Solid-phase Assay Research Articles

Thrombosis in Antiphospholipid Syndrome: Current Perspectives and Challenges in Laboratory Testing for Antiphospholipid Antibodies.

Antiphospholipid syndrome (APS) diagnosis hinges on identifying antiphospholipid antibodies (aPL). Currently, laboratory testing encompasses lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2-glycoprotein I antibodies (aβ2GPI) IgG or IgM, which are included in the APS classification criteria. All the assays needed to detect aPL antibodies have methodological concerns. LA testing remains challenging due to its complexity and susceptibility to interference from anticoagulant therapy. Solid phase assays for aCL and aβ2GPI exhibit discrepancies between different assays. Antibody profiles aid in identifying the patients at risk for thrombosis through integrated interpretation of all positive aPL tests. Antibodies targeting domain I of β2-glycoprotein and antiphosphatidylserine-prothrombin antibodies have been evaluated for their role in thrombotic APS but are not yet included in the APS criteria. Detecting these antibodies may help patients with incomplete antibody profiles and stratify the risk of APS patients. The added diagnostic value of other methodologies and measurements of other APS-associated antibodies are inconsistent. This manuscript describes laboratory parameters useful in the diagnosis of thrombotic APS and will concentrate on the laboratory aspects, clinical significance of assays, and interpretation of aPL results in the diagnosis of thrombotic APS.

Challenges in laboratory testing of patients suspected of antiphospholipid syndrome: practical implications for clinicians.

This paper focuses on the laboratory tests necessary for the diagnosis of antiphospholipid syndrome (APS). Diagnosis starts with the selection of patients suspicious of having APS. The timing related to the clinical event is important to avoid false classification of APS patients. For the interpretation of the test results of antiphospholipid antibodies (aPL) understanding of all pitfalls and interferences is necessary. Lupus anticoagulant (LA) measurement remains a complex procedure with many pitfalls and interferences. The effect of anticoagulant therapy, the main confounder of LA measurement, can be overcome by removal agents for direct oral anticoagulants (DOAC) or by considering assays as Taipan snake venom time / Ecarin clotting time not affected by antivitamin K therapy and anti-Xa DOAC. However, both procedures have limitations. Solid-phase assays for anticardiolipin antibodies (aCL) and anti-β2-glycoprotein 1 antibodies (aβ2GPI) show inter-assay differences. Diagnosis is based on the measurement of three groups of aPL: LA, aCL and aβ2GPI, of IgG and IgM isotype. This allows to make antibody profiles that helps in identifying patients at risk. Other aPL, such as antibodies against the domain I of β2GPI and anti-phosphatidylserine-prothrombin antibodies may be useful in risk stratification of APS patients and in some specific situations of patients with incomplete antibody profile, but are not needed for diagnosis. Laboratory diagnosis of APS remains challenging. To increase the diagnostic efficacy and reliability, an integrated interpretation of all results and an interpretative comment should be provided on the laboratory report. Therefore, a close interaction between clinical pathologists and clinicians is mandatory.

The sunflower jacalin Helja: biological and structural insights of its antifungal activity against Candida albicans.

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.

Different roles of the heterodimer architecture of galectin-4 in selective recognition of oligosaccharides and lipopolysaccharides having ABH antigens

The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.

Monoclonal Antibody chP3R99 Reduces Subendothelial Retention of Atherogenic Lipoproteins in Insulin-Resistant Rats: Acute Treatment Versus Long-Term Protection as an Idiotypic Vaccine for Atherosclerosis.

Atherosclerosis is triggered by the retention of apolipoprotein B-containing lipoproteins by proteoglycans. In addition to low-density lipoprotein, remnant lipoproteins have emerged as pivotal contributors to this pathology, particularly in the context of insulin resistance and diabetes. We have previously reported antiatherogenic properties of a monoclonal antibody (chP3R99) that recognizes sulfated glycosaminoglycans on arterial proteoglycans. Solid-phase assays demonstrated that chP3R99 effectively blocked >50% lipoprotein binding to chondroitin sulfate and vascular extracellular matrix invitro. The preperfusion of chP3R99 (competitive effect) resulted in specific antibody-arterial accumulation and reduced fluorescent lipoprotein retention by ~60% in insulin resistant JCR:LA-cp rats. This competitive reduction was dose dependent (25-250 μg/mL), effectively decreasing deposition of cholesterol associated with lipoproteins. In a 5-week vaccination study in insulin resistant rats with (200 μg subcutaneously, once a week), chP3R99 reduced arterial lipoprotein retention, and was associated with the production of antichondroitin sulfate antibodies (Ab3) able to accumulate in the arteries (dot-blot). Neither the intravenous inoculation of chP3R99 (4.5 mg/kg), nor the immunization with this antibody displayed adverse effects on lipid or glucose metabolism, insulin resistance, liver function, blood cell indices, or inflammation pathways in JCR:LA-cp rats. Both acute (passive) and long-term administration (idiotypic cascade) of chP3R99 antibody reduced low-density lipoprotein and remnant lipoprotein interaction with proteoglycans in an insulin-resistant setting. These findings support the innovative approach of targeting proatherogenic lipoprotein retention by chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis.

