Kinetic analysis of interactions between bispecific monoclonal antibodies and immobilized antigens using a resonant mirror biosensor

A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen–antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (K_ass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application.     

Electroconvulsive therapy in adolescent and adult psychiatric inpatients--a retrospective chart design

BACKGROUND: The knowledge available on electroconvulsive therapy (ECT) in adolescents is largely anecdotal, or based on findings from adults. The aim of the present study is to compare the use of ECT in adolescent and adult inpatients. METHODS: We retrospectively analyzed the files of all 36 adolescent (between the ages of 13 and 19) and 57 randomly selected adult inpatients (above the age of 20) treated with ECT in a university-affiliated mental heath center in Israel between 1991 and 1997. RESULTS: Sixty one percent of the adolescents improved by the end of treatment, and 53% were not hospitalized in the subsequent year. The respective percentages among adults were 83% and 49%. Whereas most adults were treated with ECT because of schizophrenic disorders, almost half of the adolescents received ECT for affective disorders. Significantly more adolescents were treated with ECT because of acute life-endangering conditions (catatonia or severe suicidal risk). No significant adverse effects were found in both groups. LIMITATIONS: Our study is based on a retrospective chart review. The adolescent and adult groups are different in psychiatric morbidity, diagnosis and outcome, have not been assessed in a blind manner, and we have not used standardized psychometric batteries for the evaluation of ECT-related memory disturbances. CONCLUSIONS: ECT may be an effective, well-tolerated and safe procedure in both adult and adolescent inpatients.     

A 10-Mb paracentric inversion of chromosome arm 2p inactivates MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred

Genomic deletions of the MSH2 gene are a frequent cause of hereditary nonpolyposis colorectal cancer (HNPCC), a common hereditary predisposition to the development of tumors in several organs including the gastrointestinal and urinary tracts and endometrium. The mutation spectrum at the MSH2 gene is extremely heterogeneous because it includes nonsense and missense point mutations, small insertions and deletions leading to frameshifts, and larger genomic deletions, the latter representing approximately 25% of the total mutation burden. Here, we report the identification and molecular characterization of the first paracentric inversion of the MSH2 locus known to cause HNPCC. Southern blot analysis and inverse PCR showed that the centromeric and telomeric breakpoints of the paracentric inversion map within intron 7 and to a contig 10 Mb 3' of MSH2, respectively. Pathogenicity of the paracentric inversion was demonstrated by conversion analysis. The patient's lymphocytes were employed to generate somatic cell hybrids to analyze the expression of the inverted MSH2 allele in an Msh2-deficient rodent cellular background. The inversion was shown to abolish MSH2 expression by both northern and western analysis. This study confirms that Southern blot analysis still represents a useful and informative tool to screen for and identify complex genomic rearrangements in HNPCC. Moreover, monoallelic expression analysis represents an attractive approach to demonstrate pathogenicity of unusual mutations in autosomal dominant hereditary conditions.     

Genetic and Environmental Determinants of Variation of Soluble Adhesion Molecules

In our research we examined the contribution of putative genetic sources on interindividual variation and cross-sectional correlations of several adhesion molecules, including intracellular (ICAM-1) and vascular cell adhesion molecules (VCAM-1) and E-selectin, in a population-based sample of ethnically homogeneous families of European origin. The plasma levels of these molecules were measured in 947 apparently healthy individuals from 217 nuclear families. Quantitative statistical-genetic analysis implementing the model fitting technique revealed significant parent/offspring and sibling correlations (p < 0.01) for all three molecules. The putative genetic effects explained 55.2 ± 7.2% (VCAM-1), 63.3 ± 7.5% (ICAM) and 63.8 ± 8.1% (E-selectin) of the variation. Common family environmental factors also significantly influenced the variation of E-selectin (13%) and VCAM-1 (28.6%). The main results of our bivariate analysis showed that the observed phenotypic correlations between ICAM-1 and VCAM-1, and between ICAM-1 and E-selectin, were mostly attributable to shared environmental factors (  r_E= 0.896  and 0.737, respectively; p < 0.01). However, the correlation between VCAM-1 and E-selectin was likely caused by common genetic effects  (r_G= 0.334, p < 0.05)  . Our results show that familial clustering of adhesion molecules is likely due to strong genetic effects, supplemented with shared environmental factors.     

DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus

Nonstructural protein 3 (NS3) of human hepatitis C virus (HCV) is a conserved multi-functional protein essential for replication and translation of viral RNA and polyprotein processing. Early T-cell response against NS3 is capable of restricting viremia. We aimed at characterizing the immunogenicity in gene immunization of the conserved regions of NS3 critical for protein folding and activity. C57BL/6 mice were injected with NS3 gene of Russian HCV 1b isolate 274933RU. Immunization did not exert any overt histological changes and had no long-term effects on the immune status of NS3 gene-recipients. The immune response in NS3 gene-recipients was screened by antibody ELISA, T-cell proliferation test and immune assays for specific cytokine production. T-lymphocytes of NS3 gene-recipients proliferated in response to peptides representing conserved regions of protease and ATPase/helicase. Stimulated T-lymphocytes produced IL-2, and in response to protease-derived peptides, also IFN-gamma. Potent and long-lasting antibody response was raised against conserved NS3 regions including "Greek-key" motif of protease, motifs II, V and polynucleotide-binding domains of ATPase/helicase. Thus, gene immunization effectively targeted conserved regions critical for NS3 protease and helicase function. In type and specificity, immune response of NS3 gene-immunized mice mimicked immunity achieved in the acute self-limiting HCV infection of human and primates and in virus-exposed healthy individuals, indicating promiscuity of NS3 as immunogen.     

