Serum Immunoreactivity Research Articles

Detection of IgG Anti-Giardia duodenalis Antibodies in Sera by Indirect Immunofluorescence and Western Blotting Assays.

Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25kDa, 27-31kDa and 45-55kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins.

Expression of concern on: Immunoreactivity of sera from low to moderate malaria-endemic areas against plasmodium vivax rPvs48/45 proteins produced in Escherichia coli and Chinese hamster ovary systems.

Expression of concern on: Immunoreactivity of sera from low to moderate malaria-endemic areas against plasmodium vivax rPvs48/45 proteins produced in Escherichia coli and Chinese hamster ovary systems.

Adhesion of Bartonella henselae to Fibronectin Is Mediated via Repetitive Motifs Present in the Stalk of Bartonella Adhesin A.

ABSTRACTAdhesion to host cells is the first and most crucial step in infections with pathogenic Gram-negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). Bartonella henselae targets the extracellular matrix glycoprotein fibronectin (Fn) via the Bartonella adhesin A (BadA) attaching the bacteria to the host cell. The TAA BadA is characterized by a highly repetitive passenger domain consisting of 30 neck/stalk domains with various degrees of similarity. To elucidate the motif sequences mediating Fn binding, we generated 10 modified BadA constructs and verified their expression via Western blotting, confocal laser scanning, and electron microscopy. We analyzed their ability to bind human plasma Fn using quantitative whole-cell enzyme-linked immunosorbent assays (ELISAs) and fluorescence microscopy. Polyclonal antibodies targeting a 15-mer amino acid motif sequence proved to reduce Fn binding. We suggest that BadA adheres to Fn in a cumulative effort with quick saturation primarily via unpaired β-strands appearing in motifs repeatedly present throughout the neck/stalk region. In addition, we demonstrated that the length of truncated BadA constructs correlates with the immunoreactivity of human patient sera. The identification of BadA-Fn binding regions will support the development of new “antiadhesive” compounds inhibiting the initial adherence of B. henselae and other TAA-expressing pathogens to host cells.IMPORTANCE Trimeric autotransporter adhesins (TAAs) are important virulence factors and are widely present in various pathogenic Gram-negative bacteria. TAA-expressing bacteria cause a wide spectrum of human diseases, such as cat scratch disease (Bartonella henselae), enterocolitis (Yersinia enterocolitica), meningitis (Neisseria meningitis), and bloodstream infections (multidrug-resistant Acinetobacter baumannii). TAA-targeted antiadhesive strategies (against, e.g., Bartonella adhesin A [BadA], Yersinia adhesin A [YadA], Neisseria adhesin A [NadA], and Acinetobacter trimeric autotransporter [Ata]) might represent a universal strategy to counteract such bacterial infections. BadA is one of the best characterized TAAs, and because of its high number of (sub)domains, it serves as an attractive adhesin to study the domain-function relationship of TAAs in the infection process. The identification of common binding motifs between TAAs (here, BadA) and their major binding partner (here, fibronectin) provides a basis toward the design of novel “antiadhesive” compounds preventing the initial adherence of Gram-negative bacteria in infections.

Identification of clinical factors related to antibody-mediated immune response to the subfornical organ.

We recently reported cases of adipsic hypernatremia caused by autoantibodies against the subfornical organ in patients with hypothalamic-pituitary lesions. This study aimed to clarify the clinical features of newly identified patients with adipsic hypernatremia whose sera displayed immunoreactivity to the mouse subfornical organ. Observational cohort study of patients diagnosed with adipsic hypernatremia in Japan, United States, and Europe. The study included 22 patients with adipsic hypernatremia but without overt structural changes in the hypothalamic-pituitary region and congenital disease. Antibody response to the mouse subfornical organ was determined using immunohistochemistry. The clinical characteristics were compared between the patients with positive and negative antibody responses. Antibody response to the mouse subfornical organ was detected in the sera of 16 patients (72.7%, female/male ratio, 1:1, 12 pediatric and 4 adult patients). The prolactin levels at the time of diagnosis were significantly higher in patients with positive subfornical organ (SFO) immunoreactivity than in those with negative SFO immunoreactivity (58.9 ± 33.5 vs. 22.9 ± 13.9 ng/ml, p < .05). Hypothalamic disorders were found in 37.5% of the patients with positive SFO immunoreactivity. Moreover, six patients were diagnosed with rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation/neural tumor syndrome after the diagnosis of adipsic hypernatremia. Plasma renin activity levels were significantly higher in patients with serum immunoreactivity to the Nax channel. The patients with serum immunoreactivity to the SFO had higher prolactin levels and hypothalamic disorders compared to those without the immunoreactivity. The clinical characteristics of patients with serum immunoreactivity to the subfornical organ included higher prolactin levels and hypothalamic disorders, which were frequently associated with central hypothyroidism and the presence of retroperitoneal tumors.

