Recombinant Molecules Research Articles

3,3′-Diindolylmethane promotes bone formation – A assessment in MC3T3-E1 cells and zebrafish

Osteoporosis is a common degenerative bone disease in middle-aged and elderly people. The current drugs used to treat osteoporosis have many side effects and low patient compliance. Phytochemotherapy may be safer and more effective. 3,3′-diindolemethane (DIM) is the digestive product of indole-3-methanol in cruciferous vegetables in the stomach, which is a kind of anti-tumor and anti-oxidation phytochemical. However, the effects of DIM on osteoblasts and the mechanism by which DIM regulates bone formation are not fully understood. The aim of this study was to investigate the effects of DIM on the bone formation of mouse preosteoblasts MC3T3-E1 and zebrafish. DIM promotes proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro, and also plays a bone promoting role by increasing the interaction between BRCA1-Associated Protein 1(BAP1) and Inositol 1,4,5-Trisphosphate Receptor(IP3R), up-regulating the expression of BAP1 and IP3R and downstream storage operation calcium entry (SOCE) related protein Recombinant Stromal Interaction Molecule 1(STIM1). The effect of DIM on osteoporosis was confirmed in zebrafish osteoporosis model, and its molecular mechanism may be related to BAP1/IP3R/SOCE signaling pathway. These findings highlight the potential therapeutic value of DIM in the prevention and treatment of osteoporosis.

Therapeutic Controlled Release Strategies for Human Osteoarthritis.

Osteoarthritis is a progressive, irreversible debilitating whole joint disease that affects millions of people worldwide. Despite the availability of various options (non-pharmacological and pharmacological treatments and therapy, orthobiologics, and surgical interventions), none of them can definitively cure osteoarthritis in patients. Strategies based on the controlled release of therapeutic compounds via biocompatible materials may provide powerful tools to enhance the spatiotemporal delivery, expression, and activities of the candidate agents as a means to durably manage the pathological progression of osteoarthritis in the affected joints upon convenient intra-articular (injectable) delivery while reducing their clearance, dissemination, or side effects. The goal of this review is to describe the current knowledge and advancements of controlled release to treat osteoarthritis, from basic principles to applications in vivo using therapeutic recombinant molecules and drugs and more innovatively gene sequences, providing a degree of confidence to manage the disease in patients in a close future.

Development of a Limosilactobacillus reuteri therapeutic delivery platform with reduced colonization potential.

Bacterial biotherapeutic delivery vehicles have the potential to treat a variety of diseases. This approach obviates the need to purify the recombinant effector molecule, allows delivery of therapeutics in situ via oral or intranasal administration, and protects the effector molecule during gastrointestinal transit. Lactic acid bacteria have been broadly developed as therapeutic delivery vehicles though risks associated with the colonization of a genetically modified microorganism have so-far not been addressed. Here, we present an engineered Limosilactobacillus reuteri strain with reduced colonization potential. We applied a dual-recombineering scheme for efficient barcoding and generated mutants in genes encoding five previously characterized and four uncharacterized putative adhesins. Compared with the wild type, none of the mutants were reduced in their ability to survive gastrointestinal transit in mice. CmbA was identified as a key protein in L. reuteri adhesion to HT-29 and enteroid cells. The nonuple mutant, a single strain with all nine genes encoding adhesins inactivated, had reduced capacity to adhere to enteroid monolayers. The nonuple mutant producing murine IFN-β was equally effective as its wild-type counterpart in mitigating radiation toxicity in mice. Thus, this work established a novel therapeutic delivery platform that lays a foundation for its application in other microbial therapeutic delivery candidates and furthers the progress of the L. reuteri delivery system towards human use.IMPORTANCEOne major advantage to leverage gut microbes that have co-evolved with the vertebrate host is that evolution already has taken care of the difficult task to optimize survival within a complex ecosystem. The availability of the ecological niche will support colonization. However, long-term colonization of a recombinant microbe may not be desirable. Therefore, strategies need to be developed to overcome this potential safety concern. In this work, we developed a single strain in which we inactivated the encoding sortase, and eight genes encoding characterized/putative adhesins. Each individual mutant was characterized for growth and adhesion to epithelial cells. On enteroid cells, the nonuple mutant has a reduced adhesion potential compared with the wild-type strain. In a model of total-body irradiation, the nonuple strain engineered to release murine interferon-β performed comparable to a derivative of the wild-type strain that releases interferon-β. This work is an important step toward the application of recombinant L. reuteri in humans.

