Human Serum IgG Research Articles

Qualification of an enzyme-linked immunosorbent assay for quantification of anti-Vi IgG in human sera.

Effective vaccines against Salmonella Typhi, targeting the Vi capsular polysaccharide, have been developed and are being introduced into routine immunization in endemic countries. Vi conjugated vaccines are also being tested in new multi-component vaccine formulations. Simple, high-throughput and cost-effective assays to quantify Vi-specific IgG in clinical sera are needed. In this study we present the development and qualification of a new anti-Vi ELISA with continuous readout, which expresses results as ELISA Unit/mL (EU/mL). We have qualified the assay in terms of precision, linearity and specificity, demonstrating performance in line with a commercially available anti-Vi ELISA. We have also calibrated the assay against the 16/138 anti-Vi international standard and established conversion factor between EU/mL and international units/mL, to allow comparability of results across studies. In summary, this new assay met all the suitability criteria and is being used to evaluate anti-Vi responses in clinical studies.

Detection of anti-enterovirus IgG in human sera by ELISA method using the KTL-510 peptide

Enterovirus (EV) infections occur frequently in humans. In some geographical areas they are more common. These viruses cause diseases with varying degrees of severity, from a simple respiratory tract infection to severe diseases. Since EVs include more than 70 serotypes currently circulating in the population, a methodology that detects most of them is needed. ELISA is a rapid, sensitive, and economical diagnostic method for the identification of EV serotypes and can also be used as a retrospective diagnostic tool or in the investigation of outbreaks of infection. Commercial EV-ELISAs often appear and gradually disappear from the market supply. We have used the KTL-510 peptide, a synthetic viral protein of poliovirus VP1, as an antigen in a peptide-based ELISA for the detection of a broader spectrum of anti-EV antibodies. We aimed to design, optimize, and standardize this in-house ELISA with the peptide, and implement the method for routine detection of anti-EV IgG in human sera. For determining the cut-off value, we used 100 patients’ sera which were previously tested negative for IgG antibodies against EVs using a commercial ELISA kit available. We monitored patients’ sera samples sent for serological testing of anti-coxsackievirus antibodies to the National Reference Center for the Identification of Enteric Viruses between 2018–2022. These serum samples were examined using a standard virus neutralization test as well as the newly developed ELISA method.

Digital Ion Trap Isolation and Mass Analysis of Macromolecular Analytes with Multiply Charged Ion Attachment.

Multiply charged ions produced by electrospray ionization (ESI) of heterogeneous mixtures of macromolecular analytes under native conditions are typically confined to relatively narrow ranges of mass-to-charge (m/z) ratio, often with extensive overlap. This scenario makes charge and mass assignments extremely challenging, particularly when individual charge states are unresolved. An ion/ion reaction strategy involving multiply charged ion attachment (MIA) to the mixture components in a narrow range of m/z can facilitate charge and mass assignment. In MIA operation, multiply charged reagent ions are attached to the analyte ions of opposite polarity to provide large m/z displacements resulting from both large changes in mass and charge. However, charge reduction of the high m/z ions initially generated under native ESI conditions requires the ability to isolate high m/z ions and to analyze even higher m/z product ions. Digital ion trap (DIT) operation offers means for both high m/z ion isolation and high m/z mass analysis, in addition to providing conditions for the reaction of oppositely charged ions. The feasibility of conducting MIA experiments in a DIT that takes advantage of high m/z ion operation is demonstrated here using a tandem 2D-3D DIT instrument. Proof-of-concept MIA experiments with cations derived from β-galactosidase using the 20- charge state of human serum immunoglobulin G (IgG, ∼149 kDa) as the reagent anion are described. MIA experiments involving mixtures of ions derived from the E. coli. ribosome are also described. For example, three components in a mixture of 70S particles (>2.2 MDa) were resolved and assigned with masses and charges following an MIA experiment involving the 20- charge state of human serum IgG.

Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides

ABSTRACT Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

High-Throughput Glycan Profiling of Human Serum IgG Subclasses Using Parallel Reaction Monitoring Peptide Bond Fragmentation of Glycopeptides and Microflow LC-MS.

