Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 µg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Infrared spectroscopy as a predictive tool for the severity of COVID-19 using patient's saliva: A strategy to avoid hyperinflammation.

Discriminate the severity level of COVID-19 disease is still a challenge. Here we investigate the capability of micro-infrared absorption spectroscopy (micro-FTIR) to probe COVID-19 severity level and predict hyperinflammation, correlating the assigned vibrational data to relevant biomolecules related to the immune system. Saliva of 184 patients was analysed by ELISA assay (Hepcidin) and micro-FTIR. Vibrational bands related to IgM and IgA can discriminate healthy from Severe individuals (sensitivity ≥ 0.749, specificity ≥ 0.945) and are less effective in discriminating Mild or Moderate individuals from the Severe group (sensitivity ≥ 0.628, specificity ≥ 0.867). Analysis of the second derivative of spectra probed increased levels of IL-6 in the saliva a key additional information for the degree of severity prediction. Because the model discriminates all the groups regarding the Severe group, it predicts an intense state of inflammation based on FTIR analysis. It is a powerful tool for predicting hyperinflammation conditions related to SARS-CoV-2 infection and may be an ally in implementing drugs or therapeutic approaches to manage COVID-19 in the Severe stage in healthcare facilities.

Salivary immunoglobulin levels and periodontal indices in Brazilian children with and without type 1 diabetes.

This cross-sectional study evaluated the association between salivary immunoglobulins, plaque index, and gingival index in Brazilian children with and without type 1 diabetes mellitus (DM1). The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist for the reporting of observational studies was followed. The DM1 group had 38 children, and an equal number of volunteers matched by sex and age were recruited as controls. Clinical examination was performed for plaque index and gingival index determination. Non-stimulated whole saliva was collected. Concentrations of IgA, IgG, and IgM were determined by ELISA test. Data were tested by the Kolmogorov-Smirnov, Mann-Whitney, and Spearman tests and a multiple linear regression model (p<0.05) was performed. Gingival index was higher in the Control (DM1: 0.16±0.17; Control: 0.24±0.23, p=0.040). In DM1, there was a correlation between IgA and age (rho=0.371, p=0.024), IgM and IgG (rho=0.459, p=0.007), and IgM and gingival index (rho=0.394, p=0.014). In DM1, multiple linear regression showed that age (p=0.041; ß=0.363), gingival index (p=0.041; ß=0.398), and plaque index (p=0.008; ß=-0.506) were good predictors of IgA levels in saliva. Thus, IgA was the only researched immunoglobulin that was directly associated with plaque and gingival indices in Brazilian children with DM1, but not in control subjects.

Interaction of Bacteriophages with the Immune System: Induction of Bacteriophage-Specific Antibodies.

In all cases when a bacteriophage makes a direct contact with a mammalian organism, it may challenge the mammalian immunological system. Its major consequence is the production of antibodies specific to the bacteriophage, particularly IgM, IgG, and IgA as the typical response. Here we present protocols applicable in studies of the ability of bacteriophage to induce specific antibodies; immunization to whole virions or to isolated phage proteins has been included. The protocols have been divided into three parts: purification, immunization, and detection (enzyme-linked immunosorbent assay, ELISA).

IMMUNOLOGICAL DISORDERS AND COLON DYSBIOSIS IN OBESE PATIENTS WITH HYPOTHYROIDISM.

