/or azygos veins with disturbed flow and formation of collateral venous channels. Studies using ultrasound in patients with multiple sclerosis (MS) have demonstrated a high prevalence of CCSVI (mean 70%; range 0-100%; N.=1496), whereas, in normal controls and patients without MS the prevalence was much lower (mean 10%; range 0-36%; N.=635). Ultrasound uses a combination of physiological measurements as well as anatomical imaging and has been used for the detection of CCSVI by different centers with variable results. A high prevalence ranging from 62% to 100% of obstructive lesions has been found by some teams in patients with MS compared with a lower prevalence of 0-25% in controls. However, absence of such lesions or a lower prevalence (16-52%) has been reported by others. This variability could be the result of differences in technique, training, experience or criteria used. The current lack of a methodology shared among experts is a confounding element in epidemiologic studies, and does not permit further Bayesan or other kind of analysis. In order to ensure a high reproducibility of Duplex scanning with comparable accuracy between centers, a detailed protocol with standard methodology and criteria is proposed. This is also necessary for training. It has been shown that inter-rater variability increases post-training (from k=0.47 to k=0.80), while within-rater reproducibility in trained operators was k=0.75. Finally, the consensus document proposes a reporting standard of Duplex measurements, and future research to answer areas of uncertainty.

Congenital adrenal hyperplasia, both in its classic (CCAH) and non-classic form (NCAH), is a morbid condition sustained by the absent or reduced function of one of the enzymes involved in cortisol biosynthesis - mainly 21 hydroxylase - associated with different levels of clinical androgenization. In a wide group of relatives of patients affected by CCAH and NCAH (no.=222) and healthy volunteers (no.=30), a clinical, hormonal and genetic evaluation was performed in order to differentiate between the condition of heterozygous mutation carrier and non-carrier of any among 21-hydroxylase gene (CYP21) mutations. This study shows that clinical presentation and basal 17α-hydroxyprogesterone (17α-OHP) are not able to differentiate between heterozygous carriers and non-carriers, whereas 17α-OHP value after ACTH bolus is significantly different between heterozygous carriers and non-carriers: p0.001 with a cut-off value of 3 ng/ml (90% sensitivity and 74,3% specificity). Moreover, our data indicate that 17α-OHP response to ACTH may be a useful tool to select subjects for genetic analysis.

To investigate the polymorphisms of the vascular endothelial growth factor (VEGF) gene in relation to female patients who developed bisphosphonate-related osteonecrosis of the jaws (BRONJ).Test subjects were 30 Italian female patients with BRONJ (Group A). Control subjects were 30 female patients with a history of intravenous bisphosphonate use without any evidence of osteonecrosis (Group B) and 125 unrelated healthy volunteers (Group C). Three single-nucleotide polymorphisms were investigated: -634 GC, occurring in 5' untranslated region (UTR); +936 CT, occurring in 3' UTR; and -2578 CA of the promoter region.The frequency of the VEGF CAC (+936/-2578/-634) haplotype was increased in patients with BRONJ, compared with female disease-negative controls [odds ratio (OR) = 2.76, 95% CI = 1.09-4.94, P = 0.039; corrected P value: P(c) = 0.117], and was also increased compared with female healthy controls (OR = 2.11, 95% CI = 1.14-3.89, P = 0.024; corrected P value: P(c) = 0.072). The CC homozygotes of -634GC of VEGF gene and AA homozygotes of -2578CA have also been significantly correlated in female patients who developed BRONJ compared with healthy controls (OR = 2.04, 95% CI = 1.12-3.70, P = 0.008; corrected P value: P(c) = 0.024).These results suggest a possible haplotype effect of VEGF polymorphisms expression in BRONJ Italian female patients. Studies with different and larger populations possibly using TagSNP to represent all haplotypes within the VEGF gene are needed to further delineate the genetic contribution of this gene to BRONJ.

Non-classical congenital adrenal hyperplasia (NCAH) is a morbid condition sustained by the reduced function of one of the enzymes involved in the adrenal steroid biosynthesis pathway, mainly the 21-hydroxylase. Different degrees of enzyme activity impairment determine different clinical pictures, with childhood or post-pubertal onset. The aim of this study was to evaluate the relationship between genotype, phenotype, and adrenal hormonal levels in a group of 66 patients affected by NCAH attending outpatient pediatric or endocrinological Clinics. Our findings show that age at pubarche/menarche was significantly younger, height SD score) and Δ bone age-chronological age were significantly higher in patients with a more severe enzyme activity impairment, while cutaneous androgenization and menstrual irregularities in post-pubertal girls were not related to the grading of genotype.

