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.

Prokaryotic expression of Staphylococcus aureus Clumping factor B and evaluation of the antiserum-mediated opsonic activity / 生物工程学报

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.

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

effect of combination of pseudomonas aeruginosa alginate and an immunomodulator protein of garlic on opsonophagocytosis in murine model

Chronic pulmonary infection in patients with cystic fibrosis is predominantly due to infection by mucoid strains of Pseudomonas aeruginosa. Mucoid P. aeriginosa is due to the production of exopolysaccharide called also alginate. Alginate in addition to interference with the clearance of lung has antiphagocytic property. Optimal killing activity of P. aeruginosa requires opsonic antibodies. Since immunomodulatory effects of garlic on enhancing phagocytic activity has been proved, in this study the effect of combination of alginate and an immunomodulator protein of garlic on production of opsonic antibodies against P. aeruginosa mucoid exopolysaccharide has been investigated. Alginate was extracted from a 72-hour culture of P. aeruginosa strain 8821M and then DNasel, RNaseA and Proteinase k were added. Subsequently, alginate was purified with gel filtration chromatography by sephacryl S-400. Female BALB/c mice aged 6-8 weeks were divided into five groups and injected subcutaneously on days 0, 7, 14 with either alginate, garlic, alginate- garlic, R10 or alginate-R10 and opsonophagocytic killing activity was calculated in each group. The purified alginate contained 34.6 micro g/ml uronic acid, 0.5 micro g/ml nucleic acid, 1.45 micro g/ml protein and 0.08 micro g/ml LPS. Opsonophagocytic killing activity after immunization with R10, alginate and their combination showed significant increases of respectively 69%, 67% and 82% comparing to the control group. Combination of P.aeruginosa alginate and immunomodulator fraction of garlic enhances immunogenicity to P.aeroginosa by the elicitation of opsonic antibodies in BALB/c mice

Determinación de valores de referencia para la microtécnica de fagocitosis y muerte intracelular de Cándida álbicans en un grupo de población sana en Bogotá / Determination of reference values for the microtechnique of phagocytosis and intracellular death of Candida albicans in a group of healthy population in Bogota

La función de las células fagocíticas es un eficiente mecanismo de protección no específico contra agentes infecciosos y de eliminación de células muertas o seniles. Este proceso lo realizan células de la inmunidad innata como los macrófagos y los polimorfonucleares neutrófilos que son especialmente efectivos durante el inicio de infecciones por bacterias extracelulares Gram positivas y Gram negativas. Los macrófagos participan en etapas más tardías de la inflamación fagocitando bacterias y restos celulares. Las técnicas de evaluación de la capacidad fagocítica han mostrado ser muy laboriosas, requieren gran cantidad de muestra sanguínea y excesiva manipulación de las células analizadas. Por el contrario, la microtécnica de muerte intracelular de Cándida para el análisis de la función fagocítica es una técnica rápida, poco dispendiosa y que requiere de muy poca cantidad de sangre. En este trabajo se analizaron 56 muestras de personas sanas con la microtécnica de muerte intracelular de Candida albicans, para hallar valores normales del índice de fagocitosis y el porcentaje de muerte intracelular, indicadores que evalúan la capacidad fagocítica de los polimorfonucleares neutrófilos. El índice de fagocitosis fue de 2.70 +/- 0.43 (media +/ - DE) con un rango de 2.03 a 3.92 y el porcentaje de muerte Intracelular fue de 31.31 +/- 5.10 (media +/- DE) con un rango de 21 a 39. No hubo diferencias por grupos de edad, género, recuento de leucocitos o porcentaje de polimorfonucleares.