Immunoreactivity of Polish Lyme Disease Patient Sera to Specific Borrelia Antigens—Part 1

The diverse clinical picture and the non-specificity of symptoms in Lyme disease (LD) require the implementation of effective diagnostics, which should take into account the heterogeneity of Borrelia antigens. According to available guidelines, laboratories should use a two-tier serological diagnosis based on the enzyme-linked immunosorbent (ELISA) screening test and confirmation of the immunoblot (IB). The aim of the study was to investigate the immunoreactivity of LD patient sera to Borrelia antigens and to attempt to identify the genospecies responsible for LD using an ELISA–IB assay combination. Eighty patients with suspected LD and 22 healthy people participated in the study. All samples were tested with ELISA and IB assays in both IgM and IgG antibodies. In the case of the ELISA assay, more positive results were obtained in the IgM class than in the IgG class. In the case of the IB assay, positive results dominated in the IgG class. Positive results obtained in the IB assay most often showed IgM antibodies against the OspC and flagellin antigens, whereas the IgG antibodies were against VlsE, BmpA, OspC, p41, and p83 antigens. The IB assay is an important part of LD serodiagnosis and should be mandatory in diagnostic laboratories.

Cancer-testis antigen ACRBP expression and serum immunoreactivity in ovarian cancer: Its association with prognosis.

IntroductionCancer testis (CT) antigens are attractive targets for cancer immunotherapy because of their expression restriction and immunogenicity. The acrosin binding protein (ACRBP) is a member of CT antigens. This study aimed to evaluate ACRBP expression and immunogenicity in ovarian cancer (OC).MethodsThe expression level of ACRBP in OC tissues, normal ovarian tissues, and cell lines was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry. We determined the levels of ACRBP antigen and antibody in serum samples collected from patients with OC and healthy donors using enzyme‐linked immunosorbent assays (ELISA), the level of ACRBP in cell‐cultured medium was also tested.ResultsACRBP mRNA and protein expressions were upregulated in OC tissues relative to normal tissue, especially highly expressed in epithelial ovarian cancer (EOC). Moreover, ACRBP expression was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage and chemosensitivity. Serological analysis showed that anti‐ACRBP antibody was detected in the sera of 16 of the 56 (28.5%) patients with OC but not in healthy donors. The area under the receiver operating characteristic curve for ACRBP antibody was 0.802 (95% confidence interval [CI]: 0.708–0.876), and the sensitivity and specificity for ACRBP antibody was 85.71% and 55.0%, respectively. Kaplan–Meier analysis revealed that the overall survival (OS) and disease‐free survival (DFS) in OC patients with high ACRBP expression were significantly lower than those with low expression (p = 0.040, p = 0.021). However, ACRBP antibody level was not associated with prognosis.ConclusionACRBP expression was upregulated in OC tissues and induced humoral immune response in patients with OC, suggesting that ACRBP is a potential prognostic biomarker and a target of tumor immunotherapy for OC.

