Soluble Immune Complexes Research Articles

Regulation of neutrophil associated RNASET2 expression in rheumatoid arthritis

Neutrophils (PMNs) are key players of innate immune responses through the release of cytoplasmic granule content and the formation of neutrophil extracellular traps (NETs). RNASET2 is an acidic ribonuclease, recently proposed as an alarmin signal associated with inflammatory responses. Here we show that, along the neutrophil maturation cascade, RNASET2 is expressed in segmented and mature PMNs. In human PMNs, RNASET2 colocalized with primary and tertiary granules and was found to be associated with NETs following PMA or Nigericin stimulation. Similarly, activation of PMNs by soluble immune complexes, a hallmark of several autoimmune diseases, also induced RNASET2-associated NETs. Genome-wide association studies recently identified RNASET2 among a cluster of genes associated with increased susceptibility to develop autoimmune diseases, including rheumatoid arthritis (RA). RNASET2 was found expressed by PMNs and macrophages infiltrating inflamed joints in a murine model of RA (K/BxN Serum-Transfer-Induced Arthritis, STIA), by immunostaining. Similar results were found in synovial biopsies of RA patients with active disease. In addition, we demonstrate that RNASET2 circulating levels correlated with the onset and the severity of disease in two mouse models of inflammatory arthritis, STIA and CIA (Collagen-Induced Arthritis) and in serum of RA patients. These results show that PMNs are an important source of RNASET2 and that its circulating levels are associated with RA development suggesting a role for RNASET2 in the pathogenesis of immune-mediated diseases.

Fcγ-receptor-IIIA bioactivity of circulating and synovial immune complexes in rheumatoid arthritis

ObjectivePrevious technical limitations prevented the proof of Fcγ-receptor (FcγR)-activation by soluble immune complexes (sICs) in patients. FcγRIIIa (CD16) is a risk factor in rheumatoid arthritis (RA). We aimed at determining...

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

Effect of JAK Inhibition on the Induction of Proinflammatory HLA-DR+CD90+ Rheumatoid Arthritis Synovial Fibroblasts by Interferon-γ.

Findings from recent transcriptome analyses of the synovium of patients with rheumatoid arthritis (RA) have revealed that 15-fold expanded HLA-DR+CD90+ synovial fibroblasts potentially act as key mediators of inflammation. The reasons for the expansion of HLA-DR+CD90+ synovial fibroblasts are unclear, but genetic signatures indicate that interferon-γ (IFNγ) plays a central role in the generation of this fibroblast subset. The present study was undertaken to investigate the generation, function and therapeutically intended blockage of HLA-DR+CD90+ synovial fibroblasts. We combined functional assays using primary human materials and focused bioinformatic analyses of mass cytometry and transcriptomics patient data sets. We detected enriched and activated Fcγ receptor type IIIa-positive (CD16+) NK cells in the synovial tissue from patients with active RA. Soluble immune complexes were recognized by CD16 in a newly described reporter cell model, a mechanism that could be contributing to the activation of natural killer (NK) cells in RA. In vitro, NK cell-derived IFNγ induced HLA-DR on CD90+ synovial fibroblasts, leading to an inflammatory, cytokine-secreting HLA-DR+CD90+ phenotype. HLA-DR+CD90+ synovial fibroblasts consecutively activated CD4+ T cells upon receptor crosslinking via superantigens. HLA-DR+CD90+ synovial fibroblasts also activated CD4+ T cells in the absence of superantigens, an effect that was initiated by NK cell-derived IFNγ and that was 4 times stronger in patients with RA compared to patients with osteoarthritis. Finally, JAK inhibition in synovial fibroblasts prevented HLA-DR induction and blocked proinflammatory signals to T cells. The HLA-DR+CD90+ phenotype represents an activation state of synovial fibroblasts during the process of inflammation in RA that can be induced by IFNγ, likely generated from infiltrating leukocytes such as activated NK cells. The induction of these proinflammatory, interleukin-6-producing, and likely antigen-presenting synovial fibroblasts can be targeted by JAK inhibition.

