Immunological Techniques Research Articles

Purification, Expression, and Characterization of Sarcoplasmic Calcium Binding Protein: A Novel Allergen of Portunus trituberculatus.

Swimming crab (Portunus trituberculatus), a crucial valuable crustacean, is a common factor causing food allergy. However, studies on allergens of P. trituberculatus are scarce. In this study, the sarcoplasmic calcium binding protein (SCP) of P. trituberculatus was expressed in Escherichia coli, purified with affinity chromatography, and the IgE-binding activity was evaluated through serological analyses. Further, the structure, physicochemical properties, and cross-reactivity were assessed via bioinformatics, immunologic, and spectroscopy techniques. The results indicated that P. trituberculatus SCP was an allergen displaying strong IgE-binding capacity, composed of 60% α-helix. It presented good immunologic and structural stability at 4-70 °C and pH 3-10, and only exhibited high IgG cross-reactivity among crustaceans, without cross-reactivity with other species tested. These results establish the foundations for further studies on SCP and are promising to promote the development of specific crustacean allergen detection and precise allergy diagnosis.

Detection of Human Paragonimiasis by ELISA Using Recombinant Paragonimus westermani Cysteine Protease 7.

Paragonimiasis is an important but neglected foodborne trematodiasis caused by Paragonimus mexicanus in Costa Rica. Immunological techniques for diagnosing this parasitosis in humans do not exist in Central America. The objective of the present study was to use recombinant Paragonimus westermani cysteine protease 7 to standardize an ELISA for the detection of antibodies against Paragonimus spp. Human sera positive for P. westermani, P. mexicanus, or Paragonimus spp., human sera infected with other helminths, as well as sera of healthy humans without parasitic infections, were analyzed. The sensitivity of the ELISA was 92.9%, and the specificity was 91.9%. This report is the first to describe the development of an ELISA for the diagnosis of Paragonimus spp. in Costa Rica and Central America. Using this ELISA in the health system of Costa Rica is recommended to detect infections.

Ps−Pt nanozyme-based synergistic signal amplification biosensor for highly sensitive colorimetric detection of protein

Immunosorbent assay is one of the most popular immunological screening techniques which has been widely used for the clinical diagnosis of alpha-fetoprotein (AFP). While traditional immunosorbent assay (ELISA) suffers from low detection sensitivity due to its low intensity of colorimetric signal. To improve the sensitivity of AFP detection, we developed a new and sensitive immunocolorimetric biosensor by combining Ps-Pt nanozyme with terminal deoxynucleotidyl transferase (TdT)-mediated polymerization reaction. The determination of AFP was achieved by measuring the visual color intensity produced by the catalytic oxidation reaction of the 3,3′,5,5′-tetramethylbenzidine (TMB) solution with Ps-Pt and horseradish peroxidase (HRP). Owing to the synergistic catalysis of Ps-Pt and horseradish peroxidase HRP enriched in polymerized amplification products, this biosensor exhibited a significant color change within 25 s in the presence of 10–500 pg/mL AFP. This proposed method allowed for the specific detection of AFP with a detection limit of 4.30 pg/mL and even 10 pg/mL target protein could be distinguished clearly by visual observation. Furthermore, this biosensor could be applied to analysis of AFP in the complex sample and could be easily extended to the detection of other proteins.

Pathophysiological Changes in the Hemostatic System and Antithrombotic Management in Kidney Transplant Recipients.

Nowadays, the main cause for early graft loss is renal graft thrombosis because kidney transplant outcomes have improved drastically owing to advances in immunological techniques and immunosuppression. However, data regarding the efficacy of antithrombotic therapy in the prevention of renal graft thrombosis are scarce. Adequate antithrombotic management requires a good understanding of the pathophysiological changes in the hemostatic system in patients with end-stage kidney disease (ESKD). Specifically, ESKD and dialysis disrupt the fine balance between pro- and anticoagulation in the body, and further changes in the hemostatic system occur during kidney transplantation. Consequently, kidney transplant recipients paradoxically are at risk for both thrombosis and bleeding. This overview focuses on the pathophysiological changes in hemostasis in ESKD and kidney transplantation and provides a comprehensive summary of the current evidence for antithrombotic management in (adult) kidney transplant recipients.

Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma.

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C18 (4.6 × 100 mm, 3.5-μm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze-thaw cycles, and 2weeks at -20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.