Evaluation of Serum Immunoreactivity to Common Indigenous Iranian Inhalation and Food Allergens in Patients with Meniere’s Disease

ABSTRACT Background A few studies investigated the relationship between allergy and Meniere disease considering complete allergen panel. We aimed to evaluate the serum immunoreactivity in patients with Meniere’s disease (MD) compared with healthy people according to common indigenous Iranian inhalation and food allergens. Methods Thirty-nine patients with MD referred to Rasoul Akram Hospital (Tehran, Iran) were evaluated and compared with a 41 membered control group. A panel of common inhalation and food allergens (using an immunoblotting method), as well as total immunoglobulin E (IgE) level (using the sandwich enzyme-linked immunosorbent assay method), were checked on the patients’ serum. Results The mean total IgE level was 193.85 ± 175.43 IU/ml in the patients with MD and 117.61 ± 138.05 IU/ml in the control group, which was significantly higher than the other subjects in the control group (P = .016). There was a significant difference between the two groups regarding inhalation allergens such as; sweet vernal grass, cultivated rye, cultivated oat, Russian thistle, goosefoot, and rough pigweed (P = .01–0.038). Patients with MD reported more reactive to food allergens such as; rye flour, hazelnut, pepper, citrus mix 2, potato, strawberry, and celery allergens. There was a significant relationship between Meniere and serum immunoreactivity to inhalation and food allergens (both P = .001). Conclusion: Serum total IgE level in patients with MD (in both inhalation and food allergens groups) was higher than the control group, and there was a relationship between MD and immunoreactivity to common indigenous inhalation and food allergens of Iran.

Human immune response to Phlebotomus sergenti salivary gland antigens in a leishmaniasis-endemic focus in Iran

ABSTRACT Salivary proteins specific antibodies have been shown to be useful biomarkers of exposure to sand fly bites. This study aimed to investigate the level, duration, and dynamics of the human immune response against the SGL of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae), and to assess the immunoreactivity of human sera with SGL components in an endemic area of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The study was carried out in 2-phase; longitudinal and cross-sectional. Sand flies were collected monthly from indoors and outdoors. In the longitudinal study, sera from healthy volunteers were collected monthly, and in the cross-sectional study, sera from healthy volunteers and patients with ACL lesion/s, were collected for immunoassay studies. The level of anti-P. sergenti saliva IgG was detected using the ELISA. Immunoreactivity of individual human sera with saliva components was also assessed by western blotting. Phlebotomus sergenti was the predominant sand fly species in the study area. The maximum and minimum percentages of IgG responses were seen in October (66%) and March (29%), respectively. Additionally, the cross-sectional study showed that 59.3% of the healthy volunteers and 80% of the patients were IgG positive. The antibody response against P. sergenti salivary gland was high during the sand fly active season and declined by the end of the activity of the vectors. Antibody response against the SGL components of P. sergenti was transient and individual-specific. Some individuals shared a strong reaction against certain individual antigens, which could be considered as vector exposure markers for further investigation. List of abbreviations ELISA: Enzyme-Linked Immunosorbent Assay; SDS PAGE: Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis; SGL: Salivary Gland Lysate; ACL: Anthroponotic Cutaneous Leishmaniasis; PBS: Phosphate Buffered Saline; BCA: Bicinchoninic Acid; PBS-T: Phosphate Buffered Saline Tween; FBS: Fetal Bovine Serum; HRP: Horseradish Peroxidase; TMB: 3,3′,5,5′-Tetramethylbenzidine; PVDF: Polyvinylidene Difluoride; SGA: Salivary Gland Antigens; OD: Optical Density; KDa: Kilodalton; VL: Visceral Leishmaniasis; CL: Cutaneous Leishmaniasis; SGs: Salivary glands

Functional MAIT Cells Are Associated With Reduced Simian-Human Immunodeficiency Virus Infection.