Pharmacological correction of immunological disorders in perimenopausal women with recurrent epithelial cervical dysplasia associated with human papillomavirus infection

Despite the successes achieved in implementing screening programs and treating diseases caused by human papillomavirus (HPV) infection, the frequency of cervical intraepithelial neoplasia (CIN) of moderate and high grades associated with high-risk human papillomavirus (HR-HPV) remains between 9% to 21%. Vaginal dysbiosis and the development of immunodeficient states due to persistent HR-HPV infection require effective immunomodulatory and antiviral therapy before cervical surgery in perimenopausal women with recurrent CIN II.The objective: to evaluate the effectiveness of antiviral and immunomodulatory therapy by determining the state of vaginal biocenosis, immunological status and papillomavirus load of the cervical mucosa before surgical treatment in perimenopausal women with recurrent CIN II.Materials and methods. A prospective study was conducted on 98 women aged 40 to 55 years old, including 60 patients with morphologically verified recurrent CIN II associated with high-risk HPV, forming the main group. The control group comprised 38 women with negative HPV test results and without significant gynecological or somatic pathology. In the main group, a complex consisting of a blend of plant extracts of sod pike grass (Deschampsia caespitosa), ground cinquefoil grass (Calamagrostis epigeios), and echinacea purpurea grass (Echinaceae purpureae) was administered orally.Vaginal microbiota was assessed using Hey-Іson criteria, and immunological status was examined in patients using solid-phase enzyme-linked immunosorbent assay to determine IgG, IgM, IgA antibodies in serum and cervical mucus, levels of interleukin-1β (IL-1β), interferons alpha (IFN-α), gamma (IFN-γ), tumor necrosis factor alpha (TNF-α). Additionally, the DNA of high-risk HPV in cervical mucosa was quantitatively assessed using real-time PCR. The investigations were conducted before and after the applied therapy.Results. The investigation of vaginal content revealed the presence of bacterial vaginosis in 65.0% of patients in the main group, the frequency of which decreased by 2 times after treatment, with the number of women with an intermediate type of vaginal content increasing by 2.8 times (51.7%) and those with normal microbiota increasing by 2.5 times. Disruptions in cellular and humoral immunity systems were identified. After treatment, there was an increase in viral load in the significant «threshold» category (3–5 lg) by 1.25 times and in the «above threshold for progression» category (>5 lg) by 2.3 times, reflecting natural HPV clearance. No direct correlation was found between viral load activity and the degree of CIN or positive morphological changes regarding regression of cervical intraepithelial lesions.Conclusions. The use of a complex containing a blend of plant extracts of sod pike grass (Deschampsia caespitosa), ground cinquefoil grass (Calamagrostis epigeios), and echinacea purpurea grass (Echinacea purpureae) improves the state of vaginal biocenosis, indicators of cellular and humoral immunity, and promotes HPV elimination, it has adequate safety and tolerability, which determines the feasibility of its use in the complex treatment of cervical intraepithelial neoplasia at the preparatory stage before surgery and in the postoperative period to prevent complications and relapses.

Clinical and Laboratory Diagnosis of Antiphospholipid Syndrome: A Review.