OBJECTIVE: The aim: To investigate the peculiarities of immunological changes and their relationship with colon dysbiosis in obese patients with HT. PATIENTS AND METHODS: Materials and methods: The examined patients included 48 patients with HT and obesity (group 1) and 34 patients with obesity (group 2). Patients under¬went fecal analysis for dysbiosis. The levels of complement, namely C3 and C4 and the concentration of immunoglobulins (IgA, Ig M, IgG) were determined by means of chromogenic analysis. RESULTS: Results: During the clinical examination, constipation and flatulence were more often diagnosed in patients of group I (58.3% and 66.7%, respectively - p<0.001), while in patients of group 2 with increased BMI without thyroid dysfunction, a tendency to diarrhea was more often found, accompanied by periodic pain along the colon (50.0% and 32.3% of patients, respectively - p<0.001). Changes in the immunological status of patients in both groups were found. In patients with HT and increase of BMI an increase in serum IgA, IgM, IgG levels were found. An increase in serum immunoglobulins (A, M and G) was also diagnosed in group 2 of examined patients too. CONCLUSION: Conclusions: 1. In patients with obesity decrease in the concentration of Bifidobacterium, Lactobacillus and increase in the number of Staphylococcus, Clostridium, Proteus and Klebsiella were detected, which is more pronounced in patients with a combination of obesity and hypothyroidism. 2. Impairment distinct of immu¬nological status in patients with hypothyroidism and obesity was diagnosed, which was manifested by increased levels of immunoglobulins, namly (A, M, G), as well as a decrease in blood serum complements (C3, C4). 3. The level of IgA, G directly depends on the decrese of Bifidobacterium, Lactobacillus and increse of Staphylococcus, Clostridium and Klebsiella in patients with obesity, which is more pronounced in patients with a combination of obesity and hypothyroidism.

Estimating the number of probable new SARS-CoV-2 infections among tested subjects from the number of confirmed cases.

OBJECTIVES: In most African countries, confirmed COVID-19 case counts underestimate the number of new SARS-CoV-2 infection cases. We propose a multiplying factor to approximate the number of biologically probable new infections from the number of confirmed cases. METHODS: Each of the first thousand suspect (or alert) cases recorded in South Kivu (DRC) between 29 March and 29 November 2020 underwent a RT-PCR test and an IgM and IgG serology. A latent class model and a Bayesian inference method were used to estimate (i) the incidence proportion of SARS-CoV-2 infection using RT-PCR and IgM test results, (ii) the prevalence using RT-PCR, IgM and IgG test results; and, (iii) the multiplying factor (ratio of the incidence proportion on the proportion of confirmed -RT-PCR+- cases). RESULTS: Among 933 alert cases with complete data, 218 (23%) were RT-PCR+; 434 (47%) IgM+; 464 (~ 50%) RT-PCR+, IgM+, or both; and 647 (69%) either IgG + or IgM+. The incidence proportion of SARS-CoV-2 infection was estimated at 58% (95% credibility interval: 51.8-64), its prevalence at 72.83% (65.68-77.89), and the multiplying factor at 2.42 (1.95-3.01). CONCLUSIONS: In monitoring the pandemic dynamics, the number of biologically probable cases is also useful. The multiplying factor helps approximating it.

Antibodies to Domain I ß2-Glycoprotein 1 in Patients with Antiphospholipid Syndrome and Systemic Lupus Erythematosus.

The role of antiphospholipid antibodies (aPL), which are not included in the Sydney diagnostic criteria, in antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) is poorly understood. The aim of this study was to determine the clinical significance of IgG antibodies for domain 1 of ß2-glycoprotein 1 (ß2-GP1), IgG anti-ß2-GP1DI, in patients with APS with and without SLE. The study included 187 patients with APS with or without SLE, 49 patients formed the comparison group, and 100 apparently healthy individuals formed the control group. IgG/IgM antibodies to cardiolipin (aCL) and IgG/IgM anti-ß2-GP1 were determined by enzyme immunoassay (ELISA) in patients with or without APS, and IgG anti-ß2-GP1DI was determined by chemiluminescence assay (CLA) in all patients and controls. IgG anti-ß2-GP1DI was detected in 37 (71%) of 52 patients with primary APS (PAPS), in 6 (50%) of 12 patients with probable APS, in 42 (71%) of 59 patients with SLE + APS, in 17 (26%) of 64 patients with SLE, in 1 (2%) of the comparison group, and in none of the control group. IgG anti-ß2-GP1DI was significantly associated with PAPS and SLE + APS compared with the patients with SLE (p = 0.0002 and 0.0001, respectively). The association of IgG anti-ß2-GP1DI with clinical manifestations of APS (thrombosis (p = 0.001) and obstetric pathology (p = 0.04)) was detected. There was a significant association of IgG anti-ß2-GP1DI with arterial thrombosis (p = 0.002) and with late gestational obstetric pathology (p = 0.01). High specificity of IgG anti-ß2-GP1DI depending on the diagnosis and clinical manifestations of APS despite low sensitivity was noted: specificity was 84% for thrombosis, 94% for obstetric pathology, and 89% for APS. Isolated IgG anti-ß2-GP1DI positivity was reported in 2% of 50 aPL-negative patients and was not associated with APS manifestations. The frequency of IgG anti-ß2-GP1DI detection was higher in the patients with APS compared to the patients with SLE, comparison group, and control (p < 0.05). Positive IgG anti-ß2-GP1DI values were significantly associated with thrombotic complications and with obstetric pathology (p = 0.002 and p = 0.01, respectively). Specificity of IgG anti-ß2-GP1DI for APS and its clinical manifestations (thrombosis and obstetric pathology) was higher than sensitivity (89, 94, and 84%, respectively).

