Lipopolysaccharides from Porphyromonas endodontalis and Porphyromonas gingivalis promote angiogenesis via Toll-like-receptors 2 and 4 pathways in vitro.

AIM: Angiogenesis contributes to the development of apical periodontitis, periodontitis, and other oral pathologies; however, it remains unclear how this process is triggered. The aim was to evaluate whether lipopolysaccharide (LPS) from Porphyromonas endodontalis and Porphyromonas gingivalis induced angiogenesis-related effects in vitro via TLR2 and TLR4. METHODOLOGY: Porphyromonas endodontalis LPS (ATCC 35406 and clinical isolate) was purified with TRIzol, whereas P. gingivalis LPS was obtained commercially. The effects of the different LPS (24 h) in endothelial cell migration were analysed by Transwell assays, following quantification in an optical microscope (40×). The effects of LPS on FAK Y397 phosphorylation were assessed by Western blotting. Angiogenesis in vitro was determined in an endothelial tube formation assay (14 h) in Matrigel in the absence or presence of either LPS. IL-6 and VEGF-A levels were determined in cell supernatants, following 24 h treatment with LPS, and measured in multiplex bead immunoassay. The involvement of TLR2 and TLR4 was assessed with blocking antibodies. The statistical analysis was performed using STATA 12® (StataCorp LP). RESULTS: The results revealed that P. endodontalis LPS, but not P. gingivalis LPS, stimulated endothelial cell migration. Pre-treatment with anti-TLR2 and anti-TLR4 antibodies prevented P. endodontalis LPS-induced cell migration. P. endodontalis LPS promoted FAK phosphorylation on Y397, as observed by an increased p-FAK/FAK ratio. Both P. gingivalis and P. endodontalis LPS (ATCC 35406) induced endothelial tube formation in a TLR-2 and -4-dependent manner, as shown by using blocking antibodies, however, only TLR2 blocking decreased tube formation induced by P. endodontalis (clinical isolate). Moreover, all LPS induced IL-6 and VEGF-A synthesis in endothelial cells. TLR2 and TLR4 were required for IL-6 induction by P. endodontalis LPS (ATCC 35406), while only TLR4 was involved in IL-6 secretion by the other LPS. Finally, VEGF-A synthesis did not require TLR signalling. CONCLUSION: Porphyromonas endodontalis and P. gingivalis LPS induced angiogenesis via TLR2 and TLR4. Collectively, these data contribute to understanding the role of LPS from Porphyromonas spp. in angiogenesis and TLR involvement.

Short Disordered Epitope of CRTAM Ig-Like V Domain as a Potential Target for Blocking Antibodies.

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order-disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order-disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.

Optimization and validation of a blocking ELISA for quantitation of anti-rabies immunoglobulins in multispecies sera.

We developed a fast, rabies virus-free, in vitro method, based on a blocking ELISA (bELISA), to detect and accurately quantify anti-rabies glycoprotein antibodies in serum of several animal species. In this method, purified rabies virus-like particles (VLPs) are used as antigen to coat the plates, while the presence of specific rabies immunoglobulins is revealed through blocking the recognition of these VLPs by a biotinylated monoclonal antibody. A quality by design approach was carried out in order to optimize the method performance, improving the sensitivity and, thereby, reducing the limit of detection of this assay. After the method validation, we confirmed that the bELISA method is able to detect a concentration of 0.06 IU/mL rabies immunoglobulins, titer lower than the 0.5 IU/mL cutoff value established as indication for correct vaccination. Further, we assessed the correlation between bELISA, the MNT, and the Platelia methods, confirming the accuracy of this new assay. On the other hand, precision was evaluated, obtaining acceptable repeatability and intermediate precision values, showing that this bELISA could be proposed as a potential alternative method, replacing the gold standard techniques in vaccination schemes and becoming a routine control technique within regional rabies surveillance programs.

