Evaluation of Opsonophagocytic Dysfunctions in Severely Burned Patients by Luminol-Dependent Chemiluminescence

We compared the luminol-dependent chemiluminescence (CL) response of peripheral blood from severely burned patients with that from normal controls to evaluate the primary defense level against bacterial infection in the patients. The CL was measured upon addition to diluted whole blood of a soluble stimulus, phorbol myristate acetate (PMA) or particulate stimuli such as bacteria or zymosan without special opsonization. In the early post-burn days, the initial rate of whole blood CL induced with the particulate stimulus was much lower than that in the normal controls, whereas the rate was higher when PMA was used as a stimulus. The number of granulocytes in the patients' blood had increased and isolated polymorphonuclear leukocytes (PMNs) from the patients exhibited higher CL responses to the particulate or soluble stimulus as compared with those of normal controls. The results suggest that the PMNs in burn patients were activated and normally mobilized in the early post-burn period but the opsonizing capacity in the blood decreased. In fact, the serum levels of complement, immunoglobulins and fibronectin were found to be lower in the blood from the patients than those from normal controls and a supplement of freshly frozen plasma of human immunoglobulin preparations restored the initial rate of the whole blood CL upon phagocytosis. The prognosis is still poor when severe infection occurs in the patients with decreased CL response of whole blood. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the CL response of PMNs from burn patients. The administration of rhG-CSF may be useful for decreasing the morbidity of severe infection following burn injury in the near future.     

Evaluation of burn blister fluid

Although edema is evident immediately after a burn, the diffusion of nutrient chemical constituents of the body is not impaired. Blister fluid, not unlike plasma or serum, contained all substances found in the body, including parenterally administered penicillin. The elevation of potassium and the cation to anion imbalance is primarily due to the Na/K cellular pump malfunction, and the destruction of the permeability of the cell membrane is most likely a direct result of complement and other cellular enzymes, which include the prostaglandins and thromboxanes. The elevated SGOT, CPK, and LDH indicated severe trauma to the cells in the immediate area of burn and possibly to the skeletal muscle. The presence of immunoglobulins indicated that high-molecular-weight proteins diffuse equally well during this edematous phase (IgM, 900,000; IgG, 190,000). Evidence of this nature strongly suggests that the integrity of the burn blister by maintained.     

The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects

Burn injury is a prevalent and traumatic event for pediatric patients. At present, the diagnosis of burn injury severity is subjective and lacks a clinically relevant quantitative measure. This is due in part to a lack of knowledge surrounding the biochemistry of burn injuries and that of blister fluid. A more complete understanding of the blister fluid biochemistry may open new avenues for diagnostic and prognostic development. Burn insult induces a highly complex network of signaling processes and numerous changes within various biochemical systems, which can ultimately be examined using proteome and metabolome measurements. This review reports on the current understanding of burn wound biochemistry and outlines a technical approach for ‘omics’ profiling of blister fluid from burn wounds of differing severity.     

Rifampicin-induced Immune Thrombocytopenia

A case is reported in which severe thrombocytopenia occurred during administration and readministration of rifampicin. The patient''s erythrocytes gave a positive direct antiglobulin test due to complement on the red cell surface; in the serum, complement-fixing antibodies were detected which were directed against the drug.Immunological studies showed antibodies, of both IgG and IgM type, capable of fixing complement to both normal and the patient''s platelets, but only in the presence of rifampicin. In addition the IgM type of antibody (but not the IgG) was capable of fixing complement to normal red cells; again only in the presence of the drug.     

Partial myeloperoxidase deficiency in preleukemia

In seven subjects with partial and apparently acquired form of myeloperoxidase (MPO) deficiency, some functional properties of neutrophils (PMNs) were studied. Five patients suffered from preleukemia, one from diabetes mellitus and one from carcinoma of the breast with bone marrow metastases. Intracellular bactericidal activity, oxygen consumption and superoxide radical production were within normal limits. In three patients with preleukemia, the serum opsonic activity was markedly reduced (less than m-3SD) in an autologous system, but normal in the presence of pooled normal serum. Decreased opsonic activity was also found when these patient's sera were assayed in the presence of normal PMNs. Since the levels of IgG and C3 were comparable in the patients' sera and the pooled serum, a deficiency of another unknown opsonin or the presence of an opsonization inhibitor has to be postulated. The partial MPO defect apparently doesn't decrease the intracellular killing of Staphylococcus aureus by PMNs. The known susceptibility to bacterial infections in preleukemia may be explained by the reduction of serum opsonization conducing to a secondary decrease of the ingestion and killing of bacteria by the PMNs.     

Serum opsonic deficiency produced by Streptococcus pneumoniae and by capsular polysaccharide antigens.