Refining Timely Diagnosis of Early Syphilis by Using Treponema pallidum PCR or IgM Immunoblotting Next to Conventional Serology for Syphilis.

Treponema pallidum subsp. pallidum is a fastidious spirochete and the etiologic agent of syphilis, a sexually transmitted infection (STI). Syphilis diagnoses and disease staging are based on clinical findings and serologic testing. Moreover, according to most international guidelines, PCR analysis of swab samples from genital ulcers is included in the screening algorithm where possible. It has been suggested that PCR might be omitted from the screening algorithm due to low added value. As an alternative to PCR, IgM serology might be used. In this study, we wanted to establish the added value of PCR and IgM serology for diagnosing primary syphilis. Added value was defined as finding more cases of syphilis, preventing overtreatment, or limiting the extent of partner notification to more recent partners. We found that both PCR and IgM immunoblotting could aid the timely diagnosis of early syphilis in ~24% to 27% of patients. PCR has the greatest sensitivity and can be applied to cases with an ulcer with suspected reinfection or primary infection. In the absence of lesions, the IgM immunoblot could be used. However, the IgM immunoblot has better performance in cases with suspected primary infection than in reinfections. The target population, testing algorithm, time pressures, and costs should determine whether either test provides sufficient value to be implemented in clinical practice.

The importance of combining serological testing with RT-PCR assays for efficient detection of COVID-19 and higher diagnostic accuracy.

Misdiagnosing suspected COVID-19 individuals could largely contribute to the viruses transmission, therefore, making an accurate diagnosis of infected subjects vital in minimizing and containing the disease. Although RT-PCR is the standard method in detecting COVID-19, it is associated with some limitations, including possible false negative results. Therefore, serological testing has been suggested as a complement assay to RT-PCR to support the diagnosis of acute infections. In this study, 15 out of 639 unvaccinated healthcare workers (HCWs) were tested negative for COVID-19 by RT-PCR and were found seropositive for SARS-CoV-2 nucleocapsid protein-specific IgM and IgG antibodies. These participants underwent additional confirmatory RT-PCR and SARS-CoV-2 spike-specific ELISA tests. Of the 15 individuals, nine participants were found negative by second RT-PCR but seropositive for anti-spike IgM and IgG antibodies and neutralizing antibodies confirming their acute infection. At the time of collection, these nine individuals were in close contact with COVID-19-confirmed patients, with 77.7% reporting COVID-19-related symptoms. These results indicate that including serological tests in the current testing profile can provide better outcomes and help contain the spread of the virus by increasing diagnostic accuracy to prevent future outbreaks rapidly.

The role of the laboratory in the diagnosis of an unusual case of Waldenstrom's macroglobulinaemia that lacked the common clinical features: A case report.

Lymphoplasmacytic lymphoma (LPL) is a neoplasm of small B lymphocytes, plasmacytoid lymphocytes and plasma cells usually involving the bone marrow (BM). A subset of LPL which is associated with IgM monoclonal gammopathy is called Waldenstrom's macroglobulinaemia (WM), and usually requires therapeutic intervention when a patient becomes symptomatic (Bone Marrow failure characterised by cytopenia or hyperviscosity syndrome). Here, we report the case of an 80-year-old female with clinically unsuspected WM who initially presented to the Emergency Department (ED) with nausea and vomiting. The patients' gastrointestinal symptoms subsequently settled and was awaiting discharge. Non-specific, borderline size significant lymph nodes on CT chest was the only substantial past medical history. The diagnosis of WM was made after the Biochemistry Biomedical Scientist (BMS) detected the presence of a Type I monoclonal cryoglobulin. A potential cryoprecipitate was suspected when repeated 'clotting' error flags occurred during routine laboratory analyses; the sample aspiration difficulties being attributed to the viscous nature of the sample. The investigation of inaccessible low volume lymphadenopathy in the elderly should include serum protein electrophoresis and immunoglobulins as this may have established an earlier diagnosis in this case. The application of good scientific principles informed the laboratory investigation and resulted in the identification of a large IgM monoclonal cryoglobulin that prompted further appropriate investigations resulting in the diagnosis of WM. This case also highlights the importance of good communication between the laboratory and clinical staff.