Anti-Budding Assay: Development of a high throughput screen to identify neutralizing antibodies that block Chikungunya virus (CHIKV) release

Objective: No licensed CHIKV vaccines or effective therapeutic agents are currently available. However, some CHIKV-specific monoclonal antibodies (mAbs) are highly effective in animal models, both prophylactically and therapeutically. This is thought to be largely mediated via blocking of CHIKV entry into cells. However, we and others have shown that inhibition of viral release/ budding is also a major mechanism for CHIKV control. We aimed to develop a high throughput in vitro screening assay to efficiently identify "antibudding"mAbs. Design and Methodology: An assay for quantification of viral budding from infected cells was optimised by varying cell line, cell density, multiplicity of infection (MOI), incubation periods, NH4Cl concentration and plate type. The assay utilized our novel, fully replication -competent, attenuated CHIKV nano-luciferase (nluc) reporter virus (CHIKV 181/25 E2nluc). The optimised assay was used to screen CHIKV+, Zika virus (ZIKV)+, CHIKV-/ZIKV- sera, and cloned memory B-cells from a CHIKV+ individual. Results: Optimal conditions involved use of rhabdomyosarcoma (RD) cells, bulk-infected at MOI 1 for 2hrs, removal of residual virus, resuspension in media containing 20mM NH4Cl, seeding at 2.5x104cells/well into 96-well plates and luminometry after 18hrs. Inter-plate coefficient of variability CV scores and Z' values remained <15% and >0.5 respectively, indicative of a valid assay. Most CHIKV+ sera displayed potent antibudding activity, two displayed no significant activity, and there was no ZIKV cross-reactivity. Of 800 memory B-cell clones, 13 exhibited significant anti-budding antibody activity. Conclusions: We developed a sensitive, reproducible, Biosafety level (BSL-2) safe, high throughput CHIKV antibudding assay useful for screening both polyclonal sera and monoclonal antibodies.

Antibody persistence and booster response in adolescents and young adults 4 and 7.5 years after immunization with 4CMenB vaccine.

BACKGROUND: Data on duration of protection against invasive meningococcal disease post-vaccination with the recombinant, 4-component, meningococcal serogroup B vaccine (4CMenB) are limited. We evaluated bactericidal activity persistence in adolescents/young adults up to 7.5 years post-primary vaccination with 4CMenB, and response to a booster dose compared with vaccine-naïve controls. METHODS: This open-label, multicenter study (NCT02446743) enrolled 15-24 year-old-previously vaccinated participants from Canada, Australia (group Primed_4y) 4 years post-priming with 4CMenB (2 doses; 0,1-month schedule), and Chile (Primed_7.5y) 7.5 years after priming with 4CMenB (2 doses; 0,1/0,2/0,6-month schedule) and vaccine-naïve participants of similar age (Naïve_4y and Naïve_7.5y groups). Primed participants received a booster dose; vaccine-naïve participants received 2 catch-up doses of 4CMenB, 1 month apart. We evaluated antibody persistence and immune responses using hSBA in terms of geometric mean titers and percentages of participants with hSBA titers ≥4, the kinetics of bactericidal activity post-booster (previously vaccinated) or post-2 doses (vaccine-naïve), and safety. RESULTS: Antibody levels declined at 4 (Primed_4y) and 7.5 (Primed_7.5y) years post-primary vaccination, but remained higher than in vaccine-naïve participants at baseline (≤44% vs ≤ 13% [fHbp]; ≤84% vs ≤ 24% [NadA]; ≤29% vs ≤ 14% [PorA]) for all vaccine antigens except NHBA (≤81% vs ≤ 79%). One month post-booster and post-second dose, 93-100% of primed and 79-100% of vaccine-naïve participants had hSBA titers ≥4 for all antigens. Kinetics of the antibody response were similar across groups with an early robust response observed 7 days post-booster/second dose. No vaccine-related serious adverse event was reported. CONCLUSION: For all antigens except NHBA, a higher proportion of primed participants had hSBA titers ≥4, at 4 and 7.5 years post-vaccination, compared with vaccine-naïve participants. A more robust immune response after booster compared to a first dose in vaccine-naïve individuals, showed effective priming in an adolescent/young adult population. No safety or new reactogenicity issues were identified.

