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.

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.

Chédiak-Higashi syndrome: presentation of seven cases.

CONTEXT: Chédiak-Higashi Syndrome (CHS) is a rare autosomal recessive disease characterized by recurrent infections, giant cytoplasmic granules, and oculocutaneous albinism. OBJECTIVE: To describe clinical and laboratory findings from CHS patients. DESIGN: Case report. SETTING: The patients were admitted into the Allergy and Immunology Unit of the Instituto da Criança, a tertiary public care institution. CASES REPORT: Seven patients had oculocutaneous albinism, recurrent infections and giant cytoplasmic granules in the leukocytes. One patient had low IgG levels and three showed impaired bactericidal activity of neutrophils. Six patients died of infectious complications during the accelerated phase. Therapy included ascorbic acid and antibiotics. Chemotherapy was used for the accelerated phase in two patients. Bone marrow transplantation (BMT) was proposed for one patient. DISCUSSION: The authors emphasize the need for early diagnosis and therapy of CHS. BMT should be indicated before the accelerated phase of the disease has developed.

Brazilian report on primary immunodeficiencies in children: 166 cases studied over a follow-up time of 15 years.

One hundred sixty-six cases of primary immunodeficiency diseases (PID) (95 males, 71 females), diagnosed according to WHO criteria, have been registered at the Children's Hospital, University of São Paulo, Brazil. The following frequencies were found: predominantly humoral defects, 60.8% (n = 101); T cell defects, 4.9% (n = 8); combined ID, 9.6% (n = 16); phagocyte disorders, 18.7% (n = 31); and complement deficiencies, 6% (n = 10). IgA deficiency was the most frequent disorder (n = 60), followed by transient hypogammaglobulinemia (n = 14), chronic granulomatous disease (n = 14), and X-linked agammaglobulinemia (n = 9). In comparison to other (national) reports, we observed higher relative frequencies of phagocyte and complement deficiencies. Recurrent infections were the cause of death in 12.7%. Allergic symptoms were observed in 41%, mainly in IgA-deficient, hypogammaglobulinemic, or hyper-IgE patients, and autoimmune disorders in 5%, predominantly in IgA and complement deficiencies. Five patients suffered from BCG dissemination; two of them died. This is the first Brazilian report on PID over an observation time of 15 years.

PIP: Over a 15-year observation period (1981-96), 166 cases of primary immunodeficiency disease (PID) were registered at the Department of Pediatrics, University of Sao Paulo, Brazil. PID was diagnosed according to World Health Organization criteria and only children with well-established deficiencies were included. The following frequencies were noted by PID classification: predominantly humoral defects (60.8%), T cell defects (4.9%), combined immunodeficiency (9.6%), phagocyte disorders (18.7%), and complement deficiencies (6%). The male to female ratio was 1.3 to 1. Immunoglobin A deficiency was the most frequent disorder (60 cases), followed by transient hypogammaglobulinemia (14 cases), chronic granulomatous disease (14 cases), and X-linked agammaglobulinemia (9 cases). Allergic symptoms occurred in 41% of cases. During the observation period, 23 children (13.8%) died, primarily of recurrent infections. Although improved diagnostic facilities have facilitated the recognition of immunodeficient children, the true incidence is likely to be higher than that detected in this study. Increased international collaboration is urged to improve the early detection of PID.

Effect of total parenteral nutrition with different lipid emulsions of human monocyte and neutrophil functions.