Molecular immunological techniques to support the differential microbiological diagnosis of acute HIV infection

This research sought to identify HIV confirmatory molecular immunological methods for the early diagnosis of acute infections, complementing the differential microbiological diagnosis. A descriptive study of a clinical case was carried out using molecular immunological analyses. The results showed that molecular immunological methods range from third generation techniques, capable of diagnosing the infection in the acute phase, to fourth generation techniques that detect the infection from the third week of infection. Confirmatory tests, such as Western Blot and LIA, presented high sensitivity and specificity. Revised molecular techniques, such as p24 detection or Polymerase Chain Reaction (PCR), reduced diagnosis time. In conclusion, molecular immunological methods allow early detection of HIV with high precision, providing diagnostic and confirmation results from the third week of infection.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

Newly outbreak of Nipah virus: epidemiology, symptoms, transmission, diagnostic testing, treatment, and global health concern

Newly outbreak of Nipah virus: epidemiology, symptoms, transmission, diagnostic testing, treatment, and global health concern

Sensitivity-enhanced nanoplasmonic biosensor using direct immobilization of two engineered nanobodies for SARS-CoV-2 spike receptor-binding domain detection

Sensitive, rapid, and easy-to-implement biosensors are critical in responding to highly contagious and fast-spreading severe acute respiratory syndrome coronavirus (SARS-CoV-2) mutations, enabling early infection screening for appropriate isolation and treatment measures to prevent the spread of the virus. Based on the sensing principle of localized surface plasmon resonance (LSPR) and nanobody immunological techniques, an enhanced sensitivity nanoplasmonic biosensor was developed to quantify the SARS-CoV-2 spike receptor-binding domain (RBD) in serum within 30 min. The lowest concentration in the linear range can be detected down to 0.01 ng/mL by direct immobilization of two engineered nanobodies. Both the sensor fabrication process and immune strategy are facile and inexpensive, with the potential for large-scale application. The designed nanoplasmonic biosensor achieved excellent specificity and sensitivity for SARS-CoV-2 spike RBD, providing a potential option for accurate early screening of the novel coronavirus disease 2019 (COVID-19).

Nanomaterials-Based Novel Immune Strategies in Clinical Translation for Cancer Therapy.

Immunotherapy shows a lot of promise for addressing the problems with traditional cancer treatments. Researchers and clinicians are working to create innovative immunological techniques for cancer detection and treatment that are more selective and have lower toxicity. An emerging field in cancer therapy, immunomodulation offers patients an alternate approach to treating cancer. These therapies use the host's natural defensive systems to identify and remove malignant cells in a targeted manner. Cancer treatment is now undergoing somewhat of a revolution due to recent developments in nanotechnology. Diverse nanomaterials (NMs) have been employed to overcome the limits of conventional anti-cancer treatments such as cytotoxic, surgery, radiation, and chemotherapy. Aside from that, NMs could interact with live cells and influence immune responses. In contrast, unexpected adverse effects such as necrosis, hypersensitivity, and inflammation might result from the immune system (IS)'s interaction with NMs. Therefore, to ensure the efficacy of immunomodulatory nanomaterials, it is essential to have a comprehensive understanding of the intricate interplay that exists between the IS and NMs. This review intends to present an overview of the current achievements, challenges, and improvements in using immunomodulatory nanomaterials (iNMs) for cancer therapy, with an emphasis on elucidating the mechanisms involved in the interaction between NMs and the immune system of the host.

Examining the Kinetics of Phagocytosis-Coupled Inflammasome Activation in Murine Bone Marrow-Derived Dendritic Cells.

In the present chapter, we describe procedures to assess NLRP3 and NLRC4 inflammasome assembly by immunofluorescence microscopy or live cell imaging, together with inflammasome activation by biochemical and immunological techniques upon phagocytosis. We also include a step-by-step guide to automating the counting of inflammasome "specks" after imaging. While our focus resides on murine bone marrow-derived dendritic cells differentiated in the presence of granulocyte-macrophage colony-stimulating factor, which results in a cell population that resembles inflammatory dendritic cells, the strategies described herein may apply to other phagocytes as well.

Prevalence of Toxocara eggs in Latin American parks: a systematic review and meta-analysis.