Spontaneous bacterial empyema in cirrhotic patients: complements 3 and 4, opsonic power and C-reactive protein as pathogenic mechanisms and diagnostic tools

Hepatic hydrothorax occurs in approximately 5-12% of patients with cirrhosis and portal hypertension and may be complicated by spontaneous bacterial empyema [SBE]. Pathogenic mechanisms of SBE still need to be investigated. The present work assesses the role of complement components [C3, C4], opsonizing power and C-reactive protein in the pathogenesis of SBE in cirrhotic patients. Twenty five cirrhotic patients with hepatic hydrothorax were randomly selected and 10 patients with hydrothorax secondary to heart failure were included as controls in the study. Pleural fluid [PF] and serum samples were analyzed for: total protein [TP], albumin, lactic dehydrogenase [LDH], glucose, polymorph nuclear leukocytic count [PMNL], complement components [C3, C4], opsonic activity [on the basis of log-kill] and high sensitive C-reactive protein [CRP]. SBE was diagnosed when pleural fluid PMNL was > 250 cells/mm[3] with a positive culture or >500 cells/ mm[3] with a negative culture after exclusion of pulmonary infections. Thirteen patients [52%] [Group I] were diagnosed as SBE and 12 patients [48%] had no SBE [Group II]. There was no significant difference between patients and controls [GIII] as regards age, gender, serum proteins, serum C3, serum WBC and effusion CRP. Levels of serum albumin, total pleural effusion proteins, PT% and opsonic activity of groups I and II were significantly lower than in GIII with no significant difference between groups I and II. Levels of serum bilirubin and C4 of groups I and II were significantly higher than group III with no significant difference between groups I and II. Level of pleural effusion C3 in group I was significantly lower than in groups II and III and level of C3 in group II was significantly lower than in group III. Level of pleural effusion C4 in group I was significantly lower than group III, but there was no significant difference between groups I and II. In hepatic patients, 7 patients [28%] belonged to Child's class B and 18 [72%] to class C. Spontaneous bacterial empyema was detected in 56% of hepatic patients with Child's class C and in 43% of Child's class B. There was no significant difference between hepatic patients with and without SBE with regard to Child-Pugh's score. In patients with SBE, levels of C3 and C4 were significantly less in pleural fluid than in serum but there was no significant difference with regard to opsonic activity. Local complement defects [especially C3] and opsonic activity in cirrhotic patients predispose to SBE. Serum CRP increases, but effusion CRP level should be reassessed as a cheap diagnostic tool

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47 PHOX. Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47 PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Functional Immunity to Cross-Reactive Serotype 6A Induced by Serotype 6B in Pneumococcal Polysaccharide Vaccine / 소아과

PURPOSE: Streptococcus pneumoniae serotype 6B and 6A are important pathogens in pneumococcal infections. It is commonly assumed that the 6B vaccines elicit antibodies cross-reacting with the 6A serotype and the cross-reactive antibodies protect against infections of 6A. To examine this assumption, we measured the opsonophagocytic capacity to serotype 6A and 6B in adults. METHODS: Twenty-four adults were immunized with pneumococcal PS vaccine that contains 6B PS. Their preimmune and postimmune sera were studied for the capacity to opsonize 6B and 6A serotypes with opsonophagocytic killing assay. RESULTS: Opsonization titers to 6B were significantly higher than those to 6A in preimmune and postimmune sera. Because significant increasesof opsonization titers were observed in adults with polysaccharide vaccines for 6A(cross-reactive) serotype as well as for 6B(vaccine) serotype, 6B PS in vaccine elicited cross-protective antibodies to 6A, but not in all cases. One adult did not have detectable levels of opsonization titers to 6A after immunization. CONCLUSION: Although 6B PS in pneumococcal PS vaccine elicits antibodies cross-reacting with 6A serotype in some adults, it may not occur always. This study should be extended to other age groups such as children and elderly people. The presence of the cross-protection should be directly determined in clinical trials of the pneumococcal vaccines as well as during the postlicensure monitoring surveys by serotyping the clinical isolates of pneumococci.