Association between Haemagglutination inhibiting antibodies and protection against clade 6B viruses in 2013 and 2015.

BACKGROUND: The epidemiology of the pandemic A(H1N1) virus has been changing as population immunity continues to co-evolve with the virus. The impact of genetic changes in the virus on human's susceptibility is an outstanding important question in vaccine design. In a community-based study, we aim to (1) determine the genetic characteristics of 2009-2015 pandemic H1N1 viruses, (2) assess antibody response following natural infections and (3) assess the correlation of A/California/07/09 antibody titers to protection in the 2013 and 2015 epidemics. METHODS: In a household transmission study, serum specimens from 253 individuals in Managua, Nicaragua were analyzed. Combined nose and throat swabs were collected to detect RT-PCR confirmed influenza infection and virus sequencing. Hemagglutination inhibition assays were performed and the protective titer for circulating H1N1pdm was determined. RESULTS: Clade 6B pandemic H1N1 viruses predominated in Nicaragua during the 2013 and 2015 seasons. Our household transmission study detected a household secondary attack rate of 17% in 2013 and 33% in 2015. Infected individuals, including vaccinees, showed an apparent antibody response to A/California/07/09. Baseline titers of A/California/07/09 antibodies were found to associate with protection in both seasons. A titer of ≥1:40 correlated to a 44% protection in children, a 29% protection in adults 15-49years old and a 51% protection in adults 50-85years old. CONCLUSION: In 2013 and 2015, antibody titers to A/California/07/09 associated with an infection risk reduction amongst exposed household contacts. This is consistent with a detectable vaccine effectiveness reported in a number of studies. Genetic changes in clade 6B viruses might have led to a reduced immunity in some whereas others might have been less affected. The use of human serologic data is important in virus characterization and if performed in a timely manner, could assist in vaccine strain selection.

Antibody-based assay discriminates Zika virus infection from other flaviviruses.

Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged recently as a global health threat, causing a pandemic in the Americas. ZIKV infection mostly causes mild disease, but is linked to devastating congenital birth defects and Guillain-Barré syndrome in adults. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to differentially detect ZIKV and dengue virus (DENV) infections, accentuating the urgent need for a specific and sensitive serological test. We previously generated a ZIKV nonstructural protein 1 (NS1)-specific human monoclonal antibody, which we used to develop an NS1-based competition ELISA. Well-characterized samples from RT-PCR-confirmed patients with Zika and individuals exposed to other flavivirus infections or vaccination were used in a comprehensive analysis to determine the sensitivity and specificity of the NS1 blockade-of-binding (BOB) assay, which was established in laboratories in five countries (Nicaragua, Brazil, Italy, United Kingdom, and Switzerland). Of 158 sera/plasma from RT-PCR-confirmed ZIKV infections, 145 (91.8%) yielded greater than 50% inhibition. Of 171 patients with primary or secondary DENV infections, 152 (88.9%) scored negative. When the control group was extended to patients infected by other flaviviruses, other viruses, or healthy donors (n = 540), the specificity was 95.9%. We also analyzed longitudinal samples from DENV-immune and DENV-naive ZIKV infections and found inhibition was achieved within 10 d postonset of illness and maintained over time. Thus, the Zika NS1 BOB assay is sensitive, specific, robust, simple, low-cost, and accessible, and can detect recent and past ZIKV infections for surveillance, seroprevalence studies, and intervention trials.

Neutrophil Extracellular Traps Stimulate Proinflammatory Responses in Human Airway Epithelial Cells.