LC-MS-based N-glycosylation profiling in four human serum IgG subclasses (IgG1, IgG2, IgG3, and IgG4) often requires additional affinity-based enrichment of specific IgG subclasses, owing to the high amino acid sequence similarity of Fc glycopeptides among subclasses. Notably, for IgG4 and the major allotype of IgG3, the glycopeptide precursors share identical retention time and mass and therefore cannot be distinguished based on precursor or glycan fragmentation. Here, we developed a parallel reaction monitoring (PRM)-based method for quantifying Fc glycopeptides through combined transitions generated from both glycosidic and peptide bond fragmentation. The latter enables the subpopulation of IgG3 and IgG4 to be directly distinguished according to mass differences without requiring further enrichment of specific IgG subclasses. In addition, a multinozzle electrospray emitter coupled to a capillary flow liquid chromatograph was used to increase the robustness and detection sensitivity of the method for low-yield peptide backbone fragment ions. The gradient was optimized to decrease the overall run time and make the method compatible with high-throughput analysis. We demonstrated that this method can be used to effectively monitor the relative levels of 13 representative glycoforms, with a good limit of detection for individual IgG subclasses.

A novel capsid-XL32-derived adeno-associated virus serotype prompts retinal tropism and ameliorates choroidal neovascularization

Gene therapy has been adapted, from the laboratory to the clinic, to treat retinopathies. In contrast to subretinal route, intravitreal delivery of AAV vectors displays the advantage of bypassing surgical injuries, but the viral particles are more prone to be nullified by the host neutralizing factors. To minimize such suppression of therapeutic effect, especially in terms of AAV2 and its derivatives, we introduced three serine-to-glycine mutations, based on the phosphorylation sites identified by mass spectrum analysis, to the XL32 capsid to generate a novel serotype named AAVYC5. Via intravitreal administration, AAVYC5 was transduced more effectively into multiple retinal layers compared with AAV2 and XL32. AAVYC5 also enabled successful delivery of anti-angiogenic molecules to rescue laser-induced choroidal neovascularization and astrogliosis in mice and non-human primates. Furthermore, we detected fewer neutralizing antibodies and binding IgG in human sera against AAVYC5 than those specific for AAV2 and XL32. Our results thus implicate this capsid-optimized AAVYC5 as a promising vector suitable for a wide population, particularly those with undesirable AAV2 seroreactivity.

Bimetallic nickel/cobalt metal-organic framework-based electrochemical sensor for trace level detection of IgG in simulated human blood serum

Electrochemical sensing is a widely used technique for the detection of biomolecules because of its low cost, high sensitivity, and versatility in terms of the various techniques that can be employed. In this study, we report a facile, cost-effective, and highly selective bimetallic Ni/Co-MOF (Nickel/Cobalt-Metal Organic Framework) on Nickel foam (NF) based electrochemical sensor for the trace-level detection of IgG (Human Immunoglobulin) antibodies in the human blood serum. In this direction, we used a simple and efficient solvothermal method to synthesize the Ni/Co-MOF, and the morphological, structural, and chemical states of the elements are studied using different material characterization techniques like TEM, FESEM, XRD, XPS, etc. The Ni/Co-MOF modified NF sensor showed a dynamic range of detection (30 fM to 10 nM) having superior sensitivity of 28 µA cm−2 mol−1 with a lower limit of detection 27.2 fM. Ni/Co-MOF/NF is highly selective over other interfering species (UA, Cl−, Ca2+, Urea, Sulphate, BSA and HAS). Real-time analysis was done using the DPV technique by the standard addition method and the sensor shows an excellent recovery percentage ̴ 97 to 98 %. Also, the addition of Ni2+ to Co enhances the electrochemical properties of a material by raising the valence of Ni2+ in Ni/Co-MOF by transferring partial electrons from Ni2+ to Co2+. The work presented here confirms Ni/Co-MOF/NF as a flexible, high-performance electrode that can be used to detect other biomolecules used in clinical diagnosis and biomedical research.

Stability assessment of anti-bacterial antibodies in immunoglobulin G-depleted serum with validated immunoassays.

