IgG Activity Research Articles

Autoimmunity and immune system dysregulation in schizophrenia: IgGs from sera of patients hydrolyze myelin basic protein.

Several different theories of schizophrenia (SCZ) were discussed; the causes of this disease are not yet clear. Using ELISA, it was shown that titers of autoantibodies against myelin basic protein (MBP) in SCZ patients are ~1.8-fold higher than in healthy individuals but 5.0-fold lower than in patients with multiple sclerosis. Several rigid criteria were checked to show that the MBP-hydrolyzing activity is an intrinsic property of SCZ IgGs. Approximately 82% electrophoretically homogeneous SCZ IgGs purified using several affinity sorbents including Sepharose with immobilized MBP hydrolyze specifically only MBP but not many other tested proteins. The average relative activity of IgGs from patients with negative symptoms was 2.5-fold higher than that of patients with positive symptoms of SCZ, and it increases with the duration of this pathology. It was shown that abzymes are the earliest statistically significant markers of many autoimmune pathologies. Our findings surmise that the immune systems of individual SCZ patients can generate a variety of anti-MBP abzymes with different catalytic properties, which can attack MBP of the myelin-proteolipid shell of axons. Therefore, autoimmune processes together with other mechanisms can play an important role in SCZ pathogenesis. MBP-hydrolyzing antibodies were previously detected in the blood of 80% to 90% of patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, some similar neuropsychiatric indicators of disease common to SLE, MS, and SCZ were described in the literature. Thus, the destruction of the myelin sheath and the production of MBP-hydrolyzing antibodies can be a common phenomenon for some different diseases.

Sublingual administration of 2-hydroxyethyl methacrylate enhances antibody responses to co-administered ovalbumin and Streptococcus mutans

Objective: The oral mucosa of patients undergoing dental procedures is often exposed to residual monomers leaking from incompletely cured acrylic resins. We investigated whether 2-hydroxyethyl methacrylate (HEMA) monomers applied to the sublingual mucosa in mice modulate the antibody responses towards co-administered ovalbumin (OVA) or live oral bacteria.Material and methods: OVA, live mouse oral commensal Lactobacillus murinus or live human oral commensal Streptococcus mutans were administered sublingually with or without HEMA to BALB/c mice on four weekly occasions. One week after the last administration, the experiment was terminated and serum antibody levels were analyzed using ELISA.Results: Significantly increased IgG and IgE anti-OVA antibody activity was found in the sera from mice immunized with OVA together with HEMA, as compared to mice immunized with OVA alone. Likewise, S. mutans together with HEMA induced an IgG anti-S. mutans antibody response that was significantly higher than the antibody response detected after application of S. mutans alone. No IgG anti-L. murinus antibody response was detected in mice immunized with L. murinus together with HEMA, as compared to the background activity.Conclusions: We report that HEMA monomers have adjuvant properties when sublingually administered in combination with OVA or S. mutans.

P.1.017 - New link between inflammation and schizophrenia: IgG with nuclease activity