Concomitant Congenital Diaphagmatic Hernia (CDH) and bilateral bacterial glomerulonephritis in a pet chinchilla (Chinchilla lanigera)

BackgroundThe domestic chinchilla has been descended from Chinchilla lanigera (long-tailed Chinchilla) or Chinchilla chinchilla (short-tailed Chinchilla). Both species of chinchilla are currently listed as endangered by the IUCN Red List of Threatened Species. Over the past 20 years, they have spread as pets and overall knowledge about their care is improving. The present case report describes a congenital diaphragmatic hernia in a Chinchilla lanigera.Case presentationA 1-year-old, 420 g female chinchilla (Chinchilla lanigera) was presented for clinical examination due to 2 days haematuria episodes and anorexia. A complete haematological analysis was performed, showing a moderate neutrophilia and severe renal involvement. X-rays showed severe intestinal meteorism affecting mostly the cecum, and a soft tissue density mass with translucent areas located in the caudal thorax, making it hard to distinguish the cardiac silhouette. A barium swallow (barium sulfate) was performed and after 20 min, radiograms were performed again, showing part of the stomach dislocated in thorax. Ultrasound was also carried out, confirming the partial stomach herniation into the thoracic cavity and a severe nephropathy. The patient was euthanized according to the owner’s wish and a complete necropsy was performed. The diagnosis was congenital diaphragmatic hernia concomitant to a severe bilateral bacterial glomerulonephritis.Discussion and conclusionsDiaphragmatic hernias can be either congenital or acquired. About CDHs in pet chinchillas, literature is still lacking. In this patient there was no history of previous traumas. No scar tissue or thickening involved margins of the pathological diaphragm window at the necropsy, supporting the hypothesis of a congenital defect. Glomerulonephritis most often results from immune-mediated mechanisms, generally after the deposition of soluble immune complexes within the glomeruli. This mechanism is favoured by a prolonged antigenemia that could occur during specific viral infections, chronic bacterial infections, chronic parasitism, autoimmune diseases and neoplasia. Few cases of nephritis are described in chinchillas (Chinchilla lanigera), mostly related to bacterial sepsis or less commonly involving fungi. The evidence of bacterial aggregates in kidneys at the histopathology, confirmed the infective aetiology. No relationship between the diaphragmatic hernia and glomerulonephritis was found in this report.

Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

Fc‐gamma receptor (FcγR) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. We developed a comprehensive reporter cell panel detecting activation of FcγRs. The reporter cell lines were integrated into an assay that enables the quantification of sIC reactivity via ELISA or a faster detection using flow cytometry. This identified FcγRIIA(H) and FcγRIIIA as the most sIC‐sensitive FcγRs in our test system. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size reproducing for the first time a complete Heidelberger‐Kendall curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC‐dependent FcγR activation has predictive capabilities regarding severity of SLE disease. The assay provides a sensitive and scalable tool to evaluate the size, amount, and bioactivity of sICs in all settings.

IgG Immune Complexes Inhibit Naïve T Cell Proliferation and Suppress Effector Function in Cytotoxic T Cells.

Elevated levels of circulating immune complexes are associated with autoimmunity and with worse prognoses in cancer. Here, we examined the effects of well-defined, soluble immune complexes (ICs) on human peripheral T cells. We demonstrate that IgG-ICs inhibit the proliferation and differentiation of a subset of naïve T cells but stimulate the division of another naïve-like T cell subset. Phenotypic analysis by multi-parameter flow cytometry and RNA-Seq were used to characterize the inhibited and stimulated T cells revealing that the inhibited subset presented immature features resembling those of recent thymic emigrants and non-activated naïve T cells, whereas the stimulated subset exhibited transcriptional features indicative of a more differentiated, early memory progenitor with a naïve-like phenotype. Furthermore, we show that while IgG1-ICs do not profoundly inhibit the proliferation of memory T cells, IgG1-ICs suppress the production of granzyme-β and perforin in cytotoxic memory T cells. Our findings reveal how ICs can link humoral immunity and T cell function.

Lung cancer induced by Benzo(A)Pyrene: ChemoProtective effect of sinapic acid in swiss albino mice.