Aim: To investigate the stability of the anti-pneumococcal (PCP) and anti-haemophilus type B (Hib) immunoglobulins (IgGs) in human IgG-depleted serum samples frozen at -20°C. Materials & methods: Modified commercially available immunoassays (ELISAs) were bioanalytically validated. These ELISAs were used to measure levels of the two anti-bacterial IgG in samples kept at -20°C for up to 15months. Human IgG-depleted serum was spiked with GAMMAGARD Liquid to obtain those samples. Results: Both ELISAs passed the validation test. Anti-PCP IgG and anti-Hib IgG were shown to be stable for at least 15months at -20°C. Conclusion: These data confirm the stability of anti-bacterial IgG in human IgG-depleted serum and support the common practice of testing frozen samples.

Electrochemical immunosensor based on carbon nanofibers and gold nanoparticles for detecting anti-Toxoplasma gondii IgG antibodies.

Anelectrochemical immunosensor based on carbon nanofibers (CNFs) and gold nanoparticles (AuNPs) was developed for detectinganti-Toxoplasma gondii antibodies (anti-T. gondii) IgG in human serum. CNFs were produced using electrospinning and carbonization processes. Screen-printed carbon electrode (SPCE) surface was modified with CNFs and AuNPs which wereelectrodeposited onto the CNFs. Then, T. gondii antigen was immobilized onto the AuNPs/CNFs/SPCE. Afterward, anti-T. gondii IgG positive serum samples were coated on the modified electrode and assessed via adding anti-human IgG labeled with horseradish peroxidase (HRP) enzyme. The morphology of SPCE, CNFs, and AuNPs/CNFs/SPCE surface was characterized using field emission scanning electron microscopy (FESEM) equipped with energy dispersive spectroscopy (EDS). Characterization of CNFs was evaluated by Raman spectroscopy and X-ray diffraction (XRD). Electrochemical characterization of the immunosensor was verified using cyclic voltammetry (CV), and electrochemical response of modified electrode for anti-T. gondii IgG was detected via differential pulse voltammetry (DPV). This immunosensor was detectedin the range 0-200 U mL-1 with a low detection limit (9 × 10-3 U mL-1). In addition, the proposed immunosensor was exhibited with high selectivity, strong stability, and acceptable reproducibility and repeatability. Furthermore, there was a strong correlation between results obtained via the designed immunosensor and enzyme-linked immunosorbent assay (ELISA) as gold standard. In conclusion, the developed immunosensor isa promising route for rapid and accurate clinical diagnosis of toxoplasmosis.

Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions

PurposeAntibody drugs are usually formulated as highly-concentrated solutions, which would easily generate aggregates, resulting in loss of efficacy. Although low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore, knowing the physical properties at low pH under high concentration conditions is important.MethodsRaman spectroscopy was used to investigate pH-induced conformational changes of antibodies at 50 mg/ml. Experiments in pH 3 to 7 were performed for human serum IgG and recombinant rituximab.ResultsWe detected the evident changes at pH 3 in Tyr and Trp bands, which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH range demonstrated that the thermal transition temperature (Tm) was highest at pH 3. Acid-treated and neutralized one showed higher Tm than that of pH 7, indicating that their extent of intermolecular interactions correlated with the Tm values. Onset temperature was clearly different between concentrated and diluted samples. Colloidal analyses confirmed the findings of the Raman analysis.ConclusionOur studies demonstrated the positive correlation between Raman analysis and colloidal information, validating as a method for evaluating antibody conformation associated with aggregation propensities.

A low-fouling electrochemical biosensor for biomarker detection in serum based on designed α/β-peptides with anti-enzymolysis and antifouling capabilities