Introduction Schizophrenia is known to be associated with the disorganization of neurotransmission processes in the brain as well as immunological disturbances [1]. Inflammation in the perinatal period can lead to a violation of synaptic plasticity and pruning of synapses in the adulthood [2]. Also, microRNAs play an important role in neuronal development. The microRNA expression profiles in the circulation and brain are altered in schizophrenia. It is shown that a change in the levels of microRNAs in the blood serum or brain leads to a violation of behavior [3]. We have shown that the formation of catalytic antibodies (abzymes) is one of the signs of immune system pathology. Catalytic antibodies hydrolyzing DNA were detected in the serum of patients with schizophrenia [4]. We assume that antibodies hydrolyzing the circulating regulatory nucleic acids can play a role in schizophrenia pathogenesis. Objectives To investigate a) microRNA-hydrolyzing activity of IgG antibodies from the serum of patients with schizophrenia; b) correlation of the microRNA-hydrolyzing activity level with clinical features of the disease; c) the cytotoxicity of isolated IgG on the human neuroblastoma cell culture IMR-32. Materials and Methods In our study, 35 patients with a verified diagnosis of simple or paranoid schizophrenia and 20 healthy donors were included. The average age in the group of patients was 35.8±8.9 years, duration of disease – 9.1±5.4 years. Electrophoretically and immunologically homogeneous IgGs were obtained by affinity chromatography of the serum proteins under conditions removing non-specifically bound proteins. The microRNA-hydrolyzing activity of IgG was detected by the level of hydrolysis of two neuro-specific fluorescently labeled microRNAs (miR-137 and miR-219a-2-3p). The products of the hydrolysis were analyzed by PAGE under denaturing conditions using laser scanner. Cytotoxicity of antibodies on human neuroblastoma cell culture IMR-32 was studied using MTT-test. Statistical analysis was carried out using the Pearson’s correlation in the Statistica 10 software. Results We have shown for the first time that IgG from all schizophrenia patients possesses microRNA-hydrolyzing activity. Using strict criteria, we proved that the observed activity is an intrinsic property of IgGs in schizophrenia. Interestingly, the microRNA-hydrolyzing activity level on miR-137 is strongly negatively correlated with the disease duration (R=–0.879). The activity decreased with increasing duration of the disease. Also, the level of RNase activity on miR-219a-2-3p was positively correlated with the values of the General Psychopathology PANSS scale and the PANSS Total score (R>0.8). It was shown that the RNase activity is metal-dependent. The kinetic parameters of nuclease reactions catalyzed by IgG were significantly lower than that for native enzymes. Besides, we detected the major hydrolysis sites of two neuro-specific microRNAs. Also, we show that IgG from schizophrenia patients with nuclease activity reduced the survival rate neuroblastoma IMR-32 cell culture by 10-28%. Conclusion We propose that immunological disorders can play an important role in schizophrenia, especially in the prodromal period. The environment and genetic predisposition can lead to inflammation and the formation of antibodies hydrolyzing the microRNAs. These antibodies can contribute to changes in the level of circulating microRNAs and schizophrenia development.

Affinity maturation of an TpoR targeting antibody in full-length IgG form for enhanced agonist activity.

It has been observed that converting scFv formatted antibodies to full-length IgG often associates with loss of affinity. We aim to address this issue in this paper by establishing an integrated affinity maturation method applying yeast display technology platform. To demonstrate that, we employed a human thrombopoietin receptor targeting antibody named 3D9 which was identified previously from a combinational antibody library in scFv-Fc fusion protein form. We have observed that significant potency loss happened when 3D9 was transformed to full-length IgG form. In this study, we tested whether the potency of the full-length IgG can be improved by affinity maturation of 3D9 using a modified Fab yeast display platform. An efficient CDR3 targeted mutagenesis strategy was designed for Fab library with pre-designed CDR diversity. Next generation sequencing was also used for evaluation of the enrichment process and investigation of sequence-function relationship of the antibody. A variant with improved affinity and higher potency was identified. The study demonstrates that the strategy we used here are efficient for optimizing affinity and activity of full-length IgGs.

In vitro evaluation of the biological activities of IgG in seven Chinese intravenous immunoglobulin preparations

The IgG activities of antigen recognition, Fc-mediated complement activation and cellular Fcγ-receptors (FcγRs) binding are critical for intravenous immunoglobulin (IVIg) immunotherapy in a variety of immune deficiency diseases. Further, these activities could be influenced by different plasma sources and the IVIg manufacturing processes of different manufacturers. This study evaluated and compared the biological activities of IgG in 7 IVIg preparations produced by different Chinese manufacturers. By using ELISA and two-dimensional immunoblotting, the binding capacity and antibody repertoire of IVIg against typical pathogenic antigens were investigated. Further, Fc-mediated complement activation and receptor binding activities were measured by the haemolysis method and flow-cytometric assay respectively. The results showed that all of the preparations tested have a broad spectrum of antibodies against the E. coli O157:H7 proteome, and each IVIg has its own distinct antibody repertoire. Compared to the European Pharmacopoeia IgG standard, the mean indices of the pathogenic antigen binding capacity, complement activation activity and FcγRs binding activity in Chinese preparations were 152%, 143% and 87%. The biological activities varied widely among the 7 IVIg preparations, and no significant differences were observed between the different batches of most IVIgs from the same manufacturer. This study will contribute to the improvement of the IVIg product quality evaluation system and an increased understanding of the variety of IgG biological activities in Chinese IVIg preparations.