Optimizing bioprocessing efficiency with OptFed: Dynamic nonlinear modeling improves product-to-biomass yield

Biotechnological production of recombinant molecules relies heavily on fed-batch processes. However, as the cells' growth, substrate uptake, and production kinetics are often unclear, the fed-batches are frequently operated under sub-optimal conditions. Process design is based on simple feed profiles (e.g., constant or exponential), operator experience, and basic statistical tools (e.g., response surface methodology), which are unable to harvest the full potential of production.To address this challenge, we propose a general modeling framework, OptFed, which utilizes experimental data from non-optimal fed-batch processes to predict an optimal one. In detail, we assume that cell-specific rates depend on several state variables and their derivatives.Using measurements of bioreactor volume, biomass, and product, we fit the kinetic constants of ordinary differential equations. A regression model avoids overfitting by reducing the number of parameters. Thereafter, OptFed predicts optimal process conditions by solving an optimal control problem using orthogonal collocation and nonlinear programming.In a case study, we apply OptFed to a recombinant protein L fed-batch production process. We determine optimal controls for feed rate and reactor temperature to maximize the product-to-biomass yield and successfully validate our predictions experimentally. Notably, our framework outperforms RSM in both simulation and experiments, capturing an optimum previously missed. We improve the experimental product-to-biomass ratio by 19% and showcase OptFed's potential for enhancing process optimization in biotechnology.

Prospects for the use of viral proteins for the construction of chimeric toxins.

One of the actively developing areas of drug development is the creation of chimeric toxins, recombinant bifunctional molecules designed to affect target cells selectively. The prevalent approach involves fusing bacterial and plant toxins with molecules that facilitate targeted delivery. However, the therapeutic use of such toxins often encounters challenges associated with negative side effects. Concurrently, viruses encode proteins possessing toxin-like properties, exerting multiple effects on the vital activity of cells. In contrast to bacterial and plant toxins, the impact of viral proteins is typically milder, presenting a significant advantage by potentially reducing the likelihood of side effects. This review delineates the characteristics of extensively studied viral proteins with toxic and immunomodulatory properties and explores the prospects of incorporating them into chimeric toxins.

Mechanism-specific chemical energy accommodation with finite-rate surface chemistry in non-equilibrium flow

During atmospheric reentry, the vehicle surface is exposed to highly non-equilibrium flow. The vehicle surface can experience heterogeneous recombination of reactive atoms, which contributes to its aerothermodynamic heating. This process is followed by chemical energy accommodation (CEA), where the released energy is either transferred to the surface or the internal energy modes of the recombined molecule. Heterogeneous recombination can be categorized into Eley–Rideal (ER) and Langmuir–Hinshelwood mechanisms, which differ in their methods of molecule formation and degrees of CEA. The complete CEA assumption may not consider the dependency of CEA on the mechanisms of heterogeneous recombination. This study aims to consider the mechanism-specific CEA for a more accurate prediction of surface heat flux. The authors implement mechanism-specific CEA within the direct simulation Monte Carlo framework using the finite-rate surface chemistry model, resolving elementary surface reactions and assigning a CEA coefficient, β, to each mechanism. The model is verified through comparisons with analytical solutions of surface coverage and validated against benchmark references. A parametric investigation of rarefied hypersonic flow over a two-dimensional cylinder is conducted under different freestream Mach and Knudsen numbers. Results show a reduction in total heat flux of up to 14.44% using mechanism-specific CEA compared to the complete CEA assumption. The reduction is attributed to the relative contribution of the ER mechanism, which can be a function of atomic partial pressure at the boundary layer.