Cancer of lung is the utmost typical cause of death and the number of cases is increasing rapidly, which has emerged as a major leading health problem. A large amount of reports suggested that Benzo(a)pyrene [B(a)P] in cigarette smoke plays the major function in an initiation of cancer of lung. Cancer prevention or chemoprevention has become a compelling approach recently for treatment of lung cancer. So, discovering a fresh candidate with reduced toxicity for targeting lung cancer is vital and urgent. Sinapic acid which is a widely extracted in various vegetables and fruit exhibits rich anti-oxidant content, anti-inflammatory and anti-tumor activity. But, the chemopreventive action of sinapic acid against lung cancer initiated by B[a]P remain unclear. Following, an in-vivo B[a]P-stimulated lung cancer in swiss albino mice and an in-vitro human lung cancer cell (A549) model were established to examine the chemopreventive activities of sinapic acid. The levels of immunoglobulins (IgG and IgM), oxidative and inflammatory markers, and tumor markers level was studied using kits and standard methods. The results showed administration of sinapic acid ameliorates the exposure of B[a]P mediated lung cancer in swiss albino mice by a decline in IgG and IgM level, leukocyte count, neutrophil function tests, soluble immune complex, lipid peroxidation, pro-inflammatory cytokines, tumor markers (AHH, LDH, GGT, 5′NT and CEA) and enhanced phagocytic index, activity index and antioxidant defense enzymes. In addition, in-vitro studies showed potential cytotoxicity against human lung cancer and exhibited a potential cytotoxic (MTT assay) and apoptotic activity by elevation of ROS production and caspase activity (caspase-3 and caspase-9). Collectively, the results, clearly specifies sinapic acid can be utilized as an effective chemo preventative agent against lung carcinogenesis.

Immunomodulatory effect of d-carvone in swiss albino mice with benzo(a)pyrene-induced lung cancer

Background: Globally, lung cancer is the second most cause of deaths, which accounts for approximately 29% of the cancer cases worldwide. d-Carvone is considered a vital constituent of many essential oils with immense pharmacological benefits. Objective: In this study, we examined the immunomodulatory effect of d-carvone in the Swiss albino mice model against benzo(a)pyrene (BaP)-induced lung carcinoma. Materials and Methods: BaP was orally administered to the mice (50 mg/kg body weight [bw] for 4 weeks [twice a week]). Post-BaP administration, d-carvone (20 mg/kg bw) was orally administered to the mice (20 mg/kg bw). We calculated the tumor incidence and performed the following measurements: lung and bw, hematological parameters, immune complexes (phagocytic and avidity indexes, nitroblue tetrazolium reduction, soluble immune complex levels, immunocompetent cells (leukocytes, lymphocytes, and neutrophils), immunoglobin (Ig) levels (IgG, IgA, and IgM), level of xenobiotics and enzymes that point toward hepatic dysfunction, carcinoembryonic antigen (CEA), and proinflammatory cytokines (PICs) in experimental and normal mice. The level of oxidative stress in the experimental animals was investigated. The lung tissues of investigational animals were examined for the histopathological changes. Results: According to the results, there was an increased level of lipid peroxidation and decreased level of antioxidant activity in the lung tumor samples. The counts of lymphocytes, polymorphonuclear cells, and macrophages were notably decreased and increased, respectively, by the d-carvone post-treatment. Moreover, BaP-induced inflammation that is indicated by the increased in the level of PICs and CEA proteins in lung samples. d-Carvone attenuated the levels of PICs and CEA. Conclusion: The results of this study reveal that d-carvone prevented the cells against BaP-induced inflammation in lung cancer without causing adverse effects.