Inactivated rotavirus vaccine by parenteral administration induces mucosal immunity in mice

To improve the safety and efficacy of oral rotavirus vaccines, we developed an inactivated rotavirus vaccine (IRV) for parenteral administration. Since it remains unknown whether parenteral vaccination can induce mucosal immunity, we performed a comprehensive assessment of immune responses to IRV in mice with an adjuvant-free dissolving polymer MN patch or by alum-adjuvanted IM injection. We demonstrated that IRV induced the expression of the gut homing receptor LPAM-1 on T and B cells in spleen and mLN of vaccinated mice. MN patch IRV vaccination induced a slight Th1 phenotype while IM vaccination induced a balanced Th1/Th2 phenotype. In addition, a dose-sparing effect was seen for rotavirus-specific serum IgG and neutralizing activity for both vaccination routes. Our study is the first to show that parenterally administered IRV can induce mucosal immunity in the gut, in addition to strong serum antibody response, and is a promising candidate vaccine in achieving global immunization against rotavirus.

Substrate specificity of healthy human sera IgG antibodies with peroxidase and oxydoreductase activities.

We have carried out an analysis of whether blood IgG antibodies can protect humans from oxidative stress by oxidizing different harmful compounds. A somewhat unexpected result was obtained. We show here for the first time that healthy human sera IgGs with the peroxidase (in the presence H2O2) efficiently oxidize different compounds: 3,3′-diaminobenzidine (1; DAB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2; ATBS), o-phenylenediamine (3; OPD), homovanillic acid (4; HVA), α-naphthol (5), 5-aminosalicylic acid (6; 5-ASA) and 3-amino-9-ethylcarbazole (7; AEC), but seven of nine IgG preparations from different volunteers cannot oxidize p-hydroquinone (8: pHQ). The average apparent kcat values in the H2O2-dependent oxidation by human IgGs decreased in the following order (min−1): ATBS (73.7) ≥ DAB (66.3) > AEC (38.0) ≥ HVA (19.8) ≥ α-naphthol (8.6) > OPD (0.62) ≥ 5-ASA (0.48) > pHQ (0.24). In the absence of H2O2 (oxidoreductase activity), the relative average kcat values decreased in the following order (min−1): DAB (52.1) ≥ ATBS (50.5) > OPD (0.25). The peroxidase average activity of human IgGs was higher than the oxidoreductase one: 1.2-, 1.5- and 2.5-fold for DAB, ATBS and OPD, respectively. It should be assumed that antibodies can oxidize in addition to the large number of other different compounds analysed by us. As a whole, the specific wide repertoire of polyclonal human IgGs oxidizing various compounds could play an important role in protecting humans from oxidative stress and serve as an additional natural system destroying H2O2 and different toxic mutagenic and carcinogenic compounds.

The effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies.

HCQ has been described as having a beneficial effect in patients with APS but its mechanism of action is unclear. We hypothesized that HCQ may have effects on subnormal angiogenesis, inflammation and haemostatic biomarkers seen in APS. The aim of our study was to assess laboratory markers [annexin A5 (AnxA5) anticoagulant activity, tissue factor (TF) levels, thromboelastography (TEG), CRP, Bb, C3a and VEGF] in HCQ-naïve patients with aPL at baseline and after commencing HCQ. Twenty-two patients with aPL [20 female, 2 male, median age 55 (range 18-70) years] had blood taken pre- and 3 months after starting HCQ 200 mg daily. Soluble TF levels were significantly reduced comparing baseline and 3 months after HCQ commencement [401.8 (152.8) vs 300.9 (108) pg/ml (P = 0.010)]. No significant changes were found in the following [reported as pre- and post-HCQ commencement, mean (s.d.)]: AnxA5 anticoagulant ratio [187.1 (29.5) vs 193 (31) (P = 0.157)], anti-domain1 β2 glycoprotein1 IgG activity [1.8 (2) vs 1.2 (1.4) μg/ml (P = 0.105)], complement C3a-des-Arg [147.8 (84.5) vs 154.4 (88.1) ng/ml (P = 0.905)], complement Bb [1.3 (0.7) vs 1.1 (0.7) μg/ml (P = 0.422)], VEGF [68.8 (40) vs 59.4 (19.6) pg/ml (P = 0.454)] and CRP [7 (3.5) vs 7 (3.9) μg/ml (P = 0.917)]. TEG results including TEG reaction time, achievement of clot firmness, TEG maximum amplitude and TEG percentage lysis 30 and 60 min after maximum amplitude showed no significant difference. HCQ significantly reduced soluble TF levels in patients with aPL. No significant change was observed in AnxA5 activity, anti-domain 1 IgG activity, TEG, CRP, complement Bb and C3a-des-Arg, and VEGF. Further studies of a larger patient cohort are needed.