Parenteral nutrition (TPN) with lipid emulsions is claimed to be associated with impaired monocyte (M) and neutrophil (N) functions. Long-chain triglycerides (LCT) and a mixture containing 50% medium-chain triglycerides (MCT) and 50% LCT, currently used in nutritional therapy with TPN, were evaluated for their ex vivo effects on human N and M chemotaxis, phagocytosis, bacterial killing, and oxidative metabolism by nitroblue tetrazolium reduction test. Cell functions were examined in a randomized, crossover, blind trial in 10 malnourished patients with gastric cancer. Prior to the operation (2 wk), central TPN (40 kcal/kg) with 25% of caloric energy provided as LCT or MCT/LCT emulsion was infused over 48 h. After the crossover period fat-free TPN was given over 48 h. Function tests were done for N and M before and after each lipid emulsion infusion. Every cell function test performed for each patient was controlled by another test done in healthy adult volunteers and the results were compared with the normal range of values previously established for a healthy adult population. All the patients completed the studies without complications. Crossover validity was statistically established. Bacterial killing was the only function reduced in neutrophils after LCT emulsion (% killed bacteria = 79.0 +/- 8.5 versus 67.4 +/- 19.2; P < 0.05), although this function remained within the normal range values in 80% of the patients. In conclusion, the lipid emulsions did not affect any monocyte functions and only moderately decreased neutrophil bacterial killing.

Effect of therapy with recombinant human interferon-gamma on the release of nitric oxide by neutrophils and mononuclear cells from patients with chronic granulomatous disease.

The aim of this study was to investigate the effect of recombinant human interferon-gamma (rHuIFN-gamma) therapy on the release of nitric oxide (NO) by neutrophils (NEU) and mononuclear cells (MON) from patients with chronic granulomatous disease (CGD). Five patients with this rare disease received rHuIFN-gamma (50 micrograms/m2 of body surface, given by subcutaneous injection three times a week) for 6 months. Clinical and laboratory evaluations were performed before and after 1 and 6 months of rHuIFN-gamma therapy. Nitric oxide release by NEU and MON was assessed by the ability of these cells to inhibit thrombin-induced washed platelet aggregation. The nitrite (NO2-) and nitrate (NO3-) levels in the supernatant of cultured NEU and MON, as well as in plasma and urine (24 h diuresis), were quantified by high-performance liquid chromatography (HPLC). Conventional immunologic tests for assessing phagocyte and lymphocyte functions and humoral immunity were also performed. Therapy with rHuIFN-gamma for 6 months did not enhance NO synthesis by NEU or MON from the patients with CGD. The urinary but not plasma levels of NO2- and NO3- were elevated after rHuIFN-gamma therapy. Phagocyte and lymphocyte functions as well as humoral immunity were not affected by rHuIFN-gamma therapy. Although few patients were available for the study, we conclude that therapy with rHuIFN-gamma for 6 months did not enhance the synthesis of NO by NEU and MON in CGD patients. Whether the increased excretion of NO2- and NO3- in the urine of CGD patients after rHUIFN-gamma therapy reflects an induction of NO-synthase in cells other than leukocytes remains to be investigated.

Influence of medium-chain triglyceride-based lipid emulsion on rat polymorphonuclear cell functions.

Intravenous lipid emulsions are used as energy and essential fatty acids sources. There are controversial reports postulating in vitro and in vivo inhibitory effects of long-chain triglycerides (LCT) upon the blood polymorphonuclear leukocytes (PMNL) functions. In the present study the in vivo and in vitro effects of LCT and a physical mixture of medium- and long-chain triglycerides (MCT/LCT) emulsions were investigated on select PMNL functions, i.e., chemotaxis, phagocytosis, and bacterial killing. Blood from 20 rats was incubated with LCT, MCT, MCT/LCT, and saline, respectively. MCT-containing emulsions exhibited an inhibitory effect on all PMNL functions investigated, whereas LCT exerted an effect on the phagocytic index only. The administration of a parenteral supply of LCT, MCT/LCT, and saline for 30 h followed by saline infusion for 14 h in discontinuous mode did not influence any of the investigated PMNL functions. Similarly, continuous infusion over 44 h at increasing infusion rates up to 1.5 mL/h did not affect the PMNL functions. The obvious difference between in vitro and in vivo response of the PMNL model emphasizes the necessity for continuous monitoring of in vivo conditions. Appropriate interpretation of the data requires continuous circumspection and consideration of trials in a clinical setting.

Application of a fluorochrome-lysostaphin assay to the detection of phagocytic and bactericidal disturbances in human neutrophils and monocytes.