FcRn augments induction of tissue factor activity by IgG-containing immune complexes

AbstractThromboembolism complicates disorders caused by immunoglobulin G (IgG)–containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), β-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

Lupus-Associated Immune Complexes Activate Human Neutrophils in an FcγRIIA-Dependent but TLR-Independent Response.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nucleic acids and nucleoproteins. Anti-dsDNA Abs are considered a hallmark of SLE, and previous studies have indicated that nucleic acid-containing immune complexes (ICs) induce B cell and dendritic cell activation in a TLR-dependent process. How ICs containing nucleic acids affect neutrophil function has not been well investigated. In this study, we report that nucleic acid-containing ICs derived from the sera of SLE patients induce human and mouse neutrophil activation through TLR-independent mechanisms. Soluble ICs containing Sm/RNP, an RNA Ag, activate human neutrophils to produce reactive oxygen species (ROS) and IL-8. In contrast, ICs containing DNA have to be immobilized to efficiently activate neutrophils. We found that deleting TLR7 or TLR9, the receptors for RNA and DNA, had no effect on mouse neutrophil activation induced by RNA-containing and immobilized DNA-containing ICs. Binding of ICs are mediated through FcγRIIA and FcγRIIIB. However, neutrophil activation induced by RNA- and DNA-containing ICs requires FcγRIIA, as blocking FcγRIIA inhibited ROS release from neutrophils. RNA-containing ICs induce calcium flux, whereas TLR7/8 ligand R848 do not. Surprisingly, chloroquine inhibits calcium flux induced by RNA-containing ICs, suggesting that this lesser known function of chloroquine is involved in the neutrophil activation induced by ICs. These data indicate the SLE-derived ICs activate neutrophils to release ROS and chemokines in an FcγRIIA-dependent and TLR7- and TLR9-independent manner that likely contributes to local tissue inflammation and damage.

Interaction with complement proteins and dendritic cells implicates LCCL domain-containing proteins (CCps) of malaria parasites in immunomodulation.

The evasion of host immune defense is critical for pathogens to invade, establish infection and perpetuate in the host. The complement system is one of the first lines of innate immune defense in humans that destroys pathogens in the blood circulation. Activation of the complement system through direct encounter with pathogens or some other agents leads to osmolysis of pathogens, clearance of soluble immune complexes and recruitment of lymphocytes at the site of activation. Although malaria parasites are not exposed to the complement system owing to their intracellular development for most part of their life cycle in the human host, the extracellular stages must face the complement system of human or mosquito or both. In a recent issue of the Biochemical Journal, Sharma et al. reported that Plasmodiumfalciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells, which implicate PfCCp1 and related proteins in immunomodulation of the host that likely benefits the parasite. PfCCp1 belongs to a multi-domain protein family that exists as multimeric protein complexes. It needs to be investigated whether PfCCp1 or its multimeric protein complexes have an immunomodulatory effect in vivo and on the mosquito complement system.

Restricted immune activation and internalisation of anti-idiotype complexes between drug and antidrug antibodies

ObjectivesTherapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although...

Diminished capacity of opsonization and immune complex solubilization, and detection of anti-C1q antibodies in sera from patients with hereditary angioedema

BackgroundHereditary angioedema (HAE) is an autosomal dominant disease caused by deficiency of C1 esterase inhibitor. Symptoms of HAE include edema, which can potentially cause suffocation. Some patients with HAE exhibit immunological abnormalities, which could prevent an accurate diagnosis. Low levels of complement components are characteristic of HAE and in other settings are thought to reduce elimination of apoptotic cells and immune complex (IC). Thus, we aimed to experimentally clarify the mechanism of immunological abnormalities using sera from HAE patients. MethodsSerum samples from 18 patients with HAE were collected when free from angioedema attack and compared with normal human pooled sera (NHPS) from 20 healthy volunteers. Opsonization was measured as the rate of phagocytosis of apoptotic Jurkat cells by macrophages differentiated from THP-1 cells incubated with serum. IC solubilization in serum was analyzed by quantifying peroxidase released from a synthetic IC composed of peroxidase and anti-peroxidase antibodies. Anti-C1q antibody levels were detected using an enzyme-linked immunosorbent assay. ResultsSerological immunological abnormalities were detected in 12 patients. Opsonization in serum samples from each patient with HAE was lower than that in NHPS (∼20% versus 70%, respectively). The rate of IC solubilization was lower in serum from HAE patients than NHPS. Some patients had high serum anti-C1q antibody levels with increased serum IC levels. ConclusionsSera from patients with HAE exhibit anti-C1q antibodies, with a lower capacity for opsonization and IC solubilization. This may be associated with immunological abnormalities and should be investigated further to facilitate accurate diagnosis of HAE.