The direct bactericidal activity of IgG antibodies isolated from serum of patients with suppurative inflammatory processes caused by Staphylococcus aureus

The bactericidal activity of polyclonal IgG against St. aureus was established. Objectives. To evaluate the activity of polyclonal IgG isolated from patients with purulent-inflammatory diseases and donors against St. aureus in plankton form, as well as the ability of immunoglobulins to destroy the exopolymeric matrix of staphylococcal biofilm. Material and methods. Using the developed method the ability of polyclonal IgG isolated from 45 patients with purulentinflammatory diseases and 16 donors was determined to have a bactericidal effect on the plankton form of St. aureus without the participation of complement and immune cells. An estimation of the ratio of viable and dead bacteria was made by laser scanning confocal microscopy using propidium iodide as a marker of dead bacteria. The ability of IgG to destroy the exopolymeric matrix of St. aureus biofilm was assessed. The relation of immunoglobulin activity to St. aureus cells and biofilm matrix with clinical and laboratory manifestations of purulent-inflammatory diseases was analyzed. Results. It was shown that IgG isolated from patients with purulent-inflammatory diseases and donors may have their own bactericidal activity against St. aureus without participation of the complement system and immune cells, while in patients with purulent-inflammatory diseases this activity is significantly higher than in persons without purulent-inflammatory processes. The ability of IgG isolated from patients with purulent-inflammatory diseases to cleave the exopolymeric matrix of S. aureus biofilm was significantly higher than in persons without purulent-inflammatory diseases. Conclusion. The findings suggest the presence of mechanisms of antibacterial activity of IgG against St. aureus, which is not associated with participation of the complement system and immune cells.

Catalase activity of IgG antibodies from the sera of healthy donors and patients with schizophrenia.

We present first evidence showing that some electrophoretically homogeneous IgGs from the sera of patients with schizophrenia (36.4%) and their Fab and F(ab)2 fragments as well as from healthy donors (33.3%) possess catalase activity. The relative catalase activity of IgGs from the sera of individual schizophrenia patients (and healthy donors) significantly varied from patient to patient, but the activity of IgGs from healthy donors is on average 15.8-fold lower than that for schizophrenia patients. After extensive dialysis of purified IgGs against EDTA chelating metal ions, the relative catalase activity of IgGs decreases on average approximately 2.5–3.7-fold; all IgGs possess metal-dependent and independent catalase activity. The addition of external Me2+ ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. The best activator of dialyzed and non-dialyzed IgGs is Co2+, the activation by Cu2+, Mn2+, and Ni2+ ions were rare and always lower than by Co2+. Every IgG preparation demonstrates several individual sets of very well expressed pH optima in the pH range from 4.0 to 9.5. These data speak for the individual repertoire of catalase IgGs in every person and an extreme diversity of abzymes in their pH optima and activation by different metal ions. It is known that antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases represent critical defense mechanisms preventing oxidative modifications of DNA, proteins, and lipids. Catalase activity of human IgGs could probably also play a major role in the protection of organisms from oxidative stress and toxic compounds.

Comparative response of IgA and IgG activity and hematological parameters among four main beef-cattle breeds infected with gastrointestinal nematodes in the warm humid tropic of Mexico

Abstract This study determined the immunoglobulin (A and G) activity against gastrointestinal nematodes (GIN) and also the hematological parameters in four beef-calf breeds naturally infected in a tropical region of Mexico. Thirty-six infected calves were used to determine the fecal nematode egg counts (FEC), the IgA and IgG activity in serum and saliva, the packed cell volume (PCV), the plasmatic protein (PP) and the differential leukocyte counts. These parameters were measured for a five-month period. ELISA assay was performed using adult worm crude antigen (AWCA) from Haemonchus contortus, Trichostrongylus colubriformis, Mecistocirrus digitatus and Cooperia punctata. The variables were analyzed using a fixed model according to the breed group. The FEC records (means ± standard deviation) for the different breeds were: Brangus (122±115), Charolais (391±507), Guzerat (294±326) and Brown Swiss (413±395). No statistical differences (P>0.05) were found among breeds. Cooperia and Haemonchus were the main identified genera in the coprocultures. Differences in hematological parameters were observed among breeds (P<0.01); Guzerat showed the highest PCV (42.6±4.7%). The lowest leukocyte counts were observed in the Charolais breed (9.2±2.4 × 109 cells L-1). Differences between Guzerat and Charolais were only detected in eosinophils, neutrophils and lymphocytes. Increased activity of IgA was observed against M. digitatus (21% OD) compared with the response against C. punctata in serum samples (13% OD). The lowest IgA activity in serum and saliva was found in Guzerat calves. Charolais and Brown Swiss showed the highest IgA activity in serum and the highest value in saliva was found in Brangus.