Differences in the direct effects of various type 2 cytokines on functions of blood eosinophils from healthy subjects.

Eosinophil inflammation often persists in the airways of severe asthmatics, even under treatment with high-dose inhaled corticosteroids. Biologics for various type 2 cytokines have been recently developed for corticosteroid-resistant, eosinophil-dominant, severe asthma. However, it is unclear whether these biologics act directly on eosinophils. In this study, we examined whether various type 2 cytokines targeted by biologics can directly modify the functions of eosinophils obtained from the peripheral blood of healthy individuals. Peripheral eosinophils of healthy subjects were purified by conventional negative-depletion methods using anti-CD16 beads to avoid the priming effect (i.e., stimulation in vitro) to the maximum extent possible. Eosinophils were stimulated with interleukin (IL)-4, IL-5, IL-13, or thymic stromal lymphopoietin (TSLP), and eosinophil adhesiveness to recombinant human-intercellular adhesion molecule (ICAM)-1 was evaluated by eosinophil peroxidase assays. The effect of these cytokines on eosinophil superoxide anion (O2 -) generation was evaluated by the superoxide dismutase-inhibitable reduction of cytochrome C. To determine whether eosinophil degranulation was induced, the concentration of eosinophil-derived neurotoxin (EDN) in the supernatant was measured using enzyme-linked immuno sorbent assay. As reported previously, at 100 pM, IL-5 increased eosinophil adhesiveness to ICAM-1, O2 - generation, and EDN release. Conversely, at concentrations up to 10 nM, IL-4, IL-13, and TSLP did not induce eosinophil adhesiveness, O2 - generation, or EDN release. Type 2 cytokines other than IL-5 do not directly affect the functions of eosinophils from healthy individuals when used at clinical concentrations. These findings suggest that eosinophils play little, or no, direct role in the effects of anti-IL-4 receptor α or anti-TSLP antibody on severe asthma.

Recombinant Protein Expression Optimizing: A Review of S/MAR, STAR, and UCOE, as a Chromatin-Modifying Element

The rapid growth of the global biopharmaceutical market in recent years indicates its importance in the biotechnology industry. The production of these medicinal products is one of the fastest-growing industries in the patient treatment field. Since 1980, processes have been developed to optimize the production of recombinant protein products upstream and downstream. In this regard, one promising approach is the engineering of expression vectors based on combinations of DNA regulatory elements found in euchromatin regions. This approach aims to achieve proper gene integration and facilitate its expression in the target cell, as epigenetic mechanisms can lead to instability of the desired gene in long-term cell cultures and gene silencing. To address this issue, genetically engineered vectors have been produced, which include components such as Matrix Attachment Regions (MARs), Scaffold Attachment Regions (SARs), Stabilizing Anti-Repressor Elements (STAREs), and Ubiquitous Chromatin Opening Elements (UCOE). These components can modify the chromatin environment to minimize gene silencing and enhance higher, more stable, and biologically active expression of recombinant molecules. In this review, we focus on different approaches and developments in the technology of expression vector engineering and their impact on increasing the production of recombinant proteins.

A New Perspective on the Biomineralization of Radicular Dentin

The biomineralization of radicular dentin involves complex molecular signaling. The exact mechanism of action remains elusive. However, the literature showed that protein binding sites for calcium ions and mineral precipitation are essential to promote the remineralization process. Dentin organic matrix possesses non-collagenous proteins, such as dentin matrix protein 1 (DMP1), that can initiate and regulate crystal nucleation. Recombinant DMP1 molecules have been thought to perform specific molecular recognition with the apatite surface, guiding calcium phosphate clusters during recruitment through the type I collagen matrix, which serves as a template for controlled biomineralization. The biological basis for the clinical benefits of this process has yet to be completely elucidated. From a clinical perspective, caries, acid erosion, or disinfection protocol treatments can cause dentin demineralization. Given this fact, dentin biomimetic remineralization strategies and treatment strategies aimed at preserving the dentin matrix have been proposed.