The zwitterionic peptides, especially those composed of glutamic (E) and lysine (K) groups have drawn enormous attention as antifouling biomaterials owing to their strong hydration capability and biocompatibility. However, the susceptibility of α-amino acid K to the proteolytic enzymes in human serum limited the broad application of such peptides in biological media. Herein, a new multifunctional peptide with favorable stability in human serum was designed, and it was composed of three sections with immobilizing, recognizing and antifouling capabilities, respectively. The antifouling section was composed of alternating E and K amino acids, but the enzymolysis-susceptive amino acid α-K was replaced by the unnatural β-K. Compared with the conventional peptide composed of all α-amino acids, the α/β-peptide exhibited significantly enhanced stability and longer antifouling performance in human serum and blood. The electrochemical biosensor based on the α/β-peptide showed a favorable sensitivity to its target IgG, with a quite wide linear range from 100 pg mL−1 to 10 μg mL−1 and a low detection limit (33.7 pg mL−1, S/N = 3), and it was promising for the detection of IgG in complex human serum. The tactic of designing antifouling α/β-peptides offered an efficient way to develop low-fouling biosensors with robust operation in complex body fluids.

The unique properties of IgG4 and its roles in health and disease.

IgG4 is the least abundant subclass of IgG in human serum and has unique functional features. IgG4 is largely unable to activate antibody-dependent immune effector responses and, furthermore, undergoes Fab (fragment antigen binding)-arm exchange, rendering it bispecific for antigen binding and functionally monovalent. These properties of IgG4 have a blocking effect, either on the immune response or on the target protein of IgG4. In this Review, we discuss the unique structural characteristics of IgG4 and how these contribute to its roles in health and disease. We highlight how, depending on the setting, IgG4 responses can be beneficial (for example, in responses to allergens or parasites) or detrimental (for example, in autoimmune diseases, in antitumour responses and in anti-biologic responses). The development of novel models for studying IgG4 (patho)physiology and understanding how IgG4 responses are regulated could offer insights into novel treatment strategies for these IgG4-associated disease settings.

Facile fabrication of hydrophilic covalent organic framework composites for highly selective enrichment of N-glycopeptides

The development of facilely synthetic materials acts an essential role in glycoproteome analysis, especially for the highly efficient enrichment of N-linked glycopeptides. In this work, a facile and timesaving route was introduced in which COFTP-TAPT served as a carrier and poly (ethylenimine) (PEI) and carrageenan (Carr) were successively coated on the surface via electrostatic interaction. The resultant COFTP-TAPT@PEI@Carr showed remarkable performance in glycopeptide enrichment with high sensitivity (2 fmol μL−1), high selectivity (1:800, molar ratio of human serum IgG to BSA digests), large loading capacity (300 mg g−1), satisfactory recovery (102.4 ± 6.0%) and reusability (at least eight times). Due to the brilliant hydrophilicity and electrostatic interactions between COFTP-TAPT@PEI@Carr and positively charged glycopeptides, the prepared materials could be applied in the identification and analysis in the human plasma of healthy subjects and patients with nasopharyngeal carcinoma. As a result, 113 N-glycopeptides with 141 glycosylation sites corresponding to 59 proteins and 144 N-glycopeptides with 177 glycosylation sites corresponding to 67 proteins were enriched from 2 μL plasma trypsin digests of the control groups and patients with nasopharyngeal carcinoma, respectively. 22 glycopeptides were identified only from the normal controls and 53 glycopeptides were detected only from the other set. The results demonstrated that this hydrophilic material was promising on a large scale and further N-glycoproteome research.

Highly-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples

Alterations of protein glycosylation can serve as sensitive and specific disease biomarkers. Labeling procedures for improved separation and detectability of oligosaccharides have several drawbacks, including incomplete derivatization, side-products, noticeable desialylation/defucosylation, sample loss, and interference with downstream analyses. Here, we develop a label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans. Our workflow provides a >45-fold increase in signal intensity compared to the conventional CZE-MS approaches used for N-glycan analysis. Qualitative and quantitative N-glycan profiling of purified human serum IgG, bovine serum fetuin, bovine pancreas ribonuclease B, blood-derived extracellular vesicle isolates, and total plasma results in the detection of >250, >400, >150, >310, and >520 N-glycans, respectively, using injected amounts equivalent to <25 ng of model protein and nL-levels of plasma-derived samples. Compared to reported results for biological samples of similar amounts and complexity, the number of identified N-glycans is increased up to ~15-fold, enabling highly sensitive analysis of sample amounts as low as sub-0.2 nL of plasma volume equivalents. Furthermore, highly sialylated N-glycans are identified and structurally characterized, and untreated sialic acid-linkage isomers are resolved in a single CZE-MS analysis.