Enzymatic activity of blood serum, abzyme activity of IgG and IgA, free serum DNA and the formation of extracellular traps by neutrophils in patients with breast neoplasms

The objective of this study included a comprehensive comparative assessment of the proteolytic, nuclease and oxidoreductase activity of serum and immunoglobulins of G and A classes, as well as the serum DNA content in patients with mammary neoplasms and the ability of their leukocytes to form neutrophil extracellular traps (NETs). To aim this, the following methods have been developed: a combined method for isolating IgG and IgA from a single serum sample, the method for fluorescent determination of serum DNA, the method of staining NETs in the culture of neutrophils with methyl green, and the method of double staining of NETs in tumor smear-imprints. Based on the results of the study it was shown that the abzymes isolated from blood serum possess proteolytic, DNase and oxidoreductase activity, which varies depending on the patient and the conditions of the reaction. Certain types of catalytic activity of serum and antibodies are different in patients with malignant and benign breast tumors, as well as in healthy individuals. Catalytic activity of serum and abzymes depends on the tumor grade as well as on histological and immunohistochemical characteristics of the tumor. Concentration of DNA in serum of breast cancer patients is reduced in comparison with healthy individuals; a negative correlation was established between the DNA content and DNase activity of sera and abzymes. The ability to form traps by neutrophils in breast cancer is significantly higher in comparison with the examined control groups (patients with benign breast tumors and healthy individuals). This finding may be used as an additional laboratory marker of the development of the tumor process in the mammary gland in women.

ANTI-HISTONE H1 IgGs POSSESS PROLIFERATIVE ACTIVITY TOWARDS HUMAN T-LEUKAEMIA CEM CELLS

Anti-histone H1 IgGs were purified from blood serum of systemic lupus erythematosus patients by precipitation of serum proteins with 50% ammonium sulfate followed by a sequential affinity chromatography on Protein G-Sepharose and histone H1-Sepharose columns. To avoid contamination with other proteins, anti-histone H1 IgGs were subjected to strongly acidic pH 2.0 during gel filtration through HPLC column. The effects of the anti-histone H1 IgGs on cell viability and cell cycle were tested by MTS-assay and flow cytometry, correspondingly. The cross-reactivity of the anti-histone H1 antibodies towards heterogenetic and cellular antigens was evaluated by Western-blot analysis. It was found that incubation of CEM cells with the HPLC-purified anti-histone H1 IgGs resulted in significant stimulation of cell growth by 46% after 48 h of incubation. These IgGs possess an antigenic poly-specificity to positively charged heterogenetic antigens and different cellular antigens. FITC-labeled and biotinylated anti-histone H1 IgGs are internalized by CEM cells and preferentially accumulated in the cytoplasm. The anti-histone H1 IgGs are shown to internalize human T-leukemia CEM and stimulate their proliferation. These IgGs are polyspecific toward cellular antigens.

Formation of multimeric antibodies for self-delivery of active monomers

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

A Comparative Study to Evaluate the Effect of Different Carbohydrates on the Stability of Immunoglobulin G during Lyophilization and Following Storage

Background: Although the stabilizing effects of cyclodextrins (CDs) on the liquid protein formulations have been proven, there is no comprehensive data on evaluation of their effects on the lyophilized antibody powders. In this study, the influence of two CD derivatives namely beta-cyclodextrin (βCD) and hydroxypropyl beta-cyclodextrin (HPβCD) was compared with trehalose and mannitol regarding the molecular and thermodynamic stability of lyophilized IgG formulations as well as its biological activity. Methods: Sugars were separately added to IgG solutions and lyophilization process was conducted. In each group of carbohydrates, the formulations with lowest amounts of aggregates were examined regarding the biological activity. The storage stability of selected formulations was subsequently determined following 1 and 3 month of storage at 45 °C. Results: Trehalose and HPβCD in the ratios of 80% showed the most stabilizing effects by control of aggregated forms in the orders of 1.02% and 0.83%, respectively. Also, it was shown that trehalose and HPβCD could incomparably preserve IgG activity in values of 100% and 96.5%. The results of DSC and SEM analysis confirmed the existence of crystalline parts in mannitol and βCD formulations of antibody. During the storage time, the lowest rate constant of aggregation was observed in formulations containing trehalose 80% (0.16/month). All prepared formulations were beta-dominant and no fragmentation was detected. Conclusion: Molecular, thermodynamic and biological stability of lyophilized IgG was more desirable in the presence of trehalose and HPβCD in comparison to mannitol and βCD.