The antiphospholipid syndrome (APS) manifests through venous or arterial thrombosis, with or without pregnancy complication alongside the continuous presence of antiphospholipid antibodies (aPL). APS classification relies on three aPL subtypes: anticardiolipin (aCL), anti-β2-glycoprotein I antibodies (anti-β2GPI), and lupus anticoagulants (LA) antibodies. Given that thrombosis and pregnancy issues are not unique to APS, the precise and reliable identification of aPL forms the basis for diagnosis. Semi-quantitative solid-phase assays identify two antibodies, aCL and anti-β2GPI, while LA detection occurs through various phospholipid-dependent coagulation assays that are based on antibody behaviour. LA, specifically, is conclusively associated with thrombosis, prompting discussions around the serological criteria for APS. Despite advancements in LA detection, the standardisation of all aPL detection assays remains imperative. The combined presence of aCL and anti-β2GPI with thrombosis inconsistently triggers concern. Initial presentations by APS patients commonly exhibit a heightened risk of stroke, miscarriages in the later stages of pregnancy, positive results of LA tests, and widespread thrombosis across multiple organs, often leading to adverse outcomes. Correctly diagnosing this condition is pivotal to avoid unnecessary long-term secondary thromboprophylaxis.

#3036 The influence of first renal graft on second graft rejection and survival: the importance of revisiting histocompatibility and clinical outcomes

Abstract Background and Aims Kidney retransplantation confers a robust survival benefit over dialysis and recent data has shown similar outcomes to first kidney transplant. The number of HLA mismatches is an important predictor of graft loss and sensitization and the presence of donor specific antibodies (DSA) -pre and -post transplant is a major risk factor for rejection and worst outcomes. However, the importance of HLA matching of first graft and the relevance of first donor DSAs on second graft outcomes is less known. Our aim was to identify risk factors to second graft acute rejection and graft loss, related to second and first graft features. Method We performed a retrospective, longitudinal study including all patients submitted to a 2nd kidney transplant between January 2008 and December 2021, excluding patients with more than 2 grafts or multi-organ transplant. Clinical and histocompatibility data from the first and second donor and recipient were collected. HLA antibodies were detected by solid phase assays and considered if MFI >2500. For subsequent kidney transplants, repeated mismatches are not allowed and unacceptable antigens are those for which the patient has pre-formed -A, -B and -DR antibodies, with MFI>1000. Biopsy proven acute rejection was defined according to Banff 2017 criteria. Follow-up was 31st December 2023 for functioning grafts or time to graft failure, with a mean time of 92 ± 53 months. Results We included 114 patients, 73 (64%) males, mostly Caucasians (96%), with a mean age of 43.9 ± 13 years at 2nd transplant. First kidney transplant was performed before 2010 for 99 patients (87%), with a median first graft survival of 82.5 [12.5-144] months and 19 patients (16.7%) presented primary disfunction due to surgical/vascular complications. After graft loss, 43 patients (37.7%) had their graft removed. For second transplant, 11 (9.6%) patients had a living donor. Induction therapy with thymoglobulin occurred in 94 patients (83%) and maintenance therapy with mycophenolic acid, calcineurin inhibitors and steroids was prescribed in 83 patients (73%). During follow-up, 20 patients (17.5%) presented biopsy proven acute rejection, 13 (65%) T cell mediated and 7 (35%) antibody-mediated, the majority during the first-year post-transplant (N = 17, 85%). The risk factors for second graft rejection are summarized in Table 1. Patients with a first graft nephrectomy had a higher risk of acute rejection (OR 3.9, 95% CI [1.4-10.9], p < 0.006) and were more susceptible to the development of second donor post-transplant DSA (58% vs 35%, p = 0.02). Death censored second allograft loss occurred in 24 patients (21%) at follow up and acute rejection was an important risk factors (OR 4.3, 95% CI [1.5-12.1], p = 0.004). First-year graft survival was 93% and a survival at follow up was 79%. On survival analysis the presence of second graft DSA was a major risk factor for graft loss, especially class 2 -DQ and -DR. First transplant total mismatch load, especially on class 1 contributed to worst graft survival. Conclusion Worst outcomes in first kidney transplant were related with an increased risk of acute rejection in second graft and 1st graft nephrectomy increases the risk of the development of 2nd graft DSA and acute rejection. First transplant mismatch load decreased second graft survival.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAPEFLM Paper

<br><b>Aim:</b> Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA).</br> <br><b>Methods:</b> A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP).</br> <br><b>Results:</b> In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations.</br> <br><b>Conclusions:</b> These recommendations are an important step to achieve high quality ANA testing.</br>

The development of anticyclic citrullinated peptide (anti-CCP) antibody following severe COVID-19.