Exploring Intensity Distributions and Sampling in SERS-Based Immunoassays.

Surface-enhanced Raman scattering (SERS) is a sensitive, widely used spectroscopic technique. However, SERS is perceived as poorly reproducible and insufficiently robust for standard applications in analytical chemistry. Here, we demonstrated that reliable SERS immunoassay quantification at low concentrations (pM range) can be achieved by careful experimental design and appropriate data analysis statistics. A SERS-based immunoassay for IgG in human serum (3.1-50.0 ng mL-1 or 20.6-333 pM) was developed as a proof of concept. Calibration curves were created using the population median of the band area, centered at 592 cm-1, of a SERS reporter (Nile Blue A). Histograms of 7200 SERS spectra show lognormal distributions. SEM images of the sensor platform confirm a correlation between the number of SERS probes (ERLs) at the surface and the SERS intensity response. The IgG immunosensor reported here presented a limit of detection of 1.11 ng mL-1 or 7.39 pM and a limit of quantification of 9.04 ng mL-1 or 60.30 pM, within a 95% confidence level. The % error of the predicted versus the actual response of a quality control (QC) sample was 0.13%. The percent error of the QC sample decreases exponentially with the number of measurements. Randomly selected spatially separated measurements provided lower QC % error than a larger number of measurements that were closely spaced. We propose that it is necessary to describe the measured populations using an appropriate sample size for good statistics and consider the interrogation of sufficiently large and well-separated areas of the sensor surface to achieve a reliable sampling.

Seroprevalence of tick-borne diseases in the Northwest Federal District of the Russian Federation

Introduction. Knowledge about tick-borne disease (TBD) distribution is necessary to improve prevention, whereas detection of human serum IgG antibodies against relevant pathogens is a method for monitoring TBD prevalence in local population. The study objective was to estimate seroprevalence of IgG antibodies against tick-borne encephalitis virus (TBEV), Borrelia burgdorferi sensu lato, Coxiella burnetii, Anaplasma phagocytophilum, and Ehrlichia chaffeensis/E. muris in healthy residents from the five territories of the Northwestern Federal District of the Russian Federation (Arkhangelsk Oblast, Leningrad Oblast, Pskov Oblast, the Republic of Komi and the Republic of Karelia). Materials and methods. In 20172019, a total of 1244 serum samples from healthy residents, not vaccinated against TBDs or other flavivirus-caused infections was studied by ELISA. Results. 21.7% of the sera samples contained IgG antibodies against a single TBD pathogen, whereas 2.1% showed signs of coinfection with two or more pathogens. The most common were IgG antibodies against TBEV (5 territories, 12.2%), followed by Borrelia burgdorferi sensu lato (5 territories, 3.5%), C. burnetii (4 territories, 2.9%), Anaplasma phagoсytophilum (3 territories, 1.6%), E. chaffeensis/E. muris (5 territories, 1.5%). The IgG antibodies were more common in men (55.2%) than in women (44.8%), being found virtually evenly in age-independent manner (from juniors under 18 to seniors over 60). Conclusion. The results of this first comprehensive serosurveillance study in the Northwestern Federal District of the Russian Federation assessing serum IgG antibodies against tick-borne diseases indicate a wide distribution of such pathogens. Moreover, infections caused by C. burnetii, Anaplasma phagocytophilum, and Ehrlichia chaffeensis/E. muris might be highly underdiagnosed.