The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

The persistent public health threat of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the need for an effective MERS-CoV vaccine. Previous studies have focused mainly on the receptor-binding domain (RBD) on the spike protein of MERS-CoV. Herein, we investigated the immunogenicity and protective potential of the recombinant N-terminal domain (rNTD) of spike proteins as a vaccine candidate. BALB/c mice vaccinated with 5 or 10μg of rNTD protein demonstrated a significant humoral immune response (serum IgG and neutralizing activity). Additionally, according to the enzyme-linked immunospot, intracellular cytokine staining, and cytometric bead array assays, significant and functional T-cell immunity was induced by 10μg of the rNTD vaccination with aluminum and CpG adjuvant. Furthermore, rNTD-immunized mice showed reduced lung abnormalities in a MERS-CoV-challenge mouse model transfected with an adenoviral vector expressing human DPP4, showing protection consistent with that found with rRBD vaccination. These data show that rNTD induced potent cellular immunity and antigen-specific neutralizing antibodies in mice and that it demonstrated protective capacity against a viral challenge, indicating that rNTD is a vaccine candidate against MERS-CoV infection.

Vaccination against H9N2 avian influenza virus reduces bronchus-associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection.

H9N2 avian influenza virus causes sporadic human infection. Since humans do not possess acquired immunity specific to this virus, we examined the pathogenicity of an H9N2 virus isolated from a human and then analyzed protective effects of a vaccine in cynomolgus macaques. After intranasal challenge with A/Hong Kong/1073/1999 (H9N2) (HK1073) isolated from a human patient, viruses were isolated from nasal and tracheal swabs in unvaccinated macaques with mild fever and body weight loss. A formalin-inactivated H9N2 whole particle vaccine derived from our virus library was subcutaneously inoculated to macaques. Vaccination induced viral antigen-specific IgG and neutralization activity in sera. After intranasal challenge with H9N2, the virus was detected only the day after inoculation in the vaccinated macaques. Without vaccination, many bronchus-associated lymphoid tissues (BALTs) were formed in the lungs after infection, whereas the numbers of BALTs were smaller and the cytokine responses were weaker in the vaccinated macaques than those in the unvaccinated macaques. These findings indicate that the H9N2 avian influenza virus HK1073 is pathogenic in primates but seems to cause milder symptoms than does H7N9 influenza virus as found in our previous studies and that a formalin-inactivated H9N2 whole particle vaccine induces protective immunity against H9N2 virus.

Activity, specificity, and titer of naturally occurring canine anti-DEA 7 antibodies.

The reported prevalence of naturally occurring anti-dog erythrocyte antigen (DEA) 7 antibodies in DEA 7-negative dogs is as high as 50%. Characterization of these antibodies may better define their importance in canine transfusion medicine. We determined in vitro activity, specificity, and titer of anti-DEA 7 antibodies in DEA 7-negative dogs. Plasma samples from 317 DEA 7-negative dogs were cross-matched with DEA 7-positive red blood cells (RBCs) using gel column technology. Agglutination occurred with DEA 7-positive RBCs but not with DEA 7-negative RBCs in 73 samples (23%), which were hence classified as containing anti-DEA 7 antibodies. These samples were evaluated for hemolytic and agglutinating activity, strength of agglutination, and antibody specificity and titers. All samples showed agglutination but none showed hemolysis. Gel agglutination was graded as 1+ for 20 samples (27%), 2+ for 49 samples (67%), 3+ for 4 samples (6%); no samples were graded 4+. The agglutination titer was <1:2 for 51 samples (73%), 1:2 for 13 samples (19%), 1:4 for 4 samples (5%), and 1:8 for 2 samples (3%). Of 16 samples treated with 2-mercaptoethanol, 11 samples (69%) contained only IgM, 4 samples (25%) exhibited only IgG activity, and 1 sample (6%) had both IgG and IgM activity. Low titers of warm, weakly agglutinating, mostly naturally occurring IgM anti-DEA 7 antibodies were found in 23% of DEA 7-negative dogs. The presence of naturally occurring anti-DEA 7 antibodies suggests that cross-matching of canine blood recipients is advisable, even at first transfusion, to minimize delayed transfusion reactions.