Mucosa-associated invariant T (MAIT) cells are recently characterized as a novel subset of innate-like T cells that recognize microbial metabolites as presented by the MHC-1b-related protein MR1. The significance of MAIT cells in anti-bacterial defense is well-understood but not clear in viral infections such as SIV/HIV infection. Here we studied the phenotype, distribution, and function of MAIT cells and their association with plasma viral levels during chronic SHIV infection in rhesus macaques (RM). Two groups of healthy and chronic SHIV-infected macaques were characterized for MAIT cells in blood and mucosal tissues. Similar to human, we found a significant fraction of macaque T cells co-expressing MAIT cell markers CD161 and TCRVα-7.2 that correlated directly with macaque MR1 tetramer. These cells displayed memory phenotype and expressed high levels of IL-18R, CCR6, CD28, and CD95. During chronic infection, the frequency of MAIT cells are enriched in the blood but unaltered in the rectum; both blood and rectal MAIT cells displayed higher proliferative and cytotoxic phenotype post-SHIV infection. The frequency of MAIT cells in blood and rectum correlated inversely with plasma viral RNA levels and correlated directly with total CD4 T cells. MAIT cells respond to microbial products during chronic SHIV infection and correlated positively with serum immunoreactivity to flagellin levels. Tissue distribution analysis of MAIT cells during chronic infection showed significant enrichment in the non-lymphoid tissues (lung, rectum, and liver) compared to lymphoid tissues (spleen and LN), with higher levels of tissue-resident markers CD69 and CD103. Exogenous in vitro cytokine treatments during chronic SHIV infection revealed that IL-7 is important for the proliferation of MAIT cells, but IL-12 and IL-18 are important for their cytolytic function. Overall our results demonstrated that MAIT cells are enriched in blood but unaltered in the rectum during chronic SHIV infection, which displayed proliferative and functional phenotype that inversely correlated with SHIV plasma viral RNA levels. Treatment such as combined cytokine treatments could be beneficial for enhancing functional MAIT cells during chronic HIV infection in vivo.

Identification of common vaginal Lactobacilli immunoreactive proteins by immunoproteomic techniques.

Lactobacilli are considered as the most important microorganisms in regulating immune system and maintaining vaginal health. The uses and benefits of Lactobacilli as probiotics, particularly the regulation of immune system, are dependent on the strain used and a comprehensive understanding of their effects on the host. Several factors have been identified in Lactobacilli that influence the immune response, such as exopolysaccharides and proteins. The current study was designed to investigate the serum immunoreactivity of healthy women against common vaginal Lactobacilli immunoreactive proteins. Three common vaginal Lactobacillus strains (L. crispatus L1, L. gasseri L9, and L. fermentum L2) were compared for immune response. The ELISA results showed that the levels of total immunoglobulin (Ig-total) antibody for L. crispatus L1, L. fermentum L2, and L. gasseri L9 were 47%, 45% and 29%, respectively. Regarding the lower prevalence of L. fermentum L2 in comparison with the other two strains, the approximately equal levels of Ig-total compared to L. crispatus L1 and more than L. gasseri L9 indicate that L. fermentum L2 has the greater antigenicity ability. Accordingly, the immunoreactive proteins of L. fermentum L2 were identified using MALDI-TOF-MS detected by SDS-PAGE and Western blotting. These proteins included 30s ribosomal protein S4 and 50s ribosomal protein L5. Antigenic epitopes on the 3D structure of these proteins was also predicted using bioinformatics analysis. The presence of antibody in serum of healthy pre-menopausal women indicates that Lactobacilli (normal flora) proteins can stimulate host immune response. Purification and further studies of the proteins may allow their potential use as an adjuvant to improve the efficacy of vaccines.

IMMUNOCHEMICAL DIAGNOSIS OF CANCER. PROTOTYPING

A prototype of the method of immunochemical detection of different types of solid cancers (primary and recurrent) in the early stages was developed. According to the initial hypothesis, the sera of patients with malignant tumors of different localization and different histological nature, contain different sets of autoantibodies (auto-Ab) of IgG class to many cancer-associated antigens (CA-AG). The content of such auto-Ab differs in cancer patients and patients with non-malignant chronic diseases, which determines the difference in serum immunoreactivity profiles of cancer patients and patients with non-malignant diseases. Confirmation of this hypothesis opens up prospects for the creation of simple and cheap laboratory methods of mass preventive examination of the population for the early detection of different cancers. The confirmation of the hypothesis was obtained. Moreover, even with non-optimal sets of test antigens, with the help of solid-phase ELISA it was possible to achieve sensitivity of 71% and specificity of 68% in the differentiation of blood sera of cancer (lung, stomach, ovary, prostate) and non-cancer patients (chronic inflammatory diseases of the lungs, stomach, ovary, prostate) and nearly 90% in the differentiation of healthy individuals from cancer patients.