This paper describes the production, characterization and in vitro activity of ethosomes containing two molecules with antiviral activity, such as acyclovir (ACY) and N1-beta-D-ribofuranosyl-pyrazole [3,4d]pyridazin-7(6p-chlorine-phenyl)-one nucleoside (N1CP). Ethosomes were prepared and morphologically characterized by Cryo-TEM. The encapsulation efficiency was 92.3 +/- 2.5% for ACY and 94.2 +/- 2.8% for N1CP. The release of the drug from vesicles, determined by a Franz cell method, indicated that both drugs were released in a controlled manner. In order to possibly guarantee the stability during long-term storage ethosome suspensions was freeze-dried. It was found that the freeze-dried ethosomes' cakes were compact, glassy characterized by low density and quick re-hydration. However, the storage time slightly influences the percentage of drug encapsulation within ethosomes showing a drug leakage after re-hydration around 10%. The antiviral activity against HSV-1 of both drugs was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that ethosomes allowed a reduction of the ED50 of N1CP evidencing an increase of its antiviral activity. However, ACY remains more active than N1CP. No differences are appreciable between drug-containing ethosomes before and after freeze-drying. Taken together these results, ethosomal formulation could be possibly proposed as mean for topical administration of anti-herpetic molecules.

Chronic fatigue (CF) severely affects patients with multiple sclerosis (MS), but its pathogenesis remains elusive and the effectiveness of available treatments is modest. We aimed to evaluate the effect on CF of the balloon dilatation of stenosing lesions affecting the main extracranial veins configuring the chronic cerebrospinal venous insufficiency (CCSVI), a condition strongly associated with MS.Thirty-one MS consecutive patients (16 males, age 46.2+/-9.4 years) with associated CCSVI and CF underwent the endovascular procedure. Fatigue was assessed using the Fatigue Severity Scale (FSS) and Modified Fatigue Impact Scale (MFIS) at baseline (T0) and one (T1), six (T6) and twelve (T12) months after the procedure. In ambulatory patients (N.=28), mobility was evaluated using the 6-min walking test at T0 and T1.and MFIS scores significantly improved from preoperative values, and the positive trend was maintained at one year (FSS: T0=5.1+/-1.0 to T12=3.5+/-1.8, P0.001; MFIS-total score: T0=34.9+/-14.8 to T12=22.5+/-13.7, P0.001; MFIS-Physical subscale: T0=21.2+/-8.0 to T12=13.5+/-9.7 P0.001; MFIS-Cognitive subscale: T0=9.2+/-9.5 to T12=6.0+/-6.3, P=0.03; MFIS-Psychosocial subscale: T0=4.5+/-2.1 to T12=2.5+/-2.1, P0.001). Six-min walking distance (6MWD) at T1 improved significantly (332+/-190m to 378+/-200m, P=0.0002). In addition, an inverted correlation between 6MWD and MFIS-physical subscale variations was found in the subgroup of patients (N.=8) with no lower limb motor impairment (r=-0.74, P=0.035).The reestablishment of cerebral venous return dramatically reduced CF perception in a group of MS patients with associated CCSVI, suggesting that CF is likely the symptom of CCSVI.

Chronic cerebrospinal venous insufficiency (CCSVI) is a syndrome described in multiple sclerosis (MS) patients, characterized by stenosis of the main extracranial veins with hampered cerebral venous outflow. In the original description echo-colour Doppler demonstrated to be an ideal non invasive tool for screening CCSVI patients, but the reproducibility was not assessed. Aim of this study is to assess the variability coefficient between trained and in not trained echo-colour Doppler operators.Thirty-six (36) subjects, matched for age and gender, were subset in 3 groups (group A, 12 healthy controls, HC; group B, 12 multiple sclerosis patients, MS; group C, 12 patients with other neurological disease, OND) underwent echo-colour Doppler screening for CCSVI according to an original protocol previously described. The inter observer variability rate was assessed by comparing respectively trained vs not trained operators, and trained vs trained operators, by using the same echo-colour Doppler equipment. In addition, by scanning 15 subjects after one month from the first session, intra observer coefficient was also assessed in trained operator.The inter observer variability rate between trained and not trained echo-colour Doppler operators, were not completely satisfactory (K coefficient 0.47 95% CI 0.27-0.68). To the contrary the inter observer agreement between trained operators was much more reliable (K coefficient 0.80 95% CI 0.59-1.01). Finally, the intra observer variability rate in trained operators was 0.93, (95% CI 0.80-1.06) confirming a highly satisfactory agreement.Echo-colour Doppler is a powerful, non-invasive and reproducible tool for screening CCSVI-MS but it needs special training.