Phase I summary of the HB0025 efficacy and safety to the heavily pretreated non-small cell lung cancer (NSCLC) population.

e14600 Background: HB0025 is a recombinant humanized bispecific molecule built on IgG1 that targets the human programmed death ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF, VEGF-A, VEGF165). Here we present the NSCLC results in Phase 1 study. Methods: This ongoing, open-label, multicenter, dose escalation and dose expansion, phase 1 trial to evaluate the safety and efficacy of HB0025 for pts with advanced solid tumors. HB0025 is administered intravenously every 14 days (Q2W). Response was evaluated by RECIST v.1.1 every 8 weeks. Results: As of Dec 25, 2023, 12 (7M/5F) NSCLC pts have received HB0025 at doses of 3mg/kg (n=1), 6mg/kg (n=2), 10mg/kg (n=2), 12mg/kg (n=1), 20mg/kg (n=5), 30mg/kg (n=1). Overall, the median age was 61.5 years, the median number of prior treatment lines was 4. The histologic types include squamous cell (Sq)-NSCLC (2 pts) without EGFR/ALK mutation and non- squamous cell (Nonsq)-NSCLC (10 pts), among of Nonsq-NSCLC 9 pts carrying EGFR/ALK mutation. The 3 pts with EGFR/ALK wild-type gene fail to prior anti-PD-1 treatment, 9 pts with EGFR/ALK gene mutation fail to prior TKI therapy. Among the 12 pts having imaging tumor assessment, the ORR was 25% and DCR was 66.7%, which included 3 partial responses (PR) ,5 stable disease (SD), and 4 progressive disease (PD) according to RECIST v1.1.For the 2 Sq-NSCLC pts, one pt achieved PR with 3mg/kg HB0025 treatment, the other pt achieved SD. For the10 Nonsq-NSCLC pts, there were 2 PR (20mg/kg and 30mg/kg treatment, respectively), 4 SD and 4 PD. Till the report date, the PR status and HB0025 treatment are ongoing to 3 PR pts, which includes 1 Sq-NSCLC pt, and 2 Nonsq-NSCLC pts which one pt doesn’t carry EGFR/ALK mutation. The Sq-NSCLC pt whose maximum tumor regression is 60% achieves PR after 4 cycles of treatment, and the duration of response (DOR) is more than 22 months. The one Nonsq-NSCLC pt whose maximum tumor regression is 74% achieves PR after 2 cycles of treatment, and the DOR is more than 11 months. The other Nonsq-NSCLC pt whose tumor regression is 32% achieves PR after 4 cycles of treatment, and the DOR is more than 2 months. Among the 5 SD pts, 1 Sq-NSCLC pt’s progression-free survival (PFS) is more than 14 months and continusous, 2 Nonsq-NSCLC pts’ PFS is more than 6 months and ongoing. For the 12 pts, 11 pts (91.7%) experienced a treatment related adverse events (TRAEs). The most common TRAEs included proteinuria (50%, 6/12), lymphocyte count decrease (50%, 6/12), blood bilirubin increased (33.3%, 4/12), and blood pressure increased (33.3%, 4/12). TRAEs ≥ grade 3 occurred in 2 (16.7%) pts. No TRAE leading to discontinuation and death. Conclusions: HB0025 monotherapy showed an acceptable safety profile and promising anti-tumor activity for prior heavily treated NSCLC, no matter in TKI-resistant population with EGFR/ALK mutation or population failed to anti-PD-1 treatment. Now Phase II studies of combination chemotherapy are ongoing. Clinical trial information: NCT04678908 .