Changes of Serum IgG Dimer Levels after Treatment with IVIg in Guillain-Barré Syndrome.

Intravenous immunoglobulins (IVIg) are standard treatment for Guillain-Barré syndrome (GBS). Their exact mechanisms of action are versatile and not fully understood. One possible mechanism is neutralization of circulating autoantibodies via binding to anti- idiotypic antibodies forming idiotype-anti-idiotype dimeric IgG immune complexes. To examine the role of immune complex formation as mechanism of action for IVIg in GBS, 34 C57Bl/6 mice were either treated with anti-ganglioside antibodies and IVIg or IVIg and PBS alone, whereas eight additional mice were treated either with anti-ganglioside autoantibodies and IVIg or anti-ganglioside autoantibodies alone. Subsequently IgG dimer formation was assessed by high performance liquid chromatography (HPLC) and enzyme- linked immunosorbent assay (ELISA). In addition, IgG dimer formation was measured in sera of eight GBS patients who were treated with IVIg. In mice, a significant increase of dimeric IgG after administration of anti-ganglioside antibodies and IVIg could be observed. Re-monomerized IgG dimers showed immunoreactivity against gangliosides and serum immunoreactivity was significantly reduced after IVIg infusion. Likewise also in GBS patients, IgG dimer formation could be detected after IVIg treatment. Our data indicate that dimeric IgG immune complexes contain anti-idiotypic antibodies and provide proof of concept that IVIg treatment in GBS results in measurable amounts of IgG dimers. Larger patient cohorts are needed to evaluate serum IgG dimer increase as a possible marker for treatment response in GBS. Graphical Abstract Mechanism of action: Intravenous immunoglobulins (IVIg) and anti-ganglioside antibodies form dimeric IgG immune complexes, preventing axonal damage in Guillain-Barré Syndrome.

Cilastatin protects against tacrolimus-induced nephrotoxicity via anti-oxidative and anti-apoptotic properties

BackgroundCilastatin (CL) is an inhibitor of dehydropeptidase-I, which is safely used in clinical practice to prevent nephrotoxicity of antibiotics. Tacrolimus (TAC) is the most important immunosuppressant in renal transplantation, but it causes considerable nephrotoxicity. We evaluated the protective effects of CL against chronic TAC-induced nephropathy.MethodsChronic nephropathy was induced by administering TAC (1.5 mg/kg/ day, subcutaneous injection) to rats on a low-salt diet for 4 weeks. CL (75 or 150 mg/kg/day, intraperitoneal injection) was concomitantly treated with TAC. Human proximal tubular cells were exposed to TAC (50 μg/mL) with or without CL (250 μg/mL). We investigated the effects of CL on TAC-induced injury in terms of renal function, tubulointerstitial fibrosis, and inflammation. The effects of CL on oxidative stress and apoptosis were evaluated in both in vivo and in vitro models of TAC nephrotoxicity.ResultsCL treatment improved TAC-induced renal dysfunction and decreased renal interstitial fibrosis (reduced expression of e-cadherin and TGFβ-1) and interstitial inflammation (decreased infiltration of ED-1-positive and osteopontin-positive cells). Compared to TAC treatment alone, CL co-treatment reduced oxidative stress (serum 8-OHdG level and immunoreactivity of 8-OHdG and 4-HHE in renal tissue) and increased renal expression of anti-oxidant enzyme, manganese superoxide dismutase. CL treatment decreased apoptotic cell death (decreased TUNEL-positive cells and reduced expression of active caspase-3) in TAC-treated kidney. In vitro CL treatment prevented tubular cell death from TAC treatment and decreased number of annexin V-positive cells were observed in cilastatin-cotreated cells.ConclusionCL has protective effects against chronic TAC-induced nephrotoxicity owing to its anti-oxidative and anti-apoptotic properties.