Tissue injury leads to the release of uric acid (UA). At high local concentrations, UA can form monosodium urate crystals (MSU). MSU and UA stimulate neutrophils to release extracellular traps (NET). Here, we investigated whether these NET could be involved in the development of inflammation by stimulating cytokine release by airway epithelial cells. We found that NET significantly increased the secretion of CXCL8/IL-8 and IL-6 by alveolar and bronchial epithelial cells. These effects were not observed when NETosis was inhibited by Diphenyleneiodonium, elastase inhibitor, or Cl-amidine. Similar findings were made with NET induced by cigarette smoke extract, suggesting that NET proinflammatory capacity is independent of the inducing stimulus. Furthermore, NET affected neither the viability and morphology of epithelial cells nor the barrier integrity of polarized cells. The epithelial stimulatory capacity of NET was not affected by degradation of DNA with micrococcal nuclease, treatment with heparin, or inhibition of the elastase immobilized to DNA, but it was significantly reduced by pretreatment with an anti-HMGB-1 blocking antibody. Altogether, our findings indicate that NET exert direct proinflammatory effects on airway epithelial cells that might contribute in vivo to the further recruitment of neutrophils and the perpetuation of inflammation upon lung tissue damage.

Annexin A1 promotes timely resolution of inflammation in murine gout.

Gout is a self-limited inflammatory disease caused by deposition of monosodium urate (MSU) crystals in the joints. Resolution of inflammation is an active process leading to restoration of tissue homeostasis. Here, we studied the role of Annexin A1 (AnxA1), a glucocorticoid-regulated protein that has anti-inflammatory and proresolving actions, in resolution of acute gouty inflammation. Injection of MSU crystals in the knee joint of mice induced inflammation that was associated with expression of AnxA1 during the resolving phase of inflammation. Neutralization of AnxA1 with antiserum or blockade of its receptor with BOC-1 (nonselective) or WRW4 (selective) prevented the spontaneous resolution of gout. There was greater neutrophil infiltration after challenge with MSU crystals in AnxA1 knockout mice (AnxA1-/- ) and delayed resolution associated to decreased neutrophil apoptosis and efferocytosis. Pretreatment of mice with AnxA1-active N-terminal peptide (Ac2-26 ) decreased neutrophil influx, IL-1ß, and CXCL1 production in periarticular joint. Posttreatment with Ac2-26 decreased neutrophil accumulation, IL-1ß, and hypernociception, and improved the articular histopathological score. Importantly, the therapeutic effects of Ac2-26 were associated with increased neutrophils apoptosis and shortened resolution intervals. In conclusion, AnxA1 plays a crucial role in the context of acute gouty inflammation by promoting timely resolution of inflammation.

Clinical experience with the use of anti-CdtB and anti-vinculin antibodies in patients with diarrhea in Mexico. / Experiencia clínica con el uso de los anticuerpos anti-CdtB y anti-vinculina en pacientes con diarrea en México.

INTRODUCTION: Circulating anti-CdtB/anti-vinculin antibodies have been validated as biomarkers to distinguish IBS-D from IBD, but there is no experience with them in Latin America. MATERIALS AND METHODS: The analysis was carried out on patients seen at a FGIDs/motility clinic over the last 7 months for diarrhea with abdominal pain and/or bloating who were tested for these antibodies. The patients were diagnosed according to the Rome III criteria or with organic disorders, and those presenting with IBS were further classified as post-infectious (PI) or non-PI-IBS. RESULTS: Thirty patients were studied. Positive biomarkers were found in IBS-D and IBS-D Overlap (58.8%) and IBS-M (33.3%), with no differences between PI-IBS (71.4%) vs. non-PI-IBS (41.7%) subjects (P=.21). There was no positivity in patients with other FGIDs or organic diarrhea, except for one with small intestinal bacterial overgrowth (SIBO). CONCLUSIONS: Our findings support the use of this test as a first-line diagnostic tool to confirm the presence of IBS-D/IBS-M according to the Rome III criteria.

Pro-inflammatory cytokines, IL-1ß and TNF-α, produce persistent compromise in tonic immobility defensive behaviour in endotoxemia guinea-pigs.

