An in vitro model mimicking the complement system to favor directed phagocytosis of unwanted cells

BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptor­ligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.

Validation of a Multiplexed Opsonophagocytic Assay for 11 Additional Pneumococcal Serotypes and Its Application to Functional Antibody Evaluation Induced by Pneumococcal Polysaccharide Vaccine

BACKGROUND: Various pneumococcal vaccines have been evaluated for immunogenicity by opsonophagocytic assay (OPA). A multiplexed OPA (MOPA) for 13 pneumococcal serotypes was developed by Nahm and Burton, and expanded to 26 serotypes in 2012. The development of new conjugate vaccines with increased valence has necessitated expanded MOPAs to include these additional serotypes. In this study, we validated this expanded MOPA platform and applied to measure antibodies against 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F) in human sera. METHODS: All materials, including serum, complement, bacterial master stocks, and HL-60 cells, were evaluated for assay optimization. Following optimization, the assay was validated for accuracy, specificity, and intra- and inter-assay precision with sera from adult donors following standard protocols. The assay was applied to evaluate functional antibodies of 42 sera immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). RESULTS: The expanded MOPA platform was specific for all serotypes, with the exception of serotype 20. The assay results were highly correlated with those obtained from single-serotype OPA, indicating acceptable accuracy. The coefficients of variation were 7%–24% and 13%–39% in tests of intra- and inter-assay precision, respectively, using three quality-control samples. A MOPA that included 11 additional serotypes in the PPV23 was established and validated with respect to accuracy, specificity, and precision. The opsonic indices of immune sera were obtained using this validated assay. CONCLUSION: The expanded MOPA will be useful for evaluation of the immunogenicity of PPV23 and future conjugate vaccine formulations.

Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products

Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0–11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635–5,706 (serotype Ia), 488–1,421 (serotype Ib), and 962–3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups.

Producción de suero de Coombs en carneros inmunizados con globulinas humana opsonizadas / Coombs serum production in sheep immunized with human globulin opsonized

Se obtuvo suero antiglobulínico (Coombs) con el empleo de un inóculo consistente en un inmunocomplejo (IC) inmunoglobulina (Ig) humana-antiglobulina humana en carnero, como opsonina para favorecer la respuesta inmune. Se inmunizaron 18 carneros divididos en 3 grupos de 6: el primero y el segundo destinados a producir anti-IgG y anti-C3, respectivamente. Estos, a su vez, subdivididos en subgrupo A: en el que se empleó el método tradicional de obtención de suero de Coombs; y B: en el que se usó el adyuvante completo de Freud en la dosis inicial y el IC en la fase de mantenimiento. Al tercer grupo solo se le administró el IC puro (subgrupo A) y en una dilución 1:200 (subgrupo B). En los carneros de los subgrupos 1B y 2B se obtuvieron títulos más elevados de anti-IgG y anti-C3dg, que en los inmunizados por el método tradicional. La respuesta de anticuerpos en los animales que se inmunizaron con los IC (3A y 3B), fue más rápida y de mayor título que las obtenidas por el método tradicional (1A y 2A) o el método combinado (1B y 2B). La respuesta en el subgrupo 3B fue más prolongada, al parecer por un efecto de dosis

An antiglobulin serum (Coombs) was obtained using a consistent inoculums in a immunocomplex (IC) the human immunoglobulin (Ig)/human antiglobulin in the seep by example, the opsonin to favor the immune response. Eighteen sheeps were immunized divided into three groups of 6 each: The first and second aimed to produce anti-IgG and anti-C3, respectively. In turn, these were divided into the A subgroup: in which we used the traditional method of Coombs's serum obtaining and B group in which we used the Freud's whole adjuvant in initial dose and the IC in the maintaining phase. Third group received the pure IC (A subgroup) and at a dilution of 1:200 (B subgroup). In sheeps from the 1B and 2B subgroups it was possible to obtain higher titration of anti-IgG and anti-C3dg than those immunized by means of the traditional method. The antibody response in animals immunized with the ICs (3A and 3B) was faster and of higher titration than those obtained by traditional method (1A and 2A) or the combined method (1B and 2B). The response in the 3B subgroup was lengthier apparently by a dose effect

A Randomized Clinical Trial of Short-term vs Long-term Therapy in the Spontaneous Bacterial Peritonitis / 대한간학회지

BACKGROUND/AIMS: The standard regimen of SBP is cefotaxime 2 g IV, every 8 hours for 10 days, and the success rate is approximately 90%. It was reported that 5-day therapy was as effective as 10-day therapy, but, generally, the 5-day therapy has not been accepted in practice. This study was done to confirm whether the short-term therapy is as effective as long-term therapy, and additionally whether the opsonin capacity influences the final output of antibiotic therapy. METHODS: Of the 27 patients who met strict criteria for SBP or culture negative neutrocytic ascites, 14 were randomized to a group receiving 5 days and 13 to a group receiving 10 days of single agent cefotaxime 2g IV every 8 hours. Many variables (clinical data, standard liver and kidney function results, ascitic fluid data, complement proteins) were obtained at admission, the 2nd day, and the last day(the 5th or 10th day) of the study. RESULTS: Hospitalization mortality(7% vs 15%), recurrence rate(21% vs 0%), infection related mortality(7% vs 0%) and therapeutic response(86% vs 92%) were not significantly different between the 5- and 10-day treatment groups. The opsonic activity was not significantly different between the recurrence(n=3) group and non-recurrence group(n=26), but the indices of opsonic activity in recurrence group showed lower tendency than those in non-recurrence group. Early response rate was significantly different between the high and low protein concentration in ascitic fluid. CONCLUSIONS: Short course treatment of SBP is as effective as long-course therapy and significantly less expensive.