Evaluation of ten (10) SARS-CoV-2 rapid serological tests in comparison with WANTAI SARS-CoV-2 ab ELISA in Burkina Faso, West Africa.

BACKGROUND: The aim of this study was to evaluate the performance of ten (10) SARS-CoV-2 serological rapid diagnostic tests in comparison with the WANTAI SARS-CoV-2 Ab ELISA test in a laboratory setting. MATERIALS AND METHODS: Ten (10) SARS-CoV-2 serological rapid diagnostic tests (RDTs) for SARS-CoV-2 IgG/IgM were evaluated with two (2) groups of plasma tested positive for one and negative for the other with the WANTAI SARS-CoV-2 Ab ELISA. The diagnostic performance of the SARS-CoV-2 serological RDTs and their agreement with the reference test were calculated with their 95% confidence intervals. RESULTS: The sensitivity of serological RDTs ranged from 27.39 to 61.67% and the specificity from 93.33 to 100% compared to WANTAI SARS-CoV-2 Ab ELISA test. Of all the tests, two tests (STANDARD Q COVID-19 IgM/IgG Combo SD BIOSENSOR and COVID-19 IgG/IgM Rapid Test (Zhejiang Orient Gene Biotech Co., Ltd)) had a sensitivity greater than 50%. In addition, all ten tests had specificity greater than or equal to 93.33% each. The concordance between RDTs and WANTAI SARS-CoV-2 Ab ELISA test ranged from 0.25 to 0.61. CONCLUSION: The SARS-CoV-2 serological RDTs evaluated show low and variable sensitivities compared to the WANTAI SARS-CoV-2 Ab ELISA test, with however a good specificity. These finding may have implications for the interpretation and comparison of COVID-19 seroprevalence studies depending on the type of test used.

Low detection rate of RT-PCR-confirmed COVID-19 using IgM/IgG rapid antibody tests in a large community sample in Lima, Peru.

BACKGROUND: Rapid IgM/IgG antibody tests were largely used in lieu of RT-PCR tests as part of COVID-19 public health response activities in Lima, Peru. To assess their utility, we explored the relationship between the time since onset of several COVID-19-related symptoms and the sensitivity of a rapid combined IgM/IgG antibody test. METHODS: We collected data from a community sample of individuals (n = 492) who received concurrent RT-PCR and rapid IgM/IgG antibody testing between May 2020 and March 2021. We estimated the sensitivity of the antibody test, against the RT-PCR test, by weeks since symptom onset via segmented regression analysis. RESULTS: The overall sensitivity of the rapid IgM/IgG antibody test was 46.7% (95% CI, 42.4-51.2%). Among 372 (75.6%) participants who reported COVID-19-related symptoms, sensitivity increased from 30.4% (95% CI, 24.7-36.6%) in week 1 after symptom onset to 83.3% (95% CI, 41.6-98.4%) in week 4. The test sensitivity increased by 31.9% (95% CI, 24.8-39.0%) per week until week 2 to 3, then decreased by - 6.0% (95% CI, - 25.7-13.7%) per week thereafter. CONCLUSION: Rapid antibody tests are a poor substitute for RT-PCR testing, regardless of presenting symptoms. This highlights the need for future pandemic planning to include timely and equitable access to gold-standard diagnostics, treatment, and vaccination.

Transfer of Injected Triamcinolone into Human Milk of a Lactating Patient Suffering from Idiopathic Granulomatous Mastitis.