A phase 1/2 study to evaluate the efficacy and safety of HB0025 monotherapy for patients (pts) with advanced renal cell carcinoma (RCC).

e14503 Background: HB0025 is a recombinant humanized bispecific molecule built on IgG1 that targets the human programmed death ligand 1 (PD-L1) (CD274) and vascular endothelial growth factor (VEGF, VEGF-A, VEGF165). In phase Ia, HB0025 monotherapy showed encouraging anti-tumor activity and safety profile in solid tumor. Herein, we aimed to explore safety and efficacy of HB0025 in advanced renal cell carcinoma pts that failed standard treatments. Methods: This ongoing, open-label, multicenter, phase 1/2 trial to evaluate the efficacy and safety of HB0025 monotherapy with 10mg/kg iv q2w for RCC pts. Response was evaluated by RECIST v.1.1 every 8 weeks. The primary endpoint was objective response-rate (ORR). Secondary endpoints included safety (CTCAE v.5.0), disease control rate (DCR), and progression free survival (PFS). Results: As of Dec 25, 2023, 17 (16M/1F) pts were enrolled and all received treatment. Median age was 58 y and 53% pts had intermediate/poor IMDC risk.6 pts were on treatment at data cutoff date. All pts had stage IV disease. 14 (82%) pts were clear cell renal cell carcinoma and the maximum duration of treatment was 7 months. 10 (59%) pts received ≥2 lines of previous systemic therapies and median follow up was 5.1 months. Among total the 15 pts having imaging tumor assessment by RECIST v1.1, 2 achieved partial responses (PR) and 9 achieved stable disease (SD) in Best of Response.1 subject who had received 2-line treatment with multiple metastases (including lung, bones, liver and adrenal gland) had achieved PR since the first tumor assessment, target lesion decreased by up to 52.1% from baseline. 1 subject who had received 2-line treatment with lung and lymph node metastasis had achieved PR since the first tumor assessment, target lesion decreased by up to 40.3% from baseline. Both two patients, duration of treatment was over 6 months and still in PR status when discontinued treatment. The ORR was 13.3% (95%CI: 1.7-40.5) and DCR was 73.3% (95%CI: 44.9-92.2). Median PFS was not reached. For all enrolled pts, 17 pts (100%) experienced a treatment related adverse events (TRAE). The most common TRAEs were proteinuria (58.8%, 10/17), hypoalbuminemia 6/9 (35.3%, 6/17), hypercholesterolemia (35.3%, 6/17), hyperuricemia (29.4%, 5/17). TRAEs ≥ grade 3 occurred in 6 (35.5%) patients and no death occurred. TRAE leading to discontinuation occurred in 3 (17.7%) pts. Conclusions: HB0025 10mg/kg monotherapy showed an acceptable safety profile and promising anti-tumor activity in prior multiple treated RCC, which supports further studies to explore the safety and efficacy of HB0025 as a monotherapy or in combination with other anti-tumor agents. HB0025 20mg/kg monotherapy study are currently ongoing. Clinical trial information: NCT06222125 .

Multiplex Detection of Seven Staphylococcal Enterotoxins Using Liquid Chromatography–Mass Spectrometry Combined with a Novel Capture Molecule

Food poisoning caused by Staphylococcal enterotoxins (SEs) is prevalent globally, making efficient detection of these toxins very important. Traditionally, liquid chromatography–mass spectrometry required immunosorbent enrichment by magnetic bead-coupled antibodies obtained by animal-specific immunization. However, this method is time-consuming and costly. In this study, two recombinant protein capture molecules were designed based on the principle of toxins binding to Major Histocompatibility Complex (MHCII) and T cell receptor (TCR) molecules. The two capture molecules are called MHCII and MHCII-D10. The design of the MHCII and TCR-D10 was achieved through searching for the binding site protein sequence of Staphylococcal enterotoxins in the relevant literature, and MHCII-D10 was to link MHCII sequence with TCR-D10 sequence using linker (G4S)3 linking peptide. These capture molecules were shown to effectively bind to seven types of toxins and to capture SEs in various matrices. The digestion time, ratio, and temperature were further optimized, reducing the overall digestion time to just 2 h. The specificity, linearity, sensitivity, precision (RSD%), and recovery of the two methods were verified by liquid chromatography–mass spectrometry. When the MHCII and MHCII-D10 captured the toxins, the limit of quantification (LOD) in the 1 × PBS, plasma, and milk matrices ranged from 1.5625 to 100 fmol/µL, with the recovery rate ranging from 18.4% to 96%. The design of these capture molecules eliminates the need for animal-specific immunization, simplifying the pre-detection process and avoiding ethical concerns. This development holds significant promise for clinical diagnosis and reference.