Toxocariasis is an infection caused in canines, felines, humans, and other vertebrates by species of the genus Toxocara, such as T. canis and T. cati. The embryonated eggs of these parasites are the main form of acquisition of the infection both for definitive hosts, such as the dog and the cat, respectively and for paratenic hosts, such as humans and other vertebrates. Toxocariasis infection in humans causes visceral larva migrans syndrome. When deposited on park soils, environmental contamination becomes a risk for environmental, human, and animal health. To systemically estimate the prevalence of Toxocara spp. eggs in park soils in Latin America. A systematic review and meta-analysis were performed to evaluate the prevalence of Toxocara eggs in park soils in Latin America, defined by copro-parasitological, molecular and immunological techniques. We searched PubMed, Scopus, Web of Sciences, Embase, LILACS and SciELO for studies published from 1900 through 28 January 2023. A meta-analysis was performed using a random-effects model to calculate the pooled prevalence and 95% confidence intervals (95% CI). Heterogeneity was measured through I2 statistics. Forty-nine studies (2,508 parks and 12,833 samples) were included, of whom 44 had a low risk of bias. The pooled prevalence of Toxocara eggs in parks in Latin America was 50.0% (95% CI: 40.0%-60.0%). Argentina had the highest prevalence of Toxocara eggs in parks (100%), followed by Brazil (66%) and Venezuela (63%). The pooled prevalence of Toxocara eggs in soil samples was 20.0% (95% CI: 14.0%-26.0%); in faecal samples, it was 13.0% (95% CI: 6.0%-23.0%). The presence of Toxocara canis eggs in public parks in Latin America is a zoonotic and public health threat for the people who go to these places, especially if children play on the ground with dirt or contaminated objects; since many pet owners and general public are not adequately informed about the mode of transmission of this parasite.

Abdominal Tuberculosis Presenting as Anemia Refractory to Standard Therapy in an Elderly Male

Abstract India bears a significant proportion of tuberculosis cases, accounting for approximately 28% of the global burden. Among the reported global cases, around 16% constitute extrapulmonary tuberculosis (EPTB). EPTB diagnosis and treatment are usually challenging. The diagnosis frequently requires the application of contemporary methods, such as acquiring extrapulmonary samples and utilizing advanced diagnostic tools that involve immunological and molecular techniques. Here, we present a case of an elderly male who presented with constitutional symptoms and severe anemia which was refractory to standard hematinic and transfusion therapy. The patient was diagnosed to be a case of abdominal tuberculosis and improved with antitubercular therapy.

A Lab-Made E-Nose-MOS Device for Assessing the Bacterial Growth in a Solid Culture Medium

The detection and level assessment of microorganisms is a practical quality/contamination indicator of food and water samples. Conventional analytical procedures (e.g., culture methods, immunological techniques, and polymerase chain reactions), while accurate and widely used, are time-consuming, costly, and generate a large amount of waste. Electronic noses (E-noses), combined with chemometrics, provide a direct, green, and non-invasive assessment of the volatile fraction without the need for sample pre-treatments. The unique olfactory fingerprint generated during each microorganism's growth can be a vehicle for its detection using gas sensors. A lab-made E-nose, comprising metal oxide semiconductor sensors was applied, to analyze solid medium containing Gram-positive (Enterococcus faecalis and Staphylococcus aureus) or Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The electrical-resistance signals generated by the E-nose coupled with linear discriminant analysis allowed the discrimination of the four bacteria (90% of correct classifications for leave-one-out cross-validation). Furthermore, multiple linear regression models were also established allowing quantifying the number of colony-forming units (CFU) (0.9428 ≤ R2 ≤ 0.9946), with maximum root mean square errors lower than 4 CFU. Overall, the E-nose showed to be a powerful qualitative-quantitative device for bacteria preliminary analysis, being envisaged its possible application in solid food matrices.

Bovine Papillomavirus Type 1 or 2 Virion-Infected Primary Fibroblasts Constitute a Near-Natural Equine Sarcoid Model.

Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 106-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four passages. Infected cells displayed typical features of equine sarcoid cells, including hyperproliferation, and loss of contact inhibition. Neither multiple passaging nor storage negatively affected cell hyperproliferation, viral DNA replication, and gene transcription, suggestive for infection-mediated cell immortalization. Intriguingly, extracellular vesicles released by BPV1-infected fibroblasts contained viral DNA that was most abundant in the fractions enriched for apoptotic bodies and exosomes. This viral DNA is likely taken up by non-infected fibroblasts. We conclude that equine primary fibroblasts stably infected with BPV1 and BPV2 virions constitute a valuable near-natural model for the study of yet unexplored mechanisms underlying the pathobiology of BPV1/2-induced sarcoids.