Phagocytosis and killing of epidemic methicillin-resistant Staphylococcus aureus by human neutrophils and monocytes

Staphylococcus aureus is a pathogen that has been associated with nosocomial infections since the preantibiotic era. Since the introduction of antibiotics in medical practice in the 1940 s, methicillin-resistant S. aureus (MRSA) strains have been emerging in various parts of the world. In view of the important role of the phagocytic system in the defense against this bacteria, we decided to study phagocytosis by neutrophils and monocytes of an epidemic MRSA strain in São Paulo, Brazil, in comparison with methicillin-sensitive strains. Complement system opsonins are fundamental for efficient ingestion of the resistant and sensitive strains by both types of phagocytes. We found no association of the opsonic requirement of the MRSA strain with the multiresistance phenotype. On the other hand, the MRSA strain was found to be more resistant to the effector mechanisms of neutrophils than both sensitive strains when opsonized with fresh serum, despite the phagocytosis results. This fact suggests that the intracellular killing of S. aureus is an additional parameter of bacterial virulence, but new approaches must be implemented to study the interactions of this MRSA strain with phagocytes in order to investigate the possible factors involved in its behavior in response to neutrophil effector mechanisms.

Functional Antibody Responses to Seven Serotypes in Pneumococcal Polysaccharide Vaccine in Children / 소아과

PURPOSE: Streptococcus pneumoniae is an important pathogen in children as well as in elderly people. The aim of this study was to assess the opsonization activity of pneumococal capsular antibody to seven serotypes in 23-valent pneumococcal polysaccharide vaccine before and after immunization in Korean children. METHODS: Nine children from 24 to 49 months of age were immunized with 23-valent polysaccha ride pneumococcal vaccine in Ewha Woman's University Tongdaemoon Hospital. From them, sera were collected before immunization as well as 1 month after immunization. Double-serotype opsono phagocytic killing assay was performed against seven serotypes(4, 6B, 9V, 14, 18C, 19F and 23F) of S. pneumoniae to get opsonization titer of these sera using dimethyl formamide differentiated HL-60 cells. RESULTS: After immunization, geometric mean opsonization titer of sera against serotype 18C(4,352) was the highest followed by serotype 9V(3,317), serotype 14(1,545), serotype 4(1,359). The opsonization titer against serotype 23F(342) was the lowest followed by serotype 19F(525), and serotype 6B (809). Fold increases of opsonization titer were higher against serotype 4(339.8), 9V(65.7), 19F(34.5), and 14(18.8) than those against serotype 23F(4.5), 6B(8.9), and 18C(9.9). CONCLUSION: The opsonization titers of postimmune sera against seven serotypes of S. pneumoniae were increased in many(not all) preschool children. But they showed variable values of opsonization titer in postimmune sera as well as in preimmune sera against different serotypes. The immune responses to pneumococcal polysaccharide in preschool children are not maturated sufficiently yet.

Phagocytosis of serum-and IgG-opsonized zymos an particles induces apoptosis through superoxide but not nitric oxide in macrophage J774A.1

Phagocytosis of serum- and IgG-opsonized zymosan (SOZ and IOZ, respectively) particles into J774A.1 macrophages induced apoptosis of the cells, accompanied by the expression of p21(WAF1), one of cyclin-dependent protein kinase (CDK) inhibitors. Furthermore, phagocytosis of SOZ and IOZ particles into macophages induced superoxide formation. Tat-superoxide dismutase (SOD), which is readily transduced into the cells using Tat-domain, protected the cells from the apoptosis induced by phagocytosis of SOZ and IOZ particles. lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) also caused the apoptosis of the cells. However, Tat-SOD could not protect the cells from LPS/IFN-gamma induced apoptosis, suggesting that apoptosis mechanisms involved are different from each other. In the present study, we determined the amounts of nitric oxide (NO) produced by SOZ, IOZ, and LPS/IFN-gamma, and found that SOZ and IOZ did not induce the generation of NO in macrophages, whereas LPS/ IFN-gamma did. The apoptosis due to phagocytosis was accompanied with the release of cytochrome c from mitochondrial membrane to cytosolic fraction. Furthermore, SOZ and IOZ induced the cleavage of procasapase-3 (35 kDa) to give rise to an active caspase-3 (20 kDa), which was blocked by Tat- SOD but not by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. On the other hand, LPS/IFN-gamma caused the activation of procaspase-3, which was blocked by PTIO but not by Tat-SOD. Taken together, phagocytosis of SOZ and IOZ particles induced apoptosis through superoxide but not NO in macrophages, accompanied with the release of cytochrome c and the activation of caspase-3.