Searching for Serum Antibodies to Neuronal Proteins in Patients With Myalgic Encephalopathy/Chronic Fatigue Syndrome

PurposeA role for the immune system in causing myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is long suspected, but few studies have looked for specific autoantibodies that might contribute to the symptoms. Our aim was to look for evidence of antibodies to neuronal proteins in patients with ME/CSF. MethodsSera samples from 50 patients and 50 healthy individuals were sent coded to the Neuroimmunology Laboratory in Oxford. Screening for antibody binding to neuronal tissue was performed on brain tissue and neuronal cultures. Specific serum antibodies were assessed by antigen-specific cell-based assays and radioimmunoassays. After antibody testing, the associations between seropositive status and clinical data were investigated. FindingsOverall, 8 patients and 11 participants were found to have some serum immunoreactivity toward neuronal or neuromuscular junction proteins, but only 1 patient and 2 participants had specific serum antibodies. Nevertheless, seropositive status in patients with ME was associated with shorter duration since onset and a more severe disease. ImplicationsThe results indicate no overall increased frequency of antibodies to neuronal proteins in ME/CSF and no evidence of a specific antibody that might be causative or contribute to clinical features in patients. However, the association of seropositive status with shorter duration of disease and more severe symptoms suggests a possible role of antibodies at onset in some patients and should be the focus of future studies.

Immunoreactivity of Gluten-Sensitized Sera Toward Wheat, Rice, Corn, and Amaranth Flour Proteins Treated With Microbial Transglutaminase.

The aim of this study was to analyze the effects of microbial transglutaminase (mTG) on the immunoreactivity of wheat and gluten-free cereals flours to the sera of patients with celiac disease (CD) and non-celiac gluten sensitivity (NCGS). Both doughs and sourdoughs, the latter prepared by a two-step fermentation with Lactobacillus sanfranciscensis and Candida milleri, were studied. In order to evaluate the IgG-binding capacity toward the proteins of the studied flours, total protein as well as protein fractions enriched in albumins/globulins, prolamins and glutelins, were analyzed by SDS-PAGE and enzyme-linked immunosorbent assay (ELISA). Results showed that while mTG modified both gluten and gluten-free flour by increasing the amount of cross-linked proteins, it did not affect the serum's immune-recognition. In fact, no significant differences were observed in the immunoreactivity of sera from CD and NCGS patients toward wheat and gluten-free protein extracts after enzyme treatment, nor did this biotechnological treatment affect the immunoreactivity of control samples or the sera of healthy patients. These results suggest that mTG may be used as a tool to create innovative gluten and gluten-free products with improved structural properties, without increasing the immune-reactivity toward proteins present either in doughs or in sourdoughs.

Composition of the Blood and Reflection of the Health State of Human Body

Blood (blood plasma) is a special all-pervading substance, functionally co-tuning all organs, tissues and cells of the body. To some extent blood is similar to the Ether of the ancients. In addition to performing house-keeping functions, blood is a medium for the transfer of huge amounts of information, which is continuously exchanged between all the compartments and structures of the macro-organism and its microbiome. This information is transmitted mostly in the form of chemical signals (peptides, micro-RNA, extracellular DNA, the products of the microbiome, antibodies, etc.), the totality of which controls lots of biological processes. Blood is not only a controlling, but also a reflecting environment: dynamic changes in the composition of this environment carry information about the smallest changes in the state of individual populations of cells, tissues, organs and the body as a whole. The prospects of practical using of information about the state of the organism, transmitted by blood and reflected in individual’s serum immunoreactivity profiles are analyzed.

Characterization of a Synovial B Cell–Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast–like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA− RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti–cit-CRT Abs were preferentially detected in ACPA+ versus ACPA− RA sera. We identified a synovial B cell–derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Development of 64Cu-NOTA-Trastuzumab for HER2 Targeting: A Radiopharmaceutical with Improved Pharmacokinetics for Human Studies.