Analytical methods to detect the Adulterants in Milk - An Overview

Milk contains whole nutrients and is consumed by the majority of population in the form of drinking as well as dietary products. Milk adulteration is one of the most common phenomena, this milk adulteration can be over looked in many countries. it shows serious health hazards leading to fatal diseases. Milk adulterants have been reported globally by adding various instances such as adding water, whey proteins, melamine, urea, detergents, starch hydrogen peroxide, boric acid. This paper presents a detailed review of common milk adulterants as well as different methods such as the chromatographic methods such as HPLC and GC coupled with mass spectrometry to detect the adulterants in milk, and immunological techniques such as ELISA and various DNA based procedures like PCR have also been used to detect the adulterants both qualitatively and quantitatively. This study is organized to be an adulterants-based study instead of a techniques-based one, where qualitative detection for most of the common adulterants are enlisted and quantitative detection methods are limited to a few major adulterants of milk. Apart from regular techniques, recent development in these detection techniques has also been reported. Nowadays milk is being adulterated in more sophisticated ways that demand for cutting- edge research for the detection of adulterants. This review intends to contribute towards the common knowledge base regarding possible milk adulterants and their detection techniques.

Prevalence of orthohantavirus in rodents: A systematic review and meta-analysis

IntroductionOrthohantaviruses are zoonotic diseases transmitted mainly by rodents, particularly mice and rats, associated with multiple outbreaks in different continents. Despite its epidemiological relevance, there is a lack of systematic reviews and meta-analyses. ObjectiveTo determine the combined global prevalence of Orthohantavirus infection in rodents. MethodsA systematic literature review was carried out in six databases (Web of Sciences, Scopus, PubMed, SciELO, Lilacs, Google Scholar) to evaluate the proportion of rodents infected with Orthohantavirus, defined by molecular and immunological techniques. The meta-analysis used a random effects model for the pooled prevalence and 95% confidence intervals (95%CI). Heterogeneity measures, Cochrane's Q, the I2 index and the tau-squared test were estimated. ResultsA total of 35,706 rodents (229 studies) were evaluated for ELISA, in which 3360 were found positive, for seroprevalence of 4.9% (95%CI 4.3–5.4%) (τ2 = 0.001; Q = 4027.708; I2 = 94.339%, p < 0.001). For PCR (N = 8812, 91 studies) it was 3.2% (95%CI 2.5–3.9%) (τ2 = 0.001; Q = 397.483; I2 = 77.358%; p < 0.001). For IFA (N = 555, 7 studies) it was 18.8% (95%CI 9.4–28.2%) (τ2 = 0.011; Q = 51.239; I2 = 88.29%, p < 0.001). At the genus level, the studies evaluated Oligoryzomys (8.98%), Reithrodontomys (8.98%), Peromyscus (8.20%), Rattus (8.20%), and Akodon (6.64%). ConclusionsThe global prevalence of Orthohantavirus is worrisome, with an increase in its report in certain regions, including Latin America. In this context, rodents have a role as reservoirs. The data of the present meta-analysis showed considerable seroprevalences with great variations by years, countries and Orthohantavirus species.

Schistosomiasis and Recurrent Arthritis: A Systematic Review of the Literature.