Comparison of diagnostic tests for determining the prevalence of failure of passive transfer in New Zealand dairy calves

AIM: To evaluate the level of agreement of three indirect testing methods with concentrations of IgG in serum, and to determine their test characteristics for diagnosing failure of passive transfer (FPT), in dairy calves in New Zealand.METHODS: From 17 July to 30 November 2015, 471 blood samples were collected from dairy calves aged 0–8 days, from 19 different commercial dairy farms in the Waikato region of New Zealand. All serum samples were tested for concentrations of IgG and total protein (TP), and gamma-glutamyl transferase (GGT) activity, and 138 samples were tested using a digital Brix refractometer. The diagnostic tests were assessed for level of agreement with concentrations of IgG using Bland-Altman plots. FPT was defined as concentration of IgG ≤10.0 g/L and receiver operating characteristic curve analysis was used to determine optimal cut-points for correctly predicting FPT. The test characteristics for TP, GGT and Brix for predicting FPT were determined using the optimal cut-points. The effect of age on the relationship between test results and concentrations of IgG was also assessed.RESULTS: Based on the limits of agreement plots, the agreement with concentrations of IgG was greatest for concentrations of TP, followed by Brix, with the lowest being GGT activity. There was an interaction between age at sampling and the association between concentrations of IgG and GGT activity (p<0.001); in calves 5–8 days of age there was a greater increase in concentrations of IgG associated with a unit increase in GGT activity than in calves <5 days of age. The optimal cut-points for diagnosis of FPT were 52 g/L for concentrations of TP, 8.8% for Brix, 250 IU/L for GGT activity in calves <5 days old, and 210 IU/L for GGT activity in calves 5–8 days old. Accuracy for predicting FPT was greatest for TP (0.95), followed by GGT in calves <5 days old (0.94) and Brix (0.92). Specificities were high for all three tests, however sensitivity varied greatly.CONCLUSION: Measurement of concentrations of TP in serum was the most accurate alternative to measuring concentrations of IgG for diagnosing FPT in non-dehydrated calves.CLINICAL RELEVANCE: Veterinary practitioners in New Zealand now have confidence in using a test that is more accurate and practical than GGT and considerably cheaper than IgG to screen for FPT.

Idiopathic thrombotic thrombocytopenic purpura: strongest risk factor for relapse from remission is having had a relapse.

Thrombotic thrombocytopenic purpura (TTP) is a rare, episodic clinical syndrome involving the production of thrombi in the microvasculature accompanied by thrombocytopenia and symptoms of organ ischemia. Idiopathic TTP develops when a patient produces antibodies that react with the protease ADAMTS13. The course after an episode is unpredictable; patients may relapse frequently or never. There is no laboratory value that can reliably predict potential relapse. To assess diagnostic and predictive values for risk of relapse, plasma samples from 27 patients with idiopathic TTP in remission were analyzed for anti-ADAMTS13 immunoglobulin (Ig)G, ADAMTS13 activity, and ADAMTS13 inhibitor titer. Patients were recruited at the Department of Hematology at the University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany. Anti-ADAMTS13 IgG was detected in 12 patients (44%); their median level of ADAMTS13 activity was nondetectable. Patients with anti-ADAMTS13 IgG had a median number of three previous relapses, whereas the 15 patients without presence of IgG (56%) had a median number of one previous relapse (p<0.001; Mann-Whitney Utest). The concentration of free anti-ADAMTS13 IgG and the levels measuring inhibitory activity (Bethesda unit) were positively correlated. A subgroup of TTP patients in remission with anti-ADAMTS13 IgG and nondetectable ADAMTS13 activity showed an increased risk for relapsing disease as demonstrated by their number of past relapses. The positive correlation we observed between anti-ADAMTS13 IgG and inhibitor levels supports the theory of ADAMTS13 inhibition as the crucial mechanism causing severe deficiency in ADAMTS13 activity in TTP.