The Mechanisms of the Anti-Inflammatory and Anti-Apoptotic Effects of Omega-3 Polyunsaturated Fatty Acids during Methotrexate-Induced Intestinal Damage in Cell Line and in a Rat Model

Background: The aim of this study was to examine the anti-inflammatory and anti-apoptotic patterns of omega-3 polyunsaturated fatty acids (n-3 PUFAs) during methotrexate (MTX) induced intestinal damage in cell culture and in a rat model. Methods: Non-treated and treated with MTX HT 29 and HCT116cells were exposed to increasing doses of n-3 PUFAs and cell viability was evaluated using PrestoBlue^® assay. Male Sprague-Dawley rats were divided into 4 experimental groups: Control rats, CONTR+n-3 PUFA rats that were treated with oral n-3 PUFA, MTX rats were treated with MTX given IP, and MTX+n-3 PUFA rats were treated with oral n-3 PUFA before and following injection of MTX. Intestinal mucosal parameters and mucosal inflammation, enterocyte proliferation and apoptosis, TNF-α in mucosal tissue and plasma (ELISA), NF-κB, COX-2, TNF-α, Fas, FasL, Fadd, Bid, Bax and Bcl-2gene and protein levels were determined 72 h following MTX injection. Results: Exposure of HT 29 and HCT116cells to n-3 PUFA attenuated inhibiting effects of MTX on cell viability. MTX-n-3 PUFA rats demonstrated a lower intestinal injury score and enhanced intestinal repair. A significant decrease in enterocyte apoptosis in MTX+n-3 PUFA rats was accompanied by decreased TNF-α, FAS, FasL, FADD and BID mRNA levels. Decreased NF-κB, COX-2 and TNF-α levels in mucosa was accompanied by a decreased number of IELs and macrophages. Conclusions: n-3 PUFAs inhibit NF-κB/COX-2 induced production of pro-inflammatory cytokines and inhibit cell apoptosis mainly by extrinsic pathway in rats with MTX-induced intestinal damage.     

Adherence to COVID-19 Nutrition Guidelines Is Associated with Better Nutritional Management Behaviors of Hospitalized COVID-19 Patients

Good nutritional support is crucial for the immune system to fight against coronavirus disease 2019 (COVID-19). However, in the context of a pandemic with a highly transmissible coronavirus, implementation of nutrition practice may be difficult. A multicenter electronic survey involving 62 dieticians was conducted, in order to understand barriers associated with dieticians’ adherence to nutrition guidelines for hospitalized COVID-19 patients in Indonesia. 69% of dieticians felt under stress when performing nutrition care, and 90% took supplements to boost their own immunity against the coronavirus. The concerns related to clinical practice included a lack of clear guidelines (74%), a lack of access to medical records (55%), inadequate experience or knowledge (48%), and a lack of self-efficacy/confidence (29%) in performing nutritional care. Half (52%) of the dieticians had performed nutrition education/counseling, 47% had monitored a patient’s body weight, and 76% had monitored a patient’s dietary intake. An adjusted linear regression showed that guideline adherence independently predicted the dieticians’ nutrition care behaviors of nutrition counselling (ß: 0.24 (0.002, 0.08); p = 0.04), and monitoring of body weight (ß: 0.43 (0.04, 0.11); p = 0.001) and dietary intake (ß: 0.47(0.03, 0.10); p = 0.001) of COVID-19 patients. Overall, adherence to COVID-19 nutrition guidelines is associated with better nutritional management behaviors in hospitalized COVID-19 patients.     

Twenty-Four-Hour Ambulatory Pulse Wave Analysis in Hypertension Management: Current Evidence and Perspectives

The predictive value of vascular biomarkers such as pulse wave velocity (PWV), central arterial pressure (CAP), and augmentation index (AIx), obtained through pulse wave analysis (PWA) in resting conditions, has been documented in a variety of patient groups and populations. This allowed to make appropriate recommendations in clinical practice guidelines of several scientific societies. Due to advances in technologies, largely operator-independent methods are currently available for estimating vascular biomarkers also in ambulatory conditions, over the 24 h. According to the acceptable accuracy and reproducibility of 24-h ambulatory PWA, it appears to be a promising tool for evaluating vascular biomarkers in daily life conditions. This approach may provide an opportunity to further improve the early cardiovascular screening in subjects at risk. However, concerning the clinical use of PWA over the 24 h in ambulatory conditions at the moment, there is no sufficient evidence to support its routine clinical use. In particular, long-term outcome studies are needed to show the predictive value of 24-h PWV, CAP, and AIx values, provided by these devices, over and beyond peripheral blood pressure, and to answer the many technical and clinical questions still open. To this regard, the VASOTENS Registry, an international observational prospective study recently started, will help providing answers on a large sample of hypertensive patients recruited worldwide.     

Hardening of Additive Manufactured 316L Stainless Steel by Using Bimodal Powder Containing Nanoscale Fraction

The particle size distribution significantly affects the material properties of the additively manufactured parts. In this work, the influence of bimodal powder containing nano- and micro-scale particles on microstructure and materials properties is studied. Moreover, to study the effect of the protective atmosphere, the test samples were additively manufactured from 316L stainless steel powder in argon and nitrogen. The samples fabricated from the bimodal powder demonstrate a finer subgrain structure, regardless of protective atmospheres and an increase in the Vickers microhardness, which is in accordance with the Hall-Petch relation. The porosity analysis revealed the deterioration in the quality of as-built parts due to the poor powder flowability. The surface roughness of fabricated samples was the same regardless of the powder feedstock materials used and protective atmospheres. The results suggest that the improvement of mechanical properties is achieved by adding a nano-dispersed fraction, which dramatically increases the total surface area, thereby contributing to the nitrogen absorption by the material.