AIM: Sepsis has been associated with acute behavioural changes in humans and rodents, which consists of a motivational state and an adaptive response that improve survival. However, the involvement of peripheral cytokines synthesized during systemic inflammation as modulators of the tonic immobility (TI) defensive behaviour remains a literature gap. Our purposes were to characterize the TI defensive behaviour in endotoxemia guinea-pigs at acute phase and after recovery from the initial inflammatory challenge. Furthermore, we investigated whether peri-aqueductal grey matter (PAG) exists as a brain structure related to this behaviour and also pro-inflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, act at this mesencephalic nucleus. METHODS: Endotoxemia was induced by lipopolysaccharide (LPS) administration in guinea-pigs. The parameters evaluated included TI defensive behaviour, survival, cytokines production, as well as neuronal activation and apoptosis in the PAG. RESULTS: Endotoxemia guinea-pigs exhibited a reduction in the duration of TI episodes, starting at 2 h after LPS administration and persisting throughout the experimental period evaluated over 7 days. Moreover, endotoxemia increased the c-FOS immunoreactivity of neurones in the ventrolateral PAG (vlPAG), as well as the caspase-3 expression. The LPS microinjection into vlPAG reproduces the same compromise, that is a decrease in the duration of TI defensive behaviour, observed after the peripheral administration. The immunoneutralization against IL-1ß and TNF-α into vlPAG reverts all the effects produced by peripheral LPS administration. CONCLUSION: Our findings confirm that vlPAG is an important brain structure involved in the behavioural alterations induced by endotoxemia, possibly changing the neuronal activity caused by pro-inflammatory cytokines produced peripherally.

Análisis de impacto presupuestal del uso de anticuerpos bloqueadores de acetilcolina receptores, comparado con la prueba de Tensilon, de estímulo repetitivo, electromiografía con electrodo de fibra única e I CE test para pacientes con miastenia gravis / Budget impact analysis of the use of acetylcholine receptor blocking antibodies, compared to the Tensilon test, repetitive stimulus, electromyography with single fiber electrode and IEC test for patients with myasthenia gravis

Tecnologías evaluadas: -Tecnologías actuales: electromiografía con electrodo de fibra única e ICE test;\r\n-Tecnología nueva: anticuerpos bloqueadores de acetilcolina receptores, prueba de Tensilon, prueba de estímulo repetitivo. Población: Esta prueba se puede aplicar a todas las edades y a todos los sexos, ya que la aparición de la enfermedad puede presentarse en toda la población. Perspectiva: Tercer pagador - Sistema General de Seguridad en Salud (SGSSS) colombiano. Horizonte temporal: El horizonte temporal de este AIP en el caso base corresponde a un año. Adicionalmente se reportan las estimaciones del impacto presupuestal para los años 2 y 3, bajo el supuesto de la inclusión en el POS en el año 1. Costos incluidos: Solo se tuvieron en cuenta los costos de las pruebas: -Electromiografía con electrodo de fibra única: $71.262,1; -Ice test: $26.223,3; Prueba completa de Tensilon: $24.000; -Prueba de estímulo repetitivo: $46.219,1; -Test de anticuerpos contra receptor de acetilcolina por RIA (ACRA): $45.416. Fuente de costos: Para todas las pruebas diagnósticas se utilizó el promedio ponderado estimado desde los registros de uso de servicios de 2014 SISPRO (módulo de prestación de servicios, mediante conexión OBDS), teniendo como corte de búsqueda la fecha del desarrollo de este impacto (20/10/2015). Todos los costos de las pruebas son ponderados por el número de unidades utilizadas que reporta la misma base de datos. Además, todas\r\nlas tecnologías son costeadas desde bases de aseguradores, para confirmación de precios. Resultados: Actualmente, el mercado se encuentra dominado por la electromiografía con electrodo de fibra única, la cual se encuentra dentro del plan de beneficios, pero por opinión de los realizadores, una vez que la prueba de acetilcolina receptores y de Lambert entre al plan de beneficios, se aumentará su participación, lo cual repercutirá en un ahorro al sistema, dado que dichas pruebas son menos costosas.(AU)

Placental Transfer of Dengue Virus (DENV)-Specific Antibodies and Kinetics of DENV Infection-Enhancing Activity in Brazilian Infants.