Background: Idiopathic granulomatous mastitis (IGM) is a chronic inflammatory condition of the mammary gland that presents as a painful mass, and it must be distinguished from both infectious mastitis and breast cancer. When diagnosed during lactation, it can result in significant distress and early weaning. Injection of triamcinolone has been used as a successful treatment method, but safety in breastfed infants has not been established. Methods: We present a case of a lactating patient who received a direct injection of triamcinolone (dosage 40 mg) in her breast to treat IGM after failure of oral corticosteroids. Breastmilk samples were expressed by the patient 0, 1, 4, and 24 hours after the procedure, and then daily for 1 week. All the samples were analyzed using liquid chromatography mass spectrometry. The patient was supported by a breastfeeding and lactation medicine clinic. Results: After injection of triamcinolone into the granulomatous mass, breast milk samples were collected and analyzed. No samples were found to contain triamcinolone. A temporary but significant decrease in milk production was noted after injection, though only a slight decrease had been noted with 6 weeks of systemic corticosteroids. With support, the patient rebuilt milk production and continued to breastfeed from both breasts. Conclusion: Triamcinolone was not found in any milk samples (≥0.78 ng/mL) following therapeutic injection of the affected breast. The patient was able to continue breastfeeding from the affected breast with intermittent symptoms.

Levels and avidities of antiphosphatidylethanolamine antibodies in patients with thrombotic events and immunologically-mediated diseases.

AIMS: Antiphosphatidylethanolamine antibodies (aPE) represent one type of antiphospholipid antibody (aPL) directed against the neutral phospholipids - phosphatidylethanolamines. The aim of this study was to evaluate levels and avidities of aPE in several groups of patients and compare them with conventional aPLs. METHODS: aPE were analysed in a cohort consisting of 68 hospitalized patients. The other cohort comprised 22 patients with immunologically-mediated diseases. The control group consisted of 20 healthy persons. ELISA methods were used for determination of aPL. Avidities of aPE were tested by modified ELISA with urea as a chaotropic agent. RESULTS: aPE IgG/IgM were significantly higher in the group of patients with venous thromboembolism than those with non-thrombotic internal disorders (P=0.02 for both Ig classes). aPE IgG/IgM elevated above cut-off values were found in 10.8% of patients with venous thromboembolism and as a single aPL in 6.5%. Levels of aPE IgG higher than our limit (>6 U/mL) were detected in 29% of patients with immunologically-mediated diseases with other positive aPL. Low-, intermediate- and high-avidity aPE IgG were found in patients of both cohorts. The avidities of aPE IgG differed from those of anticardiolipin antibodies IgG. Neither aPE IgG levels nor avidity dynamics significantly changed during follow-up. CONCLUSION: aPE may be related to venous thromboembolism and may be part of the repertoire of aPL in immunologically-mediated diseases. There are patients with thrombosis negative for conventional aPL but positive for aPE. aPE IgG may have different avidities.

Changes in colostrum ingredients of Hu sheep, as well as the missense mutation genes associated with colostrum yield.

This study focused on the changes in the composition and immune evolution in milk from birth to 144 h postpartum and the genes associated with the colostrum yield of Hu sheep. Twelve Hu sheep, which were bred carefully under animal health standards and have a litter size of two kids and similar gestation length (149 ± 1 days), were used. Lambs were transferred into their own cots to avoid interference. The compositional content (i.e., fat, protein, and lactose) and some other properties, including daily colostrum yield, DM, and SNF, were determined. In addition, immunity molecules (IgG, IgA, and IgM concentrations) received remarkable attention. The DM, SNF, fat, and protein contents were higher in the first days postpartum and then dropped quickly from the time of birth to 144 h postpartum. However, the lactose content displayed an increasing pattern and reached normal milk percentage at 48 h. The highest IgG (103.17 mg/mL), IgA (352.82 µg/mL), and IgM (2.79 mg/mL) colostrum concentrations were observed at partum, decreased quickly, and finally stabilized. The change law of concentration of IgA and IgM in colostrum are the same with IgG. Furthermore, the whole-genome resequencing was performed, and a missense variant locus in the SRC gene and two missense locus variants in the HIF1A gene were significantly associated with the colostrum yield of sheep by using the whole-genome selection signal detection analysis. In conclusions, colostrum contains abundant nutrients especially immunoglobulin, and the HIF1A gene may be used as candidate genes for colostrum yield, which has important information as a basic knowledge for the Hu sheep breeding program.