The opsonic requirements for phagocytosis of S. pneumoniae types 6, 7, 18, and 23 were determined in normal and C2 deficient serum, and in normal serum chelated with magnesium ethyleneglycoltetraacetic acid. All four strains were effectively opsonized via the alternative complement pathway, a finding suggesting that the capsular polysaccharides of these strains activated complement via the alternative pathway. Since bacteremic pneumococcal disease is often associated with circulating capsular polysaccharide, it was considered that this cellular component may activate complement in vivo and impair host defenses by producing an opsonic defect for pneumococci. To examine this hypothesis, serum was incubated with suspensions of whole S. pneumoniae types 6, 7, 18, or 23 or with purified capsular polysaccharide from each of these types, and residual complement activity and opsonic capacity were measured. Hemolytic C 3--9 complement activity and opsonic capacity for 3H-thymidine labeled Salmonella typhimurium, a species effectively opsonized via the alternative pathway, were reduced in serum following incubation. Polysaccharide concentrations as low as 1 microgram/ml inhibited serum opsonic capacity for salmonella. Whole pneumococci and pneumococcal capsular polysaccharide also inhibited the opsonic activity of human C2 deficient serum for salmonella, further evidence for activation of complement via the alternative pathway. Pneumococcal capsular polysaccharide markedly inhibited the opsonic capacity of normal serum for the homologous pneumoccal type. Thus, amounts of pneumococcal capsular polysaccharide, similar to those found in the serum of patients with pneumococcal disease, bring about decomplementation of serum via activation of the alternative pathway and inhibit pneumococcal opsonization.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately 80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunoglobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studied in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD50 of five strains of P. aeruginosa was increased greater than or equal to 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MAbs in mice challenged with approximately 10 LD50 of the homologous LPS serotype ranged from less than 0.01 mg/kg to 1.00 mg/kg.     

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

Abstract International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately vn80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunolgobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studies in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD₅₀ of five strains of P. aeuruginosa was increased ≥ 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MA0bs in mice challenged with approximately 10 LD₅₀ of the homologous LPS serotype ranged from < 0.01 mg/kg to 1.00 mg/kg.     

Opsonic activity of ascitic fluids by a chemiluminescent method

Cirrhotic ascites are highly susceptible to spontaneous bacterial infection, whereas carcinogenic ascites are seldom infected. This difference may be explained by differences in their chemotactic, bactericidal and/or opsomic activities. We measured the chemotactic and opsonic activity of ascitic fluids from 35 alcoholic cirrhotic ascites and of 12 peritoneal carcinogenic ascites. Chemotactic activity was measured by the under-agarose technique and opsonic activity by a luminol-enhanced method. Ascitic fluids from alcoholic cirrhosis had low chemotactic (62 ± 24.5% that of N-formylated peptide) and opsonic (67 ± 50% of normal serum) activities on normal human neutrophils. In contrast, ascitic fluids from peritoneal carcinoma were found to possess high opsonic activity (114 ± 49% of normal serum) and chemotactic activity similar to that of N-formylated peptide. During a 3-month follow-up, 11 spontaneous bacterial infections were observed among the first group against none in the carcinogenic group.     

Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10⁺ IL-12⁻) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10⁻IL-12⁺) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.     

Serum requirements necessary for the opsonophagocytosis ofescherichia coli O73:K92:H1

TheEscherichia coli O73:K92:H1 serotype, which possesses a capsular antigen immunochemically similar to the capsule of the group C meningococcus, is demonstrated in this study to be resistant to phagocytosis by normal human PMNs and serum and to be dependent upon immune antibody and presumably the classical complement pathway for opsonization. Using both a luminol-dependent chemiluminescence assay and an in vitro bactericidal system, we examined, in both the absence and presence of complement, the opsonic activity of IgM ang IgG antisera. Of the various antisera tested, only those sera cross-reactive with the K92 capsular antigen were found to be opsonic both in vivo and in vitro, while somatic O or lipid A antisera demonstrated no activity. In in vitro studies with capsular IgM and IgG antisera, only IgG demonstrated opsonic activity without complement, whereas IgM required complement for opsonization of O73:K92:H1. These data demonstrate that antisera directed toward capsular antigens are opsonic for this phagocytosis-resistantE. coli, and that complement is a necessity for opsonization in the absence of sufficient capsular IgG antibodies.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Escherichia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occurred in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement. Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis occurring in the first 10 min. The chemiluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

The blister and the second degree burn in guinea pigs: the effect of exposure.

A reproducible partial-thickness burn was inflicted on 100 albino guinea pigs. With the blister intact, the evaporative water loss from the burn surface was essentially the same as from normal unburned skin. When the blister was removed, the rate of water loss was initially more than 100 times normal, and it remained 20 to 50 times the normal rate throughout the first week. This high rate of water loss was associated with an increasing depth of wound destruction (from dehydration) and a thickening of the overlying crust. Microscopic examination showed that previously viable dermis was incorporated in this crust. The depth of dermal loss was negligible when the blister was left intact, and improved healing resulted. We believe it is important to leave burn blisters intact, whenever possible.     