Chronic cerebrospinal venous insufficiency (CCSVI) is associated with multiple sclerosis (MS). CCSVI is detected by transcranial and extracranial color-Doppler high-resolution examination (TCCS-ECD) and venography that permit to identify five types of venous malformations and four major (A-D) hemodynamic patterns of anomalous extracranial-extravertebral venous outflow. We investigated possible correlation between such hemodynamic patterns and both the symptoms at onset and clinical course in patients with MS and CCSVI.TCCS-ECD, selective venography and clinical records of 65 patients affected by definite MS and CCSVI were reviewed.The four hemodynamic patterns of CCSVI were unevenly (P0.0001) distributed with respect to the types of clinical presentation and course. In particular the Type A or B patterns were common in patients with onset of optic neuritis, but rare in patients presenting with spinal cord symptoms who typically showed a type D pattern. As well, the type A or type B hemodynamic were more common in patients with relapsing remitting course than in patients with secondary progressive course and rare in patients with primary progressive course. The C hemodynamic pattern was not observed in patients with primary progressive course who showed a remarkable prevalence of the type D pattern.The distribution of venous malformations and the resulting hemodynamic pattern show correlation with symptoms at onset and clinical course in patients with MS and CCSVI.

Background Parenteral Nutrition (PN) is a technique of Artificial Nutrition used to feed patients who cannot or should not get their nutrition through eating. Preparation is complicated because it involves mixing, with aseptic technique, nutritional substrates with different chemical-physical features (water, glucose, amino acids, electrolytes, trace elements and vitamins) to obtain a sterile nutritional emulsion that must be stable during preparation, storage and administration. Purpose To check the stability of nutritional formulae for parenteral nutrition. Material and methods Six nutritional formulae were prepared, three variations for each, using three kinds of lipid emulsion. Also added: long chain triglycerides (LCT) emulsion from soybean oil and middle chain triglycerides (MCT) emulsion from coconut oil; enriched lipid emulsion (LCT + MCT) from soybean, olive, coconut and fish oils; omega-3 emulsion from refined fish oil. Each formula was prepared with different calcium concentrations, which, together with viscosity, is the critical parameter that affects Zeta potential and consequently stability. Analysis was performed using a helium-neon laser granulometer on diluted samples at time 0 and after 24, 48, 72 and 96 h in order to reproduce normal storage conditions and clinical use. Results The results of the analyses of lipid particles established different stabilities, depending on the different kind of lipids and concentration of calcium: nutritional formulae prepared with LCT + MCT have proved 96 h stability, independently of calcium concentration; nutritional formulae with enriched lipids and/or LCT/MCT and lipids from refined fish oil have been proved stable only if the calcium concentration is less than 9 mEq/litre. Conclusion The results suggest a 96 h shelf life from preparation for nutritional formulae prepared with LCT + MCT. Stability is maintained only if the calcium concentration is less than 9 mEq/litre in nutritional formulae prepared with enriched lipids or with the addition of LCT + MCT and fish oil. Reference 1. SINPE Guidelines for Hospital Artificial Nutrition 2002; RINPE Year 20, S5 : Update October 2003 Reference No conflict of interest.

The crystal structure of bovine alpha-chymotrypsin (alpha-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 A resolution (R-factor = 0.18). The proteinase:inhibitor complex forms a compact dimer (two alpha-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between alpha-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P1 residue of BPTI, however, does not occupy the alpha-CHT S1 specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217.

In the present study are reported the differential DNA binding activity of the anti-tumor polyamidine tetra-p-amidinophenoxyneopentane (TAPP-H) and its 2'-halo derivative (TAPP-Br), and their effects on the binding of the recombinant Epstein-Barr virus (EBV) nuclear antigen to a synthetic oligonucleotide mimicking the target DNA sequence present in the EBV genome. In addition, the proliferation kinetics and cell cycle analysis of human leukemia K562 cells treated with TAPP-H and TAPP-Br are reported. The possible in vivo relationship between DNA binding affinity and cytotoxicity is also discussed.

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.

The HPLC assay of 1,4-dioxane in a wide range of commercially available cosmetics containing polyethoxylated surfactants is described. After solid-phase extraction using Bakerbond CN- and Bakerbond C18-cartridges, samples were directly analysed on a LiChrospher CH-8 column with an acetonitrile water eluent and UV detection at 200 nm. Of the total cosmetic products investigated, 48% were found to contain 7.3-85.9 ppm of 1,4-dioxane.