Schistosomiasis is an endemic parasitic infection found in many tropical countries and is highly prevalent in sub-Saharan Africa. It can follow different and atypical clinical patterns. In these unusual cases, diagnosis may be difficult, as symptoms are unspecific. Arthropathy can appear in parasitic infections, but making a connection between arthritis and parasitic aetiology is difficult. This review aims to summarise all cases that have reported schistosomiasis associated with arthropathy, and the different ways authors have diagnosed this disease. We present a systematic literature review of schistosomiasis associated with joint impairments, with a focus on the difficulty of differentiating between reactive arthritis and its parasitic presence in situ. Joint impairments mimicking polyarthropathy are not rare in parasitic infections. Diagnosis is difficult. On the one hand, some patients have arthritis with parasite eggs found in situ, particularly in synovial biopsy. These situations are less common and antiparasitic treatment is straightforward. On the other hand, arthritis can be associated with parasitic infections in the form of reactive arthritis due to an immunological reaction. In such cases, pathogenicity due to circulating immune complex should be suspected. Anti-inflammatory treatments such as corticosteroids or immunosuppressive therapies are ineffective in cases of schistosomal arthropathy. A joint fluid puncture appears to be necessary and parasitic examination as well as in situ immunological techniques appear to be important in order to confirm the diagnosis of schistosomal arthropathy. The frequency of articular schistosomiasis is probably underestimated and should be sought when patients have unexplained polyarthropathy, as it can be an alternative diagnosis when patients have concomitant parasitic infections. These situations are common, whereas the association between unexplained inflammatory arthritis and a concomitant parasitic infection is rarely made. Unspecific rheumatism can lead to probabilistic treatments with many side effects, and looking for a parasitic aetiology could lead to repeated antiparasitic treatments and may avoid other immunosuppressive or corticosteroid therapies. With increasing travel and global migration, physicians need to be more aware of nonspecific symptoms that may reveal an atypical presentation of a tropical disease that can be treated easily, thus avoiding inappropriate immunosuppressive treatments.

A novel PCR-free and label-free cloth-based DNA sensor for sensitive and rapid detection of Escherichia coli

BackgroundAs is known to all, pathogenic bacteria have a serious impact on human health. The development of sensitive, simple, rapid and low-cost bacterial detection method is necessary. Nowadays, some conventional methods (such as plate count, polymerase chain reaction (PCR) and immunological techniques) can not meet the above needs. This work was aimed at providing a new method for addressing these unmet needs. ResultThis study proposed a novel PCR-free and label-free DNA sensor based on multiple linear hybridization chain reaction (ML-HCR) and cloth-based closed bipolar electrochemiluminescence for sensitive and rapid detection of Escherichia coli (E. coli). The target DNA can be obtained from the E. coli genomic DNA by using the restriction enzyme instead of PCR. The auxiliary probe-triggered ML-HCR is carried out with continuous hybridization of two hairpin DNA, and as a result the double stranded DNA is formed to provide a large number of binding sites for Ru(bpy)32+. The whole detection is PCR-free and label-free, and thus the detection procedure is easier and faster. Under optimized conditions, the linear detection range was from 102 to 107 CFU/mL, and the detection limit was low to 38 CFU/mL. In addition, the proposed DNA sensor has an acceptable selectivity, stability and reproducibility, and is successfully applied to detect E. coli in milk samples with the recoveries from 96.24% to 105.98%. SignificanceThe proposed DNA sensor has broad application prospects in the fields of bacterial detection and gene diagnose. Further, this method has potential to be extended for establishing miniaturized, integrated, and automated detection system.

Prevalence of the H5N8 influenza virus in birds: Systematic review with meta-analysis

IntroductionAvian influenza viruses are members of the Orthomyxoviridae family, considered highly pathogenic (HPAI). They result from genetic variations from their low virulence predecessors. HPAI is a global problem. Large outbreaks of HAPI have significant health and economic impacts. ObjectiveThe objective of this study was to assess the prevalence of the H5N8 Influenza virus in birds, as well as to assess its variability according to the countries and years. MethodsA systematic review of the literature was carried out in six databases (Web of Sciences, Scopus, PubMed, SciELO, Lilacs and Google Scholar) to evaluate the proportion of birds infected with the H5N8 Influenza virus, by molecular and immunological techniques. A meta-analysis was performed using a random-effects model to calculate the pooled prevalence, 95% confidence intervals (95%CI). A 2-tailed 5% alpha level was used for hypothesis testing. Measures of heterogeneity were estimated and reported, including the Cochrane Q statistic, the I2 index, and the tau-squared test. In addition, bird species performed subgroup analyzes. Results152 data groups were analyzed, a combined prevalence of 1.6% (95% CI 1.3–1.9%) was found for molecular studies, and the ELISA study yielded a seroprevalence of 66.7%; those results of molecular detection varied by year, from 0.2% in 2014 to 52.6% in 2020 and 96.9% in 2015. ConclusionThe combined prevalence was substantial because large outbreaks have caused severe economic repercussions. In addition, it is considered a serious concern for public health due to its possible zoonotic activity.