Evaluation of antibody responses to pneumococcal vaccines with ELISA and opsonophagocytic assay

Antibodies to a capsular polysaccharide (PS) provide protection against Streptococcus pneumoniae which express the homologous capsular serotype, and pneumococcal vaccines are designed to induce antibodies in the capsular PS. Levels and opsonophagocytic capacity of antibodies to the capsular PS of S. pneumoniae serotype 19F were determined by sera from adults immunized with 23-valent S. pneumoniae capsular PS vaccines. Geometric means of IgG anti-19F antibody level and specific opsonic titer rise significantly after immunization. The level of anticapsular PS antibodies for S. pneumoniae 19F serotype is fairly well correlated (r2=O.63) with the opsonophagocytic activities of sera. However, 3.7% (1/27) of serum samples display strikingly less opsonophagocytic activity than expected on the basis of their antibody level. Thus, antibody level may be of general use in predicting vaccine-induced protection among adults for 19F serotype. However, the opsonic activity data suggest that antibody levels are not always indicative of functional antibody.

Fagocitosis opsónica de Histoplasma capsulatum / Opsonic phagocytosis of Histoplasma capsulatum

Objetivo: El presente trabajo hace una breve revisión de la escasa literatura existente sobre el papel que desempeñan los receptores Fcç participan en la fagocitosis de levaduras del hongo Histoplasma capsulatum por fagocitos humanos y murinos. Conclusiones: Resultados de diferentes ensayos sugieren que los receptores Fcç participan en la etapa de internalización, aunque parecen no ser los receptores preferenciales para la entrada del hongo a las células fagocíticas murinas

Serum complement functions and opsonic activity in patients with bacteremia after elective injections sclerotherapy and band ligation for oeshageal varices

Acquired deficiencies of certain complement proteins and, or impaired opsonisation activity of serum have been implicated in the pathogenesis of increased susceptibility to infections of patients with cirrhosis. One handred patients with chronic liver disease complicated with oesophageal varices were included in this study. Eighty of them were randomized to receive either endoscopic injection sclerotherapy [EIS] [40 patients] or endoscopic band ligation [EBL] [40 patients] as elective therapy for their varices. The rest of the patienit 20 were endoscoped but their varices not injected nor ligated. The there groups were well matched as regards clinical, laboratory, ultrasonograhic data, endoscopic findings and Child-Puch classification. Twenty healthy patients- apart from dyspepsia, were exposed to upper GII endoscopy and used as control group. Aerobic and anaerobic blood cutluresd were done for all members before, 30 minutes, 4 hours and 24 hours after the procedures. Throat cultures and cultures of the bands, ethanolamine, injector needle, tip of the endoscope and waer supply were cultural immediately before the procedures, Serum concentration of C3 and C4 and haemolytic complement activity [classic and alternative pathways] of serum and serum opsonic activity were determined in all patients and control group. Aerobic blood cultures were positive in 8 [20%] patients of EIS group and 7 [17.5%] patients of EBL group with a no significant difference between them. Organisms isolated were streptococcus pneumonia and coagulase negative Stahyylococci. 30 minutes positive blood cultures turned negative at 4 hours cultures. Causative organisms were also isolated from the patients throat. All anaerobic cultures were negative. Throat swab, injector needle swab and endoscopic washing swab showed positive culture in 65%, 59.5%, 20%, 28.5% and 22.8% respectively, Other swab cultures from water supply, Scelerosant agent, and bands showed no bacterial growth. Serum C3 and C4 concentration and haemolytic complement activity were significantly reduced [P <0.05 and P0.01 respectively] in patients with bacteremia compared to non bacteremic patients serum opsonic activity showed insignificant difference between bacteremic and non baceremic patients. In conclusion, low serum C3 and C4 concentaration and decreased hemolytic complement functions are major risk factors for bacteremia after both procedures

Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans

Se da cuenta de un método rápido para la cuantización del flujo citométrico de la fagocitosis en sangre periférica completamente heparinizada (HCPB), mediante la utilización de partículas de látex phycoerythrin-conjugadas de 1µm de diámetro disponibles comercialmente. El método es más rápido y presenta mayor reproducibilidad que la técnica estandar de Bjerknes' (1984) utilizando propidium iodide-teñida Candida albicans, aplicada convencionalmente a la capa leucocitica de sangre periférica pero modificada por HCPB. Tambien damos cuenta de una modificación de Bjerknes' Intracellular Killing Test para permitir su aplicación a HCPB.

We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripheral blood (HCPB), using commercially available phycoerythrin-conjugated latex particles of 1 micron diameter. The method is faster and shows greater reproducibility than Bjerknes' (1984) standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripheral blood but here modified for HCPB. We also report a modification of Bjerknes' Intracellular Killing Test to allow its application to HCPB.

Evaluación de la actividad fagocitaria y bactericida de los monocitos y heterófilos aviares contra Samonella gallinarum / Evaluation of phagocytic and bactericidal activities of avian monocytes and heterophils against Salmonella gallinarum in vitro

Se determinó, mediante ensayos in vitro, la capacidad fagocítica y bactericida de los deterófilos y de los monocitos aviares contra Salmonella gallinarum, en presencia y en ausencia de 10 por ciento de suero hiperinmune. En los ensayos de fagocitosis se observó que los hetrófilos fagocitaron al 28 ñ 4.7 por ciento de las bacterias sin opsonizar y al 45 ñ 9.9 por ciento de las bacterias opsonizadas, obteniéndose diferencias significativas (P< 0.05). En contraste, los monocitos sólo fagocitaron un 10.3 ñ 4.2 por ciento y un 11.7 ñ 3.8 por ciento de bacterias opsonizadas y sin opsonizar respectivamente (P> 0.05). En los ensayos bactericidas se observó que los heterófilos destruyeron al 90.46 ñ 3.3 por ciento de la bacteria sin opsonizar y 90 ñ 2.3 por ciento de la bacteria opsonizada (P> 0.05); sin embargo, en los monocitos se obtuvo un 10.5 ñ 6.6 por ciento y un 84.74 ñ 5 por ciento respectivamante, obteniéndose diferencias significativas (P< 0.05). Los resultados del presente estudio indican que la fagocitosis de los heterófilos fue significativamente incrementada por la opsonización; en el caso de los monocitos, no hubo un efecto significativamente mayor. Aproximadamente el 90 por ciento de las bacterias fagocitadas por los heterófilos fueron destruidos, como se determinó en el ensayo. La opsonización no incrementó significativamante el porcentaje de bacteria destruida por parte de los heterófilos, sin embargo, la psonización de Salmonella gallinarum sí favoreció la capacidad bactericida de los monocitos

Opsonophagocytosis and intracellular killing activity of neutrophils in patients with human immunodeficiency virus infection.

In this study, neutrophils isolated from asymptomatic HIV positive individuals, patients with AIDS-related complex (ARC), ARC patients receiving zidovudine (AZT) and full-blown AIDS patients were assayed for their opsonophagocytic and intracellular killing activities. Progressively decreasing opsonophagocytosis of C. albicans by neutrophils correlated with increasing severity of the disease in all groups of HIV infected individuals, as compared to neutrophils isolated from healthy controls. The intracellular killing of C. albicans by neutrophils of asymptomatic and ARC patients did not differ significantly from controls. Neutrophils of ARC patients receiving AZT and AIDS patients showed a slightly decreased killing activity in comparison to that of neutrophils from healthy controls.