Thermodynamics and kinetics for hirudin binding to human alpha-, beta- and gamma-thrombin, under experimental conditions which mimic the in vivo conditions (i.e., pH = 7.35, T = 37.0 degrees C, I = 0.1 M), indicate that the inhibitor specificity for the three species of the enzyme is different. Such a finding agrees with the human thrombin:hirudin binding geometry as revealed by X-ray crystal studies. From the therapeutic viewpoint, thermodynamics and kinetics here reported indicate that the inhibitory activity of hirudin in vivo is more effective than that shown by antithrombin III, which is generally considered the most important plasma thrombin inhibitor.

The N-alpha-L-isoleucyl-L-valine (Ile-Val) activating dipeptide, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine beta-trypsin, displays an activating effect on equilibria involved in the binding of strong ligands (i.e., n-butylamine and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen. This property has been investigated between pH 3.0 and 9.0 (I = 0.1 M) at 21.0 degrees C. The thermodynamics for the interaction of strong ligands with bovine beta-trypsin has also been studied under the same experimental conditions. The equilibria involved in the binding of the Ile-Val activating dipeptide and/or inhibitors to bovine beta-trypsin and its zymogen are described according to linkage relationships, wherefore interaction(s) between different functional and structural domains of the (pro)enzyme (i.e., the so-called Ile-Val pocket and the primary and/or secondary recognition subsite(s)), possibly involved in the bovine trypsinogen-to-beta-trypsin activation pathway, are considered.

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.

A titration method for determination of trypsin-like serine proteinase concentration has been developed by using ZArgONp and ZLysONp, two specific chromogenic amino-acid derivatives which show the characteristics of optimal active-site titrants. Active proteinase concentration has been estimated from the effect of titrant concentration on the amplitude, at time zero, of the time-course for the instantaneous release of p-nitrophenol, preceding the steady-state reaction (burst phase).

Guanidinophenyl derivatives of pyrazole have been synthesized. Their inhibitory effects on (i) bovine trypsin, bovine thrombin, porcine pancreatic kallikrein catalyzed hydrolysis of p-nitro anilide of N alpha -benzoyl-arginine and (ii) blood coagulation and platelet aggregation, were investigated. The kinetic behaviour of all compounds conformed to that of a reversible competitive inhibition pattern, and they were also found to act in vitro as inhibitors of platelet aggregation induced by ADP.

The inhibitory effect of benzamidine as well as of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H) and TAPP-halo derivatives (with Cl, Br or I) on (i) the catalytic properties of purified plasma serine proteinases (notably, factor Xa, factor VIIa, thrombin and plasmin), (ii) blood coagulation, and (iii) platelet aggregation was investigated in vitro. For all the enzyme/inhibitor systems examined, the inhibition patterns were strictly competitive, and titrations conformed to simple equilibria. The inhibitory effect of TAPP-H and TAPP-halo derivatives is higher, by at least 10-fold, than that of benzamidine, which binds at the primary specificity subsite (S1) of serine proteinases and is commonly taken as a molecular inhibitor model. The high inhibitory effect of aromatic tetraamidines has been interpreted taking into account an additional productive binding for a second benzamidine or halo-benzamidine moiety to the enzyme surface. As a whole, the data reported here indicate that aromatic amidines inhibit the plasma serine proteinases involved in different steps of haemostasis (coagulation and platelet aggregation) as well as clot lysis under physiological-like conditions in vitro.

The synthesis of Leu-enkephalinamide, Leu-enkephalin-1-adamantylamide, Leu-[D Ala2]enkephalinamide and Leu-[D Ala2]enkephalin-1-adamantylamide are described. Preliminary results of pharmacological tests in vitro are reported.

Ornithine transcarbamylase (OCT) is one of the urea-cycle enzymes in mitochondria which is involved in metabolic disorders in man. Reye's syndrome, characterized by encephalopathy and fatty infiltration of the liver, is one of such diseases. During this syndrome a reduction of OCT specific activity was observed, probably due to increased proteolytic degradation or to alteration in the enzyme conformation. It has recently been found that OCT administration to patients with liver disorders, contributes to illness resolution. Since the therapeutic properties of the enzymes are usually related to their structure, this paper reports some findings obtained by spectroscopic measurements on OCT structure and conformation stability in solution.