Effect of Scoparia dulcis on noise stress induced adaptive immunity and cytokine response in immunized Wistar rats

BackgroundNoise acts as a stressor and is reported to have impact on individual health depending on nature, type, intensity and perception. Modern medicine has no effective drugs or cure to prevent its consequences. Being an environmental stressor noise cannot be avoided; instead minimizing its exposure or consuming anti-stressor and adaptogens from plants can be considered. ObjectivesThe present study was carried out to evaluate the anti-stressor, adaptogen and immunostimulatory activity of Scoparia dulcis against noise-induced stress in Wistar rat models. Material and methodsNoise stress in rats was created by broadband white noise generator, 100 dB A/4 h daily/15 days and S. dulcis (200 mg/kg b.w.) was administered orally. 8 groups of rats were used consisting of 6 animals each; 4 groups for unimmunized and 4 groups for immunized. For immunization, sheep red blood cells (5 × 109 cells/ml) were injected intraperitoneally. ResultsSub-acute noise exposed rats showed a significant increase in corticosterone and IL-4 levels in both immunized and unimmunized rats whereas lymphocytes, antibody titration, soluble immune complex, IL-4 showed a marked increase with a significant decrease in IL-2, TNF-α, IFN-γ cytokines only in unimmunized rats. Immunized noise exposed rats presented increased leukocyte migration index and decreased foot pad thickness, IL-2, TNF-α, IFN-γ with no changes in the lymphocytes. ConclusionS. dulcis (SD) has normalized and prevented the noise induced changes in cell-mediated and humoral immunity and it could be the presence of anti-stressor and immuno stimulant activity of the plant.

Study on the immunological safety of universal plasma in the Chinese population in vitro

BackgroundThe prepared procedure for universal plasma in the Chinese population has been developed. However, the immunological safety with the level of antibodies, soluble immune complexes and complements is necessary to investigate. MethodsThe universal plasma was pooled at the optimal ratio of A:B:AB=6:2.5:1.5 at 22°C for 1 hour. The titer of IgM antibodies was detected by saline agglutination, and the titer of IgG antibodies was detected by a Polybrene test after IgM destroyed by 2-mereaptoethanol. The hemolysis extent of RBC was investigated by an extracorporal hemolysis test, and the concentration of free-hemoglobin was determined by the ortho-tolidine method. The levels of CIC-C1q, C3b and TCC (C5-9) were tested using an enzyme linked immunosorbent assay (ELISA). ResultsThe titer of IgM and IgG in universal plasma was lower than 2 and 4, respectively. The hemolysis of the universal plasma with A, B and AB group RBCs was negative with values of 5.5, 6.8 and 5.7, respectively. The level of CIC-C1q and TCC (C5-9) in universal plasma was comparable to that in A or B type pooled plasma, but CIC-C1q was lower than that and TCC (C5-9) was higher than that in AB type pooled plasma. The level of complement C3b was comparable to that in A type pooled plasma, but lower than that in B type pooled plasma and higher than that in AB type pooled plasma. ConclusionThe results of this study demonstrated that the immunological levels were within an acceptable range and confirmed the safety in vitro.