The dysregulated immune response is one of the cardinal features of severe coronavirus disease 2019 (COVID-19). This study was conducted to clarify the occurrence of autoantibodies (AABs) associated with systemic autoimmune rheumatic diseases (SARDs) in hospitalized patients with a moderate, severe, and critical form of COVID-19. The serum samples obtained from 176 hospitalized COVID-19 patients were investigated in this study, including patients with moderate (N = 90), severe (N = 50), and critical (N = 36) forms of COVID-19. Also, the serum samples collected from healthy subjects before the COVID-19 pandemic were used as controls (N = 176). The antinuclear antibodies (ANAs), antidouble-stranded DNA (anti-dsDNA), cytoplasmic-anti neutrophil cytoplasmic antibody (c-ANCA), perinuclear ANCA (p-ANCA), antiphospholipid antibodies (aPLs), and anticyclic citrullinated peptide (anti-CCP) occurrence was evaluated using a solid-phase enzyme-linked immunosorbent assay (ELISA). The results showed that the occurrence of ANAs, anti-dsDNA, anti-CCP, c-ANCA, and p-ANCA was significantly higher in the COVID-19 patients compared to serum obtained from healthy subjects (p < .0001, p < .0001, p < .0001, p < .05, and p < .001, respectively). The positive number of anti-CCP tests increased significantly in severe COVID-19 compared to the moderate group (p < .01). Our study further supports the development of autoantibodies related to systemic autoimmune rheumatologic diseases. To the best of our knowledge, this is the first study with a large sample size that reported the occurrence of anti-CCP in a severe form of COVID-19.

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1.

Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥.

Abstract 6716: DLL3 TriTCE Co-Stim: A next generation trispecific T cell engager with integrated CD28 costimulation for the treatment of DLL3-expressing cancers

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with a poor prognosis and high unmet medical need. Current standards of care, including chemotherapy, targeted therapy and immunotherapy, are associated with limited duration of response. Furthermore, SCLC is characterized by an immunosuppressive microenvironment and poor T cell infiltration which present additional treatment challenges. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is aberrantly overexpressed in SCLC and other neuroendocrine tumors, but minimally expressed in normal tissues, and is a new therapeutic target. Bispecific T cell engagers (TCE) targeting DLL3 have entered the clinic and demonstrated promising anti-tumor activity in SCLC patients; however, we believe there is room to improve on the rate and depth of response. To address treatment challenges and enhance the durability and sustainability of T cell activation, we have engineered a trispecific co-stimulatory T cell engager (TriTCE Co-stim) molecule that optimally engages CD3 and CD28 and redirects and enhances cytotoxic T cell responses to DLL3-expressing tumor cells while maintaining a desired safety profile. A panel of DLL3xCD3xCD28 trispecific antibodies (Abs) was engineered with a conventional anti-CD28 agonist paratope in a variety of Ab formats, geometries, paratope affinities and specificities. Geometry and design features of the tri-specific Abs were informed by prior TriTCE Co-stim molecule optimization1. AzymetricTM and EFECTTM platforms were used to facilitate heterodimeric multi-chain antibody assembly with silenced Fc gamma function. Lead TriTCE Co-stim Abs were selected for enhanced DLL3-dependent antitumor cytotoxic activity and increased T cell function including T cell activation, proliferation and cytokine production, compared to competitor bispecific TCEs. DLL3-independent activity and cytokine release were assessed with human PBMCs in solid and solution phase assays to evaluate the potential for cytokine release induction. In summary, we have identified multiple TriTCE Co-Stim Ab formats with improved DLL3-dependent cytotoxicity and T cell activation over competitor bispecific TCEs across multiple DLL3-expressing tumors. By screening various Ab formats, geometries and paratope affinities, we have selected lead DLL3 TriTCE Co-stim Abs displaying target-dependent activation of T cells with potent anti-tumor activity which may translate to improved and more durable antitumor responses in the treatment DLL3-expressing cancers. Citation Format: Peter Repenning, Desmond Lau, Diana C. Hernaez, Alec Robinson, Diego P. Escanda, Mariana Rocha, Begonia S. Moreno, John Zhang, Polly Shao, Nichole Escalante, Lisa Newhook, Purva Bhojane, Chayne L. Piscitelli, Paul A. Moore, Thomas Spreter Von Kreudenstein, Nina E. Weisser. DLL3 TriTCE Co-Stim: A next generation trispecific T cell engager with integrated CD28 costimulation for the treatment of DLL3-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6716.