1H NMR spectra of the porcine pancreatic secretory trypsin inhibitor (PSTI) have been recorded vs. pH and temperature. Of the two tyrosines, one titrates with a pK of 11.25, while the resonances from the other are pH insensitive in the investigated range 4.8 less than or equal to pH less than or equal to 12. This is consistent with PSTI having one Tyr solvent exposed (Tyr-20) and the other buried (Tyr-31). The resonances from the lysyl epsilon-CH2 protons titrate with a pK of 10.95. The titration is accompanied by a pronounced line broadening, which starts near pH 8.5. Between pH 11.5 and pH 12 the epsilon-CH2 resonances recover their low pH line width. Titration curves for the lysines and Tyr-20 reflect single proton ionization equilibria, suggesting that these residues do not interact among themselves. On the basis of double resonance experiments, combined with analysis of chemical shifts, spin-spin couplings, and line widths, all methyl resonances are identified and followed as functions of pH and temperature. The gamma-CH3 doublet from the N-terminal Thr-1 is assigned by comparison between spectra of forms I and II of the inhibitor, the latter lacking the first four residues of form I. The beta-CH3 resonance from Ala-7 is also assigned. Proton resonance parameters of methyl groups are shown to afford useful NMR probes for the characterization of local nonbonded interactions, microenvironments, and mobilities.

The values of pre-steady-state and steady-state parameters for the beta-trypsin catalyzed hydrolysis of Z-Arg-ONp and Z-Lys-ONp are superimposable between pH 2.4 and 8. At variance, the kinetic parameters for the beta-kallikrein-B catalyzed hydrolysis of Z-Arg-ONp are more favourable than those observed for Z-Lys-ONp and depend on different pKa values. The different primary specificity and the catalytic behaviour of beta-trypsin and beta-kallikrein-B reflect structural differences at their S1 subsite, especially at level of the 226 residue as well as the 217-220 segment.

The catalytic properties of bovine tyrpsin (EC 3.4.21.4) have been investigated using a synthetic chromogenic substrate: alpha-CBZ-L-lysine-p-nitrophenyl ester (ZLNPE). The use of ZLNPE allows the determination of trypsin down to a concentration of 2 . 10(-9) M. Steady-state and pre-steady-state data have been in the framework of the minimum three-step mechanism: (Formula: see text). The pH-dependence of the kinetic parameters shows that at acid pH values (congruent to 2.6) the k+3 step is rate limiting in catalysis, whereas for pH values higher than 4.8 k+2 becomes rate limiting. This change in rate-limiting step with pH illustrates the danger in the assumption that kcat vs. pH profiles for protease action on substrates with good leaving groups are equivalent to k+3 vs. pH profiles.

Ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids (namely, glycine, alanine, valine, leucine and phenylalanine) were synthesized and their inhibitory effect on the bovine trypsin and porcine pancreatic kallikrein catalyzed hydrolysis of p-nitroanilides of amino acids was investigated, at pH 8.1 and 37 degrees, in parallel with the effect of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids and benzamidine. For both proteinases, the inhibitory effect of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids is independent of the aminoacidic side chain, is closely similar to that of benzamidine (which binds at the S1 subsite of the proteinases examined and is commonly taken as a molecular inhibitor-model), and is higher by at least 10-fold than that of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids (depending on the aminoacidic residue). On structural grounds, the peculiar inhibitory behaviour of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids has been related to the interaction of the positively charged substituent at the N-position with the Asp189 residue present in the primary specificity subsite (S1) of bovine trypsin and porcine pancreatic kallikrein. The consistently lower affinity for porcine pancreatic kallikrein of all the inhibitors considered, as compared to bovine trypsin, may be related to the marked structural differences of the primary specificity subsite of these two serine proteinases.

The inhibitory effect of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H), TAPP-halo derivatives (with Cl, Br or I) and benzamidine on the Ancrod, bovine Factor Xa, and human plasmin catalyzed hydrolysis of esters and anilides of amino acids was investigated, at pH 8.1 and 37 degrees, and compared with that shown from these compounds on bovine thrombin and porcine pancreatic kallikrein catalysis. The inhibitory effect of TAPP-H and TAPP-halo derivatives on the proteinases considered, involved, to different extents, in blood coagulation and clot lysis, is higher by at least 10-fold, than that of benzamidine, which binds at the primary specificity subsite (S1) of serine endopeptidases and is commonly taken as a molecular inhibitor model. The high inhibitory effect of aromatic tetra-amidines has been interpreted taking into account an additional productive binding for a second benzamidine or halo-benzamidine moiety to the enzyme surface. Moreover, the data reported here allowed us to clarify the inhibition mechanism (in vitro) of TAPP-H on blood coagulation, induced by the "cancer coagulation factor" produced by the Walker carcinoma in Wistar rats and on the fibrinogen-to-fibrin conversion, and to identify some serine proteinases which act as targets for aromatic tetra-amidines.