Unlocking the Power of Molecular Cloning: Revolutionizing Medical Microbiology Procedures

The revolutionary realm of molecular cloning, encompassing the creation of recombinant DNA molecules, has ignited a wave of progress within the life sciences. The advent of potent tools has facilitated the manipulation of DNA, resulting in an extraordinary surge in the versatility and breadth of applications in recombinant DNA technology. The once complex task of cloning genes has now been simplified, triggering a veritable explosion of insights into gene functionality. This has been achieved through the seamless fusion of multiple DNA fragments or the utilization of interchangeable gene cassettes, culminating in a state of unparalleled agility and expediency. In the 1970s, when restriction endonucleases, enzymes that cut DNA molecules selectively were discovered, molecular cloning technology has grown exponentially in application and intricacy, resulting in influential DNA manipulation tools. Recent decades have seen an explosion in our understanding of gene function due to the simplicity and efficiency of molecular cloning. It is expected that emerging technologies will offer superior potentials, such as stitching together multiple DNA fragments in under a few hours and transforming the resulting plasmids into bacteria, or the use of swappable genes, which can be easily moved between different paradigms, maximizing promptness and flexibility. It has been proved that cloning techniques provide a gold standard technique for polymicrobial infection, recombinant cytokines, antimicrobial peptides, epidemiology and gene therapy due to the limitations of culture-based methods. Due to molecular cloning technique, recombinant antigens are now being used to monitor patients against clinical infections. As a result of laboratory techniques that permit in vitro chemical synthesis of any DNA construct specified in silico, molecular cloning will likely undergo a paradigm shift in the coming future. As a result of these advances, DNA clones can be constructed faster and iteratively, which will speed up the growth of new vaccines, gene therapy vectors, and recombinant proteins. Here I present a detailed overview of the latest applications of molecular cloning techniques in medical microbiology.

Use of a Tissue-Targeting Recombinant Human Interleukin-22 Fusion Molecule (F-652) for the Treatment of Advanced Refractory Lower GI Acute Gvhd

Use of a Tissue-Targeting Recombinant Human Interleukin-22 Fusion Molecule (F-652) for the Treatment of Advanced Refractory Lower GI Acute Gvhd

MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients' HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

Особливості анестезіологічної допомоги хворим на холодову кропивницю (огляд літератури)

Cold urticaria (CU) is an allergic reaction that manifests itself as hives-like rashes or red spots in response to general or local cooling of the body. Th e disease can be acquired or hereditary, and in the cold season it can affect all segments of the population. Th is pathological condition, at first glance, does not seem to be a very dangerous variant of a local cold injury, but in persons who are prone to exposure to low temperatures, especially with a burdened cold history, it may be accompanied by chronicity of the process and complicated by neurovasculitis, obliterating endarteritis and secondary Raynaud’s syndrome, may decrease the quality of life of the victims and become a cause of disability. The pathophysiology of CU is largely unknown, but it is likely to be related to immunoglobulin E (IgE) and mast cell activation. Cooling has been reported to induce the release of neutrophilic and eosinophilic chemotactic factors, prostaglandin D2, and tumor necrosis factor (TNF-α). Less common immunologic fi ndings in patients with CU include cryoglobulinemia consisting of monoclonal IgG and mixed IgG/IgM and IgG/IgA cryoglobulin types. The mechanisms of development of CU are mainly determined by the formation of cryoglobulins (cold hemolysins) and subsequent degranulation of mast cells. Th e diagnosis of CU depends on the patient’s history and the results of cold provocation tests. Patients with CU are recommended first of all not to overcool, to take warm showers, to wear warm clothes and a hat, and not to consume cold food and drinks. Treatment options include second-generation H1 antihistamines and glucocorticosteroids. New promising option is omalizumab, a humanized monoclonal antibody derived from a recombinant DNA molecule that targets and selectively binds to circulating IgE and affects mast cells function. In patients with CU undergoing general anesthesia, premedication including antihistamines and corticosteroids is recommended, along with strict maintenance of perioperative normotermia.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