Detection of Antibodies against Toxoplasma from Human Serum Sample using ELISA.

BACKGROUND: Toxoplasmosis is a common worldwide zoonotic infection affecting warm blooded animals and humans caused by Toxoplasma gondii. Clinical features range from mildfebrile illness and lymphadenopathy in the immunocompetent host to encephalitisin the immunosuppressed host (E.g. HIV infected patients). Congenital infection can also occur. For effective control and treatment of toxoplasmosis, accurate detection of T. gondii infection is important. OBJECTIVE: In this study, ELISA detecting anti-toxoplasma antibodies IgM and IgG has been used for diagnosing Toxoplasmosis in patients. METHODS: Enzyme-linked immunosorbentassay (ELISA) was carried out in serum samples collected from 40 patients to detect anti-toxoplasma IgG and IgM antibodies as a part of work up in suspected cases. Relevant clinical history was also taken. RESULTS: Of the total 40 samples taken, only one sample came positive for IgM and 9 came positive for IgG antibody. All patients who were seropositivefor T. gondii antibodies had HIV infection. Five patients were in the age group between 30 to 40 years. CONCLUSION: From the limited data available in this study, it may be recommended to screen for T. gondii antibodies in HIV patients.

Age-dependent nasal immune responses in non-hospitalized bronchiolitis children.

Bronchiolitis in children is associated with significant rates of morbidity and mortality. Many studies have been performed using samples from hospitalized bronchiolitis patients, but little is known about the immunological responses from infants suffering from mild/moderate bronchiolitis that do not require hospitalization. We have studied a collection of nasal lavage fluid (NLF) samples from outpatient bronchiolitis children as a novel strategy to unravel local humoral and cellular responses, which are not fully characterized. The children were age-stratified in three groups, two of them (GI under 2-months, GII between 2-4 months) presenting a first episode of bronchiolitis, and GIII (between 4 months and 2 years) with recurrent respiratory infections. Here we show that elevated levels of pro-inflammatory cytokines (IL1ß, IL6, TNFα, IL18, IL23), regulatory cytokines (IL10, IL17A) and IFNγ were found in the three bronchiolitis cohorts. However, little or no change was observed for IL33 and MCP1, at difference to previous results from bronchiolitis hospitalized patients. Furthermore, our results show a tendency to IL1ß, IL6, IL18 and TNFα increased levels in children with mild pattern of symptom severity and in those in which non RSV respiratory virus were detected compared to RSV+ samples. By contrast, no such differences were found based on gender distribution. Bronchiolitis NLFs contained more IgM, IgG1, IgG3 IgG4 and IgA than NLF from their age-matched healthy controls. NLF from bronchiolitis children predominantly contained neutrophils, and also low frequency of monocytes and few CD4+ and CD8+ T cells. NLF from infants older than 4-months contained more intermediate monocytes and B cell subsets, including naïve and memory cells. BCR repertoire analysis of NLF samples showed a biased VH1 usage in IgM repertoires, with low levels of somatic hypermutation. Strikingly, algorithmic studies of the mutation profiles, denoted antigenic selection on IgA-NLF repertoires. Our results support the use of NLF samples to analyze immune responses and may have therapeutic implications.

A performance comparison between fluorescent immunoassay and immunochromatography for rapid dengue detection in clinical specimens.

Dengue virus (DENV 1-4) infection has been a global health threat where no specific treatment is currently available. Therefore, a rapid and accurate diagnosis is critical for an appropriate management as it could reduce the burden of severe clinical manifestation. Currently, dengue immunochromatography (IC) is commonly used to primarily differentiate acute febrile illnesses. Fluorescent immunoassay (FIA) utilized a highly sensitive detection system and claimed 70-100% sensitivity and 83.5-91.7% specificity for dengue infection in a preliminary report. This report recruited samples with acute febrile illnesses sent for dengue screening and tested IC and FIA in parallel. The performance of both tests was verified by a definitive diagnosis retrieved from combinatorial reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) for IgM and IgG confirmation tests. Results showed that the viral nonstructural protein (NS1) performance of FIA was slightly higher than IC with the sensitivity, specificity, PPV, NPV, agreement, kappa, and its standard error at 79.11, 92.28, 86.81, 87.31, 352 (87.13%), 0.725 ± 0.035, respectively; whereas those of the IC were at 76.58, 92.28, 86.43, 85.98, 348 (86.14%), 0.703 ± 0.037, respectively. Moreover, the IgM and IgG performance of FIA had higher specificity, PPV, and agreement than the IgM IC performance, suggesting that the FIA was more specific but less sensitive for antibody detection. No correlation was observed in IgM and IgG levels of ELISA and FIA assays. In conclusion, the FIA and IC were highly sensitive, specific, and substantially agreed in NS1 detection but moderately agreed in IgM and IgG detection.