烧伤早期伴发脓毒症患者血清PCT、hs-CRP、COR及WBC的动态变化及临床意义

目的:探讨烧伤早期伴发脓毒症患者血清降钙素原(PCT)、超敏C反应蛋白(hs-CRP)、皮质醇(COR)和白细胞计数(WBC)水平并分析其临床价值。方法:回顾性分析2014年12月至2018年1月本院收治的63例烧伤早期患者的临床资料,其中19例患者伴发脓毒症(脓毒症组),44例患者未伴发脓毒症(无脓毒症组),另选取30例于本院进行体检的志愿者作为健康对照组,依据病情严重程度将脓毒症患者分为脓毒症休克组(n=4)、严重脓毒症组(n=7)、一般脓毒症组(n=8),依据预后情况分为预后良好组(n=13)和预后不良组(n=6)。动态检测并对比所有研究对象血清PCT、hs-CRP、COR和WBC水平。结果:无脓毒症组、脓毒症组血清PCT、hs-CRP、COR和WBC水平高于健康对照组,且脓毒症组高于无脓毒症组(P<0.05)。烧伤后7 d、11 d、15 d,脓毒症组血清PCT、hs-CRP、COR和WBC水平均高于无脓毒症组(P<0.05)。脓毒症休克组血清PCT、hs-CRP、COR和WBC水平高于严重脓毒症组、一般脓毒症组,且严重脓毒症组患者高于一般脓毒症组患者(P<0.05)。预后不良组血清PCT、hs-CRP、COR和WBC水平高于预后良好组(P<0.05)。结论:血清PCT、hs-CRP、COR和WBC水平在烧伤早期伴发脓毒症患者中水平异常升高,与病情严重程度有关,上述指标对预后判断有一定的临床参考价值。     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

Abstract We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Eschericia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occured in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement, Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis iluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

重症监护室烧伤感染患者脓毒症的病原菌分布及影响因素研究

目的:探讨重症监护室(ICU)烧伤感染患者脓毒症的发生情况及病原菌分布,并分析其危险因素,为防治ICU烧伤感染患者脓毒症提供理论依据。方法:收集我院2016年7月-2019年7月期间的86例ICU烧伤感染患者的临床资料,依据是否发生脓毒症,将其分为脓毒症组和非脓毒症组,比较两组的基本资料等情况,分析ICU烧伤感染患者脓毒症的发生率及病原菌分布,并分析ICU烧伤感染患者脓毒症发生的影响因素。结果:86例ICU烧伤感染患者中有33例发生脓毒症,发生率为38.37%;ICU烧伤感染患者脓毒症病原菌主要是革兰阴性杆菌(占54.55%),其次是革兰阳性球菌(占42.42%);单因素结果分析发现,烧伤至入院时间、合并糖尿病、吸入性损伤、首次手术时间以及烧伤面积与ICU烧伤感染患者脓毒症的发生有关(P<0.05);多因素Logistic回归分析发现,首次手术时间>2d、TBSA>50%是ICU烧伤感染患者脓毒症发生的独立危险因素(P<0.05)。结论:ICU烧伤感染患者易发生脓毒症,首次手术时间>2d以及TBSA>50%是其发生的独立危险因素,临床应采取针对性预防措施,以降低ICU烧伤感染患者脓毒症发生率。     

Effects of long‐distance running on serum opsonic activity measured by chemiluminescence

Exhaustive exercise such as long‐distance running has been shown to increase susceptibility to infection. In order to investigate whether serum opsonic activity plays a role in such conditions, we utilized luminol‐dependent and lucigenin‐dependent chemiluminescence (LmCL and LgCL). We took serum samples from 24 male marathon runners before and after running 30 km. Neutrophils were isolated from the peripheral blood of healthy volunteers. Serum opsonic activity was examined by measuring neutrophil ROS stimulated with zymosan particles opsonized by the serum samples. Immunoglobulin and complement levels in the serum were also measured. After a 30 km run, the maximum light emission was increased and the time to reach the maximum light emission was shortened significantly (p < 0.05) in LmCL. However, there were no significant changes in the immunoglobulin and complement levels. The increase of ROS production may suggest that serum opsonic activity is accelerated after running 30 km. Thus, serum opsonic activity might not play a significant role in the susceptibility to infection after long‐distance running. Copyright © 2003 John Wiley & Sons, Ltd.     

Bacteremia caused by viridans streptococci in 71 children.

A review of the hospital records of 71 patients from whose blood viridans streptococci were isolated showed that in 13 cases the patient''s illness was definitely related to the bacteremia: 4 patients had endocarditis, 3 had pneumonia, 2 had peritonitis and 1 each had meningitis, a scalp wound infection, sinusitis and otitis media. The bacteremia may have contributed to the two deaths among these 13 patients. In 45 cases the viridans streptococci may have contributed to the patient''s illness: 15 patients had an infection of the lower respiratory tract and 7 an infection of the upper respiratory tract, 8 were neonates with suspected septicemia, 3 had soft tissue infections, 3 had leukemia and sepsis, and 9 had miscellaneous infections; the bacteremia was unrelated to the two deaths in this group. In another 13 cases the viridans streptococci could not be related to the patient''s illness. The species most frequently isolated were Streptococcus mitis, S. sanguis II and S. MG-intermedius. The outcome of the bacteremia was generally good, even among the 11 patients not treated with antibiotics. When viridans streptococci are cultured from a single blood sample, further samples of blood and, if feasible, specimens from the associated focus of infection should be obtained for culture; further blood cultures are especially important in cases of suspected endocarditis.