COMPARISON OF THE RESULTS OF DETECTION OF ANTIBODIES TO HEPATITIS C VIRUS REAGENTS FROM DIFFERENT MANUFACTURERS

We conducted a comparative characteristic analysis of the four kits Anti-HCV Abbott (AbHCV), Anti-HCV II Roche (RoHCV), Vector-best Anti-HCV spectrum ELISA (VeHCVsp) and Vector-best screening ELISA (VeHCVsc) to select screening and confirming method measurement antibody to hepatitis C virus (anti-HCV) considering Se, Sp.We evaluated 321 samples in the three stages. At the first stage after examining 214 primary positive in screening samples we received conflicting results AbHCV, VbHCVsp and RocHCV with discrepancies in 12.6% of cases. In the second phase, we selected 67 samples with conventionally border values cut-off that is “weakly positive” or “doubtful negative” results. Differences for this group was 17.9%. In the third phase, we selected 40 conventionally border results samples (CBRS). We used for comparison immunoblotting method “Recomline HCV IgG”, Microgen diagnostic (MGHCV). We evaluated Se with positive and doubtful MGHCV results, Sp - with negative results. Se: AbbHCV = 75,0%, RocHCV = 60,0%, VbHCV = 50,0%. Sp: AbbHCV = 5,0%, RocHCV = 75,0%, VbHCV = 95,0%. To understand the causes of discrepancies of various kits, we conducted an analysis HCV antigens (Core 1, Core 2, Helicase, NS3, NS4, NS5). The uncertainty of the results for CBRS is determined by different design of recombinant molecules used each kit. Therefore, at the stage of screening samples for HCV antibodies will always be samples with conflicting results. The best decision for screening is a combination of primary screening and retesting of positive samples to use kits with the “opposite” benefits.

Recombinant DNA technology: revolutionizing regenerative medicine and empowering nanotechnology strategies

In the past century, the concept of controlling gene expressions to enhance desirable traits in living organisms through recombinant DNA technology was merely a concept. However, in recent times, this field has made significant advancements, offering unique benefits to human life. Recombinant DNA technology allows for the safe, accessible, and abundant production of crucial proteins needed for addressing various health issues. Through laboratory methods of genetic manipulation, scientists generate recombinant DNA molecules by merging genetic material from various origins that wouldn’t naturally occur within organisms. Although the chemical structure of DNA is the same across all organisms, the nucleotide sequences vary. The application of recombinant DNA technology extends to diverse fields such as regenerative medicine, nanotechnology, and tissue engineering, allowing for the production of proteins with specific characteristics and effectiveness. This article will delve into the prevalent applications of recombinant DNA technology within fundamental research, highlighting its crucial role in contemporary endeavours across the realms of biological and biomedical sciences, particularly within the fields of regenerative medicine and nanotechnology.

Extravascular factor IX pool fed by prophylaxis is a true hemostatic barrier against bleeding

BackgroundFactor (F)IX can bind to type IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. ObjectivesThe aim of the study was to evaluate the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds. MethodsThe capacity of 4 different FIX molecules (plasma-derived and recombinant) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knockout mice. ResultsA specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50 to 100 IU/kg, while plasma-derived FIX generated a FIX signal at all doses, including 50 IU/kg. Such a signal was also detected after five 100 IU/kg daily infusions of recombinant FIX, suggesting that FIX can accumulate in the extravascular space during prophylaxis. The extravascular procoagulant activity of FIX, assessed in saphenous vein bleeding assays, was significantly higher in hemophilia B mice after these 5 days of prophylaxis compared to a single infusion of 100 IU/kg of FIX and assessment of FIX activity 7 days later. ConclusionTaken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection toward bleeding.