THE INFLUENCE OF ADJUVANTS ON THE EXPRESSION OF PRO-INFLAMMATORY CYTOKINES DURING SPINAL ANESTHESIA

Objective. To investigate the influence of adjuvants (fentanyl and dexmedetomidine) on TNF-α and IL-6 expression during spinal anesthesia based on bupivacaine.&#x0D; Materials and Methods. A total of 150 patients with an average age of 41.3±8.2 years undergoing endoscopic biportal discectomy were examined. Patients were divided into groups: Group 1 received intrathecally 2.5 ml hyperbaric bupivacaine; Group 2 received intrathecally 2.5 ml hyperbaric bupivacaine and 25 μg fentanyl; Group 3 received intrathecally 2.5 ml hyperbaric bupivacaine and 5 μg dexmedetomidine. Peripheral venous blood was collected 1 hour before surgery, at 12 and 24 hours postoperatively, to measure TNF-α and IL-6 levels through solid-phase enzyme-linked immunosorbent assay. Results. The expression of pro-inflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6)) may play a crucial role in modulating postoperative inflammatory reactions. However, contemporary scientific literature presents conflicting data on the influence of anesthetics on cytokine release.&#x0D; Groups 1 and 2 showed a significant increase in TNF-α levels at all observation stages. In Group 3, a significant elevation in TNF-α was observed only 12 hours postoperatively, with no statistically significant difference in the subsequent 24 hours. Inter-group analysis of TNF-α dynamics revealed the smallest increase in Group 3, while Group 1 had significantly higher values compared to Group 3 and Group 2. In all study groups, there was a progressive, statistically significant increase in IL-6 levels within 24 hours postoperatively. However, Group 1 exhibited consistently higher values, with statistically significant differences compared to Group 2 and Group 3. In Groups 2 and 3, statistically significant inter-group differences in IL-6 were noted 12 hours postoperatively, but there was no statistical significance in IL-6 dynamics 24 hours postoperatively.&#x0D; Conclusion. Intrathecal use of bupivacaine with dexmedetomidine had a lesser impact on postoperative TNF-α and IL-6 expression during endoscopic biportal discectomy.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Utility of insulin-like growth factor-1 (IGF-1) in growth hormone-treated short children – An experience from a tertiary level center

Introduction: Very few studies have been conducted in low-middle-income countries to assess the response to growth hormone (GH) therapy by measuring the improvement in insulin-like growth factor-1 (IGF-1) concentrations. Objectives: The objective is to assess IGF-1 concentrations in patients receiving GH therapy to correlate the height increment with respect to increase in the IGF-1 concentrations, including IGF-1 Z-scores as a measure of safety profile. Methods: Clinical and anthropometric data were extracted retrospectively from hospital records of children aged 0–18 who received GH and had IGF-1 measured during the study period. Serum IGF-1 concentrations were analyzed by a solid-phase enzyme-linked immunosorbent assay. Patients with GH deficiency (GHD) and multiple pituitary hormone deficiency (MPHD) were grouped as group A, while participants receiving GH therapy for non-GHD/MPHD were clubbed as group B. Results: We report a significant positive correlation between an increase in IGF-1 values and increase in height. The improvement in height Z-scores was significantly higher in participants of group A as compared to B, with no significant difference in increase in IGF-1 Z-scores between the two groups. A total of 18.75% of subjects had IGF-1 Z-scores between +2 and +3, and these were largely subjects belonging to group B. Conclusion: The increment in height and height velocity Z-scores in the patients on GH correlated with increase in the IGF-1 concentrations, particularly in GHD/MPHD group of patients.

Characteristics of two zoonotic swine influenza A(H1N1) viruses isolated in Germany from diseased patients