The purpose of this study was to develop 64Cu-labeled trastuzumab with improved pharmacokinetics for human epidermal growth factor receptor 2 (HER2). Methods: Trastuzumab was conjugated with SCN-Bn-NOTA and radiolabeled with 64Cu. Serum stability and immunoreactivity of 64Cu-NOTA-trastuzumab were tested. Small-animal PET imaging and biodistribution studies were performed in a HER2-positive breast cancer xenograft model (BT-474). The internal dosimetry for experimental animals was determined using the image-based approach with the Monte Carlo N-particle code. Results:64Cu-NOTA-trastuzumab was prepared with high radiolabeling yield and radiochemical purity (>98%) and showed high stability in serum and good immunoreactivity. Uptake of 64Cu-NOTA-trastuzumab was highest at 48 h after injection as determined by PET imaging and biodistribution results in BT-474 tumors. The blood radioactivity concentrations of 64Cu-NOTA-trastuzumab decreased biexponentially with time in both mice with and mice without BT-474 tumor xenografts. The calculated absorbed dose of 64Cu-NOTA-trastuzumab was 0.048 mGy/MBq for the heart, 0.079 mGy/MBq for the liver, and 0.047 mGy/MBq for the spleen. Conclusion:64Cu-NOTA-trastuzumab was effectively targeted to the HER2-expressing tumor in vitro and in vivo, and it exhibited a relatively low absorbed dose due to a short residence time. Therefore, 64Cu-NOTA-trastuzumab could be applied to select the right patients and right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread.

Neutrophil extracellular traps deficiency aggravates Citrobacter rodentium infection and the associated intestinal inflammation

Abstract Peptidyl arginine deiminase-4 (PAD4) is required for the formation of neutrophil extracellular traps (NETs), which is part of the host antimicrobial innate immune response. However, the role of NETs in the pathogenesis and clearance of Citrobacter rodentium (a well-characterized mouse enteropathogen) infection remains unclear. In this study, we challenged the PAD4-deficient (Pad4KO; NETs-deficient) and WT littermates with C. rodentium. Luminal colonization of C. rodentium in Pad4KO mice peaked between 11-14 days post-infection (pi), whereas WT mice cleared the infection with no detectable level of the pathogen in fecal shedding after 7-10 days. Bacterial dissemination to spleen and mesenteric lymph node was significantly higher in Pad4KO mice at day 8 and 28 pi compared to WT. Additionally; Pad4KO mice displayed splenomegaly, colomegaly and elevated colonic and systemic pro-inflammatory markers (lipocalin 2 and serum amyloid A). Histological analysis demonstrated that transmissible colonic hyperplasia and goblet cell depletion were more pronounced in Pad4KO mice. Pad4KO mice also exhibited epithelial barrier dysfunction as indicated by their heightened serum immunoreactivity to bacterial proteins. Flow cytometric analysis revealed a substantial reduction in IL-10-producing macrophages and Th17 cells in the lamina propria and spleens of Pad4KO mice compared to WT mice. However, colonic expression of IL-17A and IL-22 were highly elevated in Pad4KO mice on day 8 pi, but were attenuated on day 28 both in Pad4KO and WT mice. Taken together, our findings highlight that NETs are indispensable in promoting clearance of C. rodentium and resolving the associated intestinal inflammation.

Altered gastrointestinal motility involving autoantibodies in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that, in addition to motor, sensory, and cognitive symptoms, also causes constipation, which is poorly understood. Here, we characterize gastrointestinal (GI) dysmotility in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and evaluate whether autoantibodies target the enteric nervous system (ENS) and cause dysmotility. EAE was induced in male SJL and B6 mice. GI motility was assessed in vivo and ex vivo in wild type (WT) and B cell-deficient mice. MS and EAE serum was used to survey potential targets in the ENS and changes in the ENS structure were characterized using immunohistochemistry. EAE mice developed accelerated gastric emptying and delayed whole GI transit with reduced colonic motility. Fecal water content was reduced, and colonic migrating myoelectrical complexes (CMMC) and slow waves were less frequent. Colons from EAE mice exhibited decreased GFAP levels in glia. Sera from MS patients and from EAE mice targeted ENS neurons and glia. B-cell deficiency in EAE protected against colonic dysmotility. Consistent with symptoms experienced in MS, we demonstrate that EAE mice widely exhibit features of GI dysmotility that persisted in the absence of extrinsic innervation, suggesting direct involvement of ENS neurocircuitry. The absence of GI dysmotility in B cell-deficient mice with EAE together with EAE and MS serum immunoreactivity against ENS targets suggests that MS could be classified among other diseases known to induce autoimmune GI dysmotility.