BACKGROUND: Maternal-fetal transferred dengue virus (DENV)-specific antibodies have been implicated in the immunopathogenesis of dengue during infancy. METHODS: A prospective birth cohort was established in a dengue-endemic area in the Northeast Region of Brazil. DENV-specific immunoglobulin G (IgG) and DENV-1-4 serotype-specific neutralizing antibody (NAb) levels were assessed in 376 paired maternal and umbilical cord blood samples. The kinetics of enhancing activity by maternally acquired DENV antibodies was determined in serum samples from children enrolled in the cohort. RESULTS: Mothers were mostly immune to DENV-3 alone (53.7%) or combined with DENV-4 (30.6%). Levels of DENV-specific IgG, DENV-3 NAbs, and DENV-4 NAbs were significantly higher in newborns than in their respective mothers. Mothers immune to a single serotype transferred greater levels of DENV-specific IgG (P = .02) and DENV-3 NAbs (P = .04) than mothers immune to multiple DENV serotypes. Maternally acquired DENV-3 NAbs disappeared in >90% of the children by the age of 4 months. The peak enhancing activity was detected by the age of 2 months (P < .0001) and rapidly declined by the age of 4 months (P = .0035). CONCLUSIONS: Unlike Asian infants, the enhancing activity of DENV infection by maternally transferred DENV antibodies occurs at earlier ages in Brazilian children. These findings might explain the low occurrence of severe dengue among infants in our setting.

Differential replicative ability of clinical dengue virus isolates in an immunocompetent C57BL/6 mouse model.

BACKGROUND: Several experimental animal models have been used to study the pathogenesis of dengue disease; however, most of the studies used laboratory-adapted viruses, which lack the virulence of viruses circulating in humans. The aim of this study was to analyze the ability of clinical Dengue virus (DENV) isolates (D2/BR/RP/RMB/09 and D3/BR/SL3/02) to infect immunocompetent C57BL/6 mice. METHODS: Two strategies of intraperitoneal infection, which were based on the concept of the antibody dependent enhancement phenomenon, were used. In one strategy, the animals were inoculated with macrophages infected in vitro with dengue viruses, which were incubated with enhancing antibodies, and in the other strategy, the animals were inoculated with a complex of enhancing antibodies and dengue viruses. RESULTS: The D3/BR/SL3/08 isolate showed a higher ability of infection (virus RNA was more frequently detected in the serum and in several organs) in the experimental model compared to both the D2/BR/RP/RMB/2009 isolate and a laboratory adapted DENV-1 strain (Mochizuki strain), regardless of the infection strategy used. The main features of the D3/BR/SL3/08 isolate were its neuroinvasiveness and the induction of an extended period of viremia. Enhancing antibodies did not influence on the infection of animals when macrophages were used, but the level of viremia was increased when they were used as a complex with a D3/BR/SL3/02 isolate. DISCUSSION: We showed that DENV isolates could infect immunocompetent C57BL/6 mice, which have has been previously used to study some aspect of dengue disease when infected with laboratory adapted strains. DENV genome was detected in the same organs found in humans when autopsy and biopsy samples were analyzed, showing that C57BL/6 mice reproduce some aspects of the DENV tropism observed in humans. The main difference observed between the D3/BR/SL3/02 and D2/BR/RP/RMB/2009 clinical isolates was the neuroinvasive ability of the first one. Neuroinvasiveness has been described in some DENV infected cases and is common for other members of the Flavivirus genus. CONCLUSIONS: These results suggest that C57BL/6 mice can be used as an experimental model to evaluate virulence differences among DENV clinical isolates.

Immunomodulatory and clinical effects of the "tiaomian III decoction" in patients with blood blocking antibody deficiency and recurrent spontaneous abortion.