Experimental induction of autoantibodies to IgG by pigeon IgY

Abstract Background IgG is a frequent target of autoantibodies in a variety of experimental and clinical settings. Interestingly, the possible antigens (Ag) that may induce such a break of tolerance have not been fully studied. Since our previous observations showed the frequent co-occurrence of antibodies (Abs) to pigeon IgY and self-reactivity to IgG in hypersensitivity pneumonitis secondary to avian exposure, we decided to evaluate experimentally the role of IgY in this kind of autoimmune reactivity. Objective To study whether pigeon IgY induces anti-IgG reactivity in an experimental model in which foreign and self-immunoglobulins are phylogenetically related. Methods IgY was purified from pigeon (Columba livia) eggs, prepared in Freund’s adjuvant and immunized to Balb/c mice. Abs production to foreign (IgY) and self-antigen (mouse IgG) were evaluated by ELISA and Western blot. Tregs (CD4+CD25+Foxp3+) were also studied by flow cytometry since the first evidences suggested a T-cell dependent Ab reactivity. Results IgG with anti-IgY activity occurred 2 weeks after immunization. Autoantibodies were predominantly IgG and bind Fc fractions of IgG. Immunoglobulins to self-IgG showed a gradual increase and they persisted for 3 weeks. A correlation between high levels of autoantibodies and elevated Tregs was also found. Conclusions Pigeon IgY induced autoantibody production, it was predominantly IgG, follows a kinetic similar to soluble immune complexes formation, and bind the Fc fraction of IgG. T-cell analyses suggested a Tregs dependent reactivity. The break of tolerance to self-IgG in mammals has not been previously studied using a phylogenetically related Ag.

Effect of Kalpaamruthaa on immunosuppression of both humoral and cell-mediated immunity in DMBA-induced mammary tumours of rats

The present study was carried out to elucidate the protective effect of Kalpaamruthaa on improving 7,12-dimethylbenz(a)anthracene (DMBA)-induced immunosuppression of both humoral and cell-mediated immunity in mammary carcinoma-induced rats. Breast cancer was induced in rats by administering DMBA orally (25 mg/rat) as a single dose. After 90 days of induction, SA (200 mg/kg body weight) and KA (300 mg/kg body weight) were administered for 14 days, by gastric intubation. Several immunotoxicological assays such as T cell rosette delayed type hypersensitivity (DTH) response, migration inhibition factor (MIF) assay, lymphocyte proliferation assay, plaque forming cell (PFC) assay and haemagglutination assay, plaque forming cell (PFC) assay, serum soluble immune complex and cytokine production, T and B cell mitogenesis induced by Con A and nonspecific cell-mediated immunity were evaluated using phagocytosis activity and NBT reduction. In cancer-induced animals (group II), the leukocyte migration inhibition declined markedly (p < 0.001), the levels of cytokines IFN-γ and IL-2 were significantly decreased (p < 0.001) and also the antibody titre level (p < 0.001) was significantly reduced when compared with control rats. A marked decline in PFC (p < 0.001) and serum soluble immune complex (PEG) formation (p < 0.001) was also observed. Hence, the present study clearly demonstrates the immunoprotective effect of KA.

Unique ligand-binding property of the human IgM Fc receptor.

The IgM Fc receptor (FcμR) is the newest FcR, and coligation of FcμR and Fas/CD95 on Jurkat cells with agonistic IgM anti-Fas mAb was shown to inhibit Fas-induced apoptosis. The ligand-binding activity of human FcμR was further examined. FcμR-mediated protection from apoptosis was partially blocked by addition of 10(4) molar excess of IgM or its soluble immune complexes, but it could be inhibited by addition of 10-fold excess of IgM anti-CD2 mAb. This suggests that FcμR binds more efficiently to the Fc portion of IgM reactive with plasma-membrane proteins than to the Fc portion of IgM in solution. The former interaction occurred in cis on the same cell surface, but not in trans between neighboring cells. This cis engagement of FcμR resulted in modulation of Ca(2+) mobilization via CD2 on Jurkat cells or BCRs on blood B cells upon cross-linkage with the corresponding IgM mAbs. Several functional changes were observed with FcμR mutants: 1) significant increase in IgM ligand binding in the cytoplasmic tail-deletion mutant, 2) enhanced cap formation in FcμR upon IgM binding at 4°C with a point mutation of the transmembrane His to Phe, and 3) less protective activity of FcμR in IgM anti-Fas mAb-mediated apoptosis assays with a point mutation of the membrane-proximal Tyr to Phe. These findings show the importance of the cis engagement of FcμR and its critical role in receptor function. Hence, FcμR on B, T, and NK cells may modulate the function of surface proteins recognized by natural or immune IgM Abs on the shared membrane cell surface.