Interspecies transmission of influenza A viruses (IAV) from pigs to humans is a concerning event as porcine IAV represent a reservoir of potentially pandemic IAV. We conducted a comprehensive analysis of two porcine A(H1N1)v viruses isolated from human cases by evaluating their genetic, antigenic and virological characteristics. The HA genes of those human isolates belonged to clades 1C.2.1 and 1C.2.2, respectively, of the A(H1N1) Eurasian avian-like swine influenza lineage. Antigenic profiling revealed substantial cross-reactivity between the two zoonotic H1N1 viruses and human A(H1N1)pdm09 virus and some swine viruses, but did not reveal cross-reactivity to H1N2 and earlier human seasonal A(H1N1) viruses. The solid-phase direct receptor binding assay analysis of both A(H1N1)v showed a predominant binding to α2–6–sialylated glycans similar to human-adapted IAV. Investigation of the replicative potential revealed that both A(H1N1)v viruses grow in human bronchial epithelial cells to similar high titers as the human A(H1N1)pdm09 virus. Cytokine induction was studied in human alveolar epithelial cells A549 and showed that both swine viruses isolated from human cases induced higher amounts of type I and type III IFN, as well as IL6 compared to a seasonal A(H1N1) or a A(H1N1)pdm09 virus. In summary, we demonstrate a remarkable adaptation of both zoonotic viruses to propagate in human cells. Our data emphasize the needs for continuous monitoring of people and regions at increased risk of such trans-species transmissions, as well as systematic studies to quantify the frequency of these events and to identify viral molecular determinants enhancing the zoonotic potential of porcine IAV.

Methodological aspects of anti-nuclear antibodies detection: EFLM, EASI, ICAP AND RAMLD recommendations

This is a review of international and Russian recommendation for the study of anti-nuclear antibodies (ANA) in autoimmune inflammatory rheumatic diseases (AIRD) and autoimmune liver diseases (ALD), including a description of the most important methodological aspects. The main purpose of laboratory diagnostics of AIRD and ALD is to obtain objective information about the presence and immunopathological changes, which is an important tool for early diagnosis, assessment of activity, severity, prognosis of the disease and the effectiveness of therapy. The positive results of ANA determination are the main laboratory markers of AIRD and ALD, being among the diagnostic criteria for diseases. The ‘gold standard’ and primary screening method for determining ANA in serum is the indirect immunofluorescence assay (IFA). Antigen-specific solid phase assays methods are used as confirmatory tests. Standardization of the ANA determination contributes for reducing the intra- and inter-laboratory variability of the results, helps to optimize the interaction between laboratory specialists and clinicians in matters of prescribing and clinical interpretation of ANA tests. Solving the problem of ANA detection standardization is important because of the growing number of laboratories performing these tests and an increased referring for this investigation from rheumatologists and another medical specialist.

Dynamic roles of neutrophil extracellular traps in cancer cell adhesion and activation of Notch 1-mediated epithelial-to-mesenchymal transition in EGFR-driven lung cancer cells.

Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils that may modulate different steps of the metastatic cascade. The aim of our study was to investigate how NETs can modulate the adhesion properties of cancer cells and whether cell exposure to NETs can activate the epithelial-to-mesenchymal transition (EMT) program thus enhancing the migratory and invasive properties of tumor cells. Different cancer cell lines were subjected to a solid-phase adhesion assay using NET-coated plates with or without the addition of antibodies against α5β1 or CCDC25 receptor. After 1-4 h of incubation, adherent cells were expressed as the percentage of total cell number. To test EMT occurrence, cells were treated with NETs for up to 48 h and then the levels of E-cadherin, vimentin, Snail, Slug, Zeb 1 and Twist 1 along with levels of Notch 1 and cleaved Notch 1 were determined by western blotting. Untreated and NET-treated cells were subjected to migration assays using 24-multiwell plates with transwell and FBS as chemoattractant. Cancer cell adhesion to NET-coated plates varied between 30% and 92.7% and was significantly higher than that obtained in uncoated plates. The addition of antibodies against α5β1 or CCDC25 caused a strong reduction of cell adhesion to NETs. The prolonged exposure of EGFR-driven cancer cell lines to NETs caused the activation of the EMT program through the upregulation and cleavage of Notch 1 and was confirmed by the enhanced expression of EMT markers. The consequent loss of the epithelial phenotype induced a strong reduction of the expression of the oncogene driver. Cell migration was significantly enhanced in NET-treated cells as compared to untreated cells. Our findings reveal the dynamic role of NETs that may provide a DNA and fibronectin rich environment for binding of many cancer cells at distant sites where the prolonged exposure to NETs triggers the EMT through the activation of Notch 1 signaling pathway with the subsequent enhancement of migratory and invasive properties of cancer cells. Furthermore, our findings provide an example of how an immune/inflammatory microenvironment may directly modulate the sensitivity of cancer cells to oncogene targeted agents.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.