We studied the immunomodulatory and clinical effects of the empirical formula "tiaomian III decoction" on maternal blood blocking antibody deficiency and recurrent spontaneous abortion. Sixty-one patients with blocking antibody deficiency were divided in the experimental group (N = 31), who took tiaomian III decoction, and the control group (N = 30), who received active immunotherapy with paternal lymphocytes; both treatments lasted 3 months. Blocking antibodies, anti-idiotypic antibodies, interleukin, T-lymphocyte subsets, and macrophage colony-stimulating factor (M-CSF) were tested. After treatment, the positive conversion rate reached 87.1 and 86.7% in the experimental and control groups, respectively. After treatment, CD4 levels decreased while CD8 levels increased in both groups. The CD4/CD8 ratio was higher than normal and increased significantly from pre-treatment (P < 0.05). IL-10 and M-CSF levels increased significantly in both groups (P < 0.05). The 1-year conception rates of the experimental and control groups were 58.1 and 46.7%, respectively (P < 0.05). The results show the tiaomian III decoction can increase the positive conversion rate of maternal blocking antibodies and promote the production of IL-10 and M-CSF. Thus, it strengthens the maternal body's protection of the fetus and maintenance of conception. The higher conception rate of the experimental group demonstrates the positive clinic efficacy of the tiaomian III decoction on maternal blood blocking antibody deficiency and recurrent spontaneous abortion.

Blocking ELISA using recombinant NcSRS2 protein for diagnosing bovine neosporosis.

Neospora caninum is the etiologic agent of neosporosis, which leads to economic impacts on cattle industry. The reference method for serodiagnosis of neosporosis is the indirect fluorescent antibody test (IFAT). However, IFAT is laborious, expensive, and is not practicable in high throughput screening. In order to facilitate the serological diagnosis of neosporosis, we developed a blocking enzyme-linked immunosorbent assay (b-ELISA) based on NcSRS2 recombinant protein (rNcSRS2) and polyclonal antibodies against rNcSRS2 (b-ELISA/rNcSRS2). Compared to IFAT, b-ELISA/rNcSRS2 showed 93.7 % accuracy (98.7 % sensitivity and 88.7 % specificity), suggesting its potential as diagnostic assay to detect N. caninum antibodies in cattle sera.

Modulation of immune response by RAGE and TLR4 signalling in PBMCs of diabetic and non-diabetic patients.

Diabetes is associated with increased glucose levels and accumulation of glycated products. It is also associated with impairment in the immune response, such as increased susceptibility to infections. In this study, we assessed the possible interactions between TLR4 and RAGE signalling on apoptosis and on the expression of inflammatory cytokines in PBMC from individuals with and without diabetes. PBMCs were isolated from seven diabetic patients and six individuals without diabetes and stimulated in vitro with bacterial LPS (1 µg/ml) associated or not with BSA-AGE (200 µg/ml). This stimulation was performed for 6 h, both in the presence and in the absence of inhibitors of TLR4 (R. sphaeroides LPS, 20 µg/ml) and RAGE (blocking monoclonal antibody). Apoptosis at early and late stages was assessed by the annexin-V/PI staining using flow cytometry. Regulation of TNF-α and IL-10 gene expression was determined by RT-qPCR. PBMCs from diabetes patients tended to be more resistant apoptosis. There were no synergistic or antagonistic effects with the simultaneous activation of TLR4 and RAGE in PBMCs from either diabetes or non-diabetes group. Activation of TLR4 is more potent for the induction of TNF-α and IL-10; RAGE signalling had a negative regulatory effect on TNF-α expression induced by LPS. TLR and RAGE do not have relevant roles in apoptosis of PBMCs. The activation of TLR has greater role than RAGE in regulating the gene expression of IL-10 and TNF-α.

Semaphorin 3F and neuropilin-2 control the migration of human T-cell precursors.

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.

The effect of antidrug antibodies on the sustainable efficacy of biologic therapies in rheumatoid arthritis: practical consequences.

Biologic therapies, predominantly TNF-α inhibitors, have revolutionized the treatment of rheumatoid arthritis (RA). However, their clinical utility can be limited by the development of antidrug antibodies (ADAs). Immunogenicity is a complex phenomenon related to various drug, disease, and patient characteristics, and may be more common with the monoclonal antibodies than with etanercept, a soluble TNF receptor-Fc immunoglobulin fusion protein. Neutralizing antibodies - those that hinder bioactivity by preventing drug molecules from binding to TNF - are correlated with reduced serum drug concentrations, loss of therapeutic response, adverse events, and treatment discontinuation. Cost-effective use of these agents will depend on further research into drug and ADA assays, and how they should guide dose reduction or switching strategies.

Allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens: an in vitro study.

One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%-51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.