The Structural Characterization and Immunomodulatory Activity of Polysaccharides from Pleurotus abieticola Fruiting Bodies.

Polysaccharides obtained from mushrooms have been reported to possess immunomodulatory properties. In this study, a water-soluble polysaccharide was purified from the fruiting bodies of Pleurotus abieticola, entitled PAPS1. After its composition and structural analysis, the immunomodulatory activity was investigated in immunosuppressed mice induced by cyclophosphamide (CTX) at a dosage of 70 mg/kg by intraperitoneal injection for 7 days. After 28 days of intragastric administration, PAPS1 alleviated cyclophosphamide (CTX)-induced histopathological damage and increased the expressions of splenic CD4, CD8, CD56 and IgM in the serums of immunosuppressed mice. PAPS1 suppressed the oxidative stress indicated by preventing the increases in ROS and MDA levels. According to the intestinal microflora analysis, PAPS1 regulated 11 bacteria at the gene level, including Helicobacter and Paraprevotella, which are related to immunity and oxidative capacity. Compared with CTX-treated mice, significant increases in immune-related cytokines, such as interleukin (IL)-2, IL-6 and IL-12 in the serums of mice treated with PAPS1, were observed. Finally, PAPS1 can strongly increase the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins. In conclusion, PAPS1-boosted immunity may be related to its suppression on oxidative stress via enhancing the activity of Nrf2 signaling. Thus, PAPS1 can be investigated as a candidate for immunomodulatory therapy.

Measles Virus and Subacute Sclerosing Panencephalitis.

BACKGROUND: Subacute Sclerosing Panencephalitis (SSPE) mainly affects children and young people. It is a rare, chronic progressive degenerative form of cerebral inflammation with various infectious noxa, which develops for years after a primary, uncomplicated infection, and the highest percentage can be caused by measles virus and in rare cases by rubella. The aim of the present study is to investigate in the laboratory the role of measles virus in the development of neurological symptoms and diseases of the CNS. METHODS: A total of 46 clinical materials (23 sera samples and 23 CSF) obtained from 23 patients with neurological symptoms and diagnoses: "SSPE" (in 10 patients) and "Encephalitis" (in 13 patients), in the period January 2011 - December 2020 were tested in the National Reference Laboratory (NRL) "Measles, mumps and rubella" at National Centre of Infectious and Parasitic Diseases (NCIPD), Sofia, Bulgaria. Serological (indirect ELISA test for the detection of specific measles IgG/IgM antibodies in serum samples and cerebrospinal fluid) and molecular (RT-PCR for the demonstration of viral RNA) methods were used. RESULTS: The study was performed by parallel testing of serum samples and CSF from each patient. Positive results for measles IgG antibodies in sera were found in 21 patients. Presence of measles IgG antibodies in CSF was demonstrated in four children with diagnosis SSPE (two children at 4 years, one child at 4 years and 6 months, and one at 11 years old). All children with positive laboratory results for SSPE had evidence of MeV infection before 2 years of age. The patients with SSPE had high antibody titers (CSF > 230 U/mL) in their CSF. Patients with positive anti-Measles IgG in the CSF were also found to have positive results for protective measles IgG in the serum samples and their IgG titers were nearly twice as high compared to other patients' sera. The presence of specific measles IgM antibodies was not demonstrated in the tested specimens. RT-PCR test was performed for all samples, and the presence of viral RNA was not detected. CONCLUSIONS: The measles infection can be a reason for developing serious complications affecting CNS in all age groups. SSPE itself is extremely difficult to diagnose, which is why laboratory confirmation of any clinical case is a necessary condition for effective disease surveillance.