Antibodies in the breastmilk of COVID-19 recovered women

ObjectiveHuman milk contains antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which may serve as a protective factor through passive immunization in infants. The objective of this study was to measure the levels of anti-SARS-CoV-2 IgG and IgA in human milk and serum after a SARS-CoV-2 infection.DesignBreast milk and serum samples from 72 lactating mothers with confirmed SARS-CoV-2 asymptomatic or symptomatic infection were collected 1-229 days after the onset of clinical symptoms related to COVID-19. Seventeen mothers with no history of COVID-19 served as a control group. Enzyme-Linked ImmunoSorbent Assay was performed to analyze antibodies against SARS-CoV-2.ResultsSARS-CoV-2-IgA human milk antibodies were detected in mothers and their concentrations were consistently higher than SARS-CoV-2-IgG antibodies. The serum and breastmilk samples of women with COVID-19 was characterized by a higher concentration of anti-RBD IgA and IgG than the serum from the control group without COVID-19. No statistically significant difference was observed between the antibody levels in the serum samples obtained from symptomatic and asymptomatic women exposed to SARS-CoV-2 and between the antibody level and the time from a positive SARS-CoV-2 test result over the period studied.ConclusionOur results confirm the presence of SARS-CoV-2 IgA and IgG antibodies in the breastmilk of COVID-19 recovered women and the possibility of these antibodies in providing specific immunologic benefits to breastfeeding infants such as protection against the virus transmission and severity of the acquired COVID-19 disease.

Immunoglobulin G N-glycan Biomarkers for Autoimmune Diseases: Current State and a Glycoinformatics Perspective.

The effective treatment of autoimmune disorders can greatly benefit from disease-specific biomarkers that are functionally involved in immune system regulation and can be collected through minimally invasive procedures. In this regard, human serum IgG N-glycans are promising for uncovering disease predisposition and monitoring progression, and for the identification of specific molecular targets for advanced therapies. In particular, the IgG N-glycome in diseased tissues is considered to be disease-dependent; thus, specific glycan structures may be involved in the pathophysiology of autoimmune diseases. This study provides a critical overview of the literature on human IgG N-glycomics, with a focus on the identification of disease-specific glycan alterations. In order to expedite the establishment of clinically-relevant N-glycan biomarkers, the employment of advanced computational tools for the interpretation of clinical data and their relationship with the underlying molecular mechanisms may be critical. Glycoinformatics tools, including artificial intelligence and systems glycobiology approaches, are reviewed for their potential to provide insight into patient stratification and disease etiology. Challenges in the integration of such glycoinformatics approaches in N-glycan biomarker research are critically discussed.

Loss of Dihydroxyacid Dehydratase Induces Auxotrophy in Bacillus anthracis.

Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that ΔilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to nonsupplemented wild type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and ΔilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis, and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts. IMPORTANCE Anthrax disease, caused by B. anthracis, can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.

Clinical and immunological features and prognosis of different phenotypes of occupational asthma

Introduction. Occupational asthma is a complex and heterogeneous disease. Development of personalized treatment and prevention tactics becomes feasible due to phenotyping, which means identifying of markers to combine cases of occupational asthma with similar manifestations (clinical, instrumental, laboratory) and prognosis to phenotypes. The study aims to determination and comparative analysis of spirographic and immunological parameters for different phenotypes of occupational asthma. Materials and methods. In this study, we included 170 patients with different phenotypes of occupational asthma and 50 participants in control group. The spirographic examination was performed using computer spirograph with determination of the following parameters: forced vital capacity (FVC), forced expiratory volume during the first second (FEV1), Tiffeneau-Pinelli index (FEV1/FVC), peak expiratory flow (PEF), maximal expiratory flow at 75%, 50%, 25% of the forced vital capacity (MEF75%VC, MEF50%VC, MEF25%VC). Quantification of the immunoglobulins IgA, IgM, and IgG in human serum was conducted by Mancini method; levels of IgE, C-reactive protein and fibronectin were determined using a solid-phase enzyme immunoassay. Fibrinogen concentration in plasma was measured by an automatic coagulometer. Haptoglobin concentration in serum was determined by spectrophotometry. Results. According to spirometry and laboratory results obtained, there is a strong evidence, that the phenotype "occupational asthma - occupational chronic obstructive pulmonary disease" has the lowest values in pulmonary function tests and the most significant changes in immunoglobulins, fibronectin and acute phase proteins levels among other studied phenotypes of occupational asthma. Conclusions. Dynamic determination of spirometric, immunological parameters, fibronectin and proteins of the acute phase of inflammation in workers at risk for the formation of occupational bronchial asthma may be recommended to be carried out once every 6 months, followed by the isolation of disease phenotypes. This will optimize diagnostics, therapeutic and preventive tactics, as well as predict the course of this pathology.