The evaluation of phagocytic and microbicidal activities of the blood neutrophils has been recognized as one of the important tools for investigating phagocytic dysfunctions in patients with recurrent infections. In the present study, these activities were examined in neutrophils and monocytes from healthy adults and patients affected by primary phagocytic dysfunctions by using a modified fluorochromic microbicidal assay, discriminating simultaneously the extracellular adherence, ingestion and intracellular killing of Staphylococcus aureus Cowan I. The assay employs acridine orange staining, as described in Bellinati-Pires et al. (1989) (AO assay), but was modified by the addition of an alternative leukocyte treatment with 0.5 U/ml of lysostaphin (LS) for 5 min at 37 degrees C, after phagocytosis (AO-LS assay). The LS treatment was standardized to eliminate staphylococci adhered to the outer surface of the phagocytes without affecting the determination of intracellular live or dead bacteria, as demonstrated in normal neutrophils and monocytes. Our purpose in this study was to compare AO and AO-LS assays in order to evaluate the effect of LS on the determination of actually ingested staphylococci and to provide a means for improving the fluorochromic assay for detecting phagocytic defects, as well as bactericidal disturbances. By using the AO-LS assay, decreased ingestion of staphylococci by neutrophils in Chediak-Higashi Syndrome (CHS) was demonstrated. However, increased staphylococci adherence, as well as ingestion, was observed in neutrophils or monocytes from chronic granulomatous disease (CGD) patients, comparing AO and AO-LS assays. Bactericidal defect, which is a common feature in CHS and CGD, was detected in neutrophils or monocytes in both assays. We emphasize that such alterations were deduced by comparing the patients' results with those obtained from their respective normal controls and with the normal range of values previously established for 160 healthy adults. No alteration was observed in hyper IgE syndrome phagocytes. Despite the possible penetration of LS into the leukocytes, as stated in other studies, we concluded that a short period of phagocyte incubation with this enzyme increased the sensitivity of the fluorochromic assay to detect phagocytic defect without affecting the determination of the bactericidal activity. Moreover, comparations between AO and AO-LS assays may be important in the study of the initial pathways of staphylococci phagocyte interaction, including adherence by non-phagocytic receptors.

Functional alterations of human neutrophils by medium-chain triglyceride emulsions: evaluation of phagocytosis, bacterial killing, and oxidative activity.

Medium-chain triglyceride (MCT) and long-chain triglyceride (LCT) emulsions currently used in nutritional therapy were evaluated for their in vitro effect on neutrophil oxidative metabolism, phagocytosis, and bacterial killing activities. Neutrophils from healthy adult male volunteers were assessed after blood incubation with commercially available fat emulsions containing LCT, MCT, or a mixture of 50% MCT and 50% LCT at a final triglyceride concentration of 20 mg/ml. It was observed that MCT-containing emulsions stimulated nitroblue tetrazolium (NBT) dye reduction by neutrophils as determined by a cytochemical NBT test performed directly on whole blood. This effect was dose dependent. However, after lipid removal by cell washing, the MCT-treated neutrophils showed decreased production of hydrogen peroxide (H2O2) and NBT reduction in response to bacterial lipopolysaccharide or phorbol myristate acetate stimuli as well as impaired phagocytosis and killing of Staphylococcus aureus. In contrast, the LCT emulsion did not alter any of the neutrophil functions evaluated. The present data suggest that MCTs elicit the oxidative metabolism of neutrophils, probably by phagocytosis of fat particles and, depending on the lipid concentration, this effect may not be reversible, leading to impairment of the cellular response to subsequent membrane stimuli.

Delayed phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a fluorochrome assay. Ultrastructural aspects.

The few studies already published about phagocyte functions in Chediak-Higashi syndrome (CHS) has stated that neutrophils present slow rate of bacterial killing but normally ingest microorganisms. In the present study, both phagocytosis and killing of Staphylococcus aureus were verified to be delayed in neutrophils from two patients with CHS when these functions were simultaneously evaluated by a fluorochrome phagocytosis assay. Electron microscopic examination showed morphologic differences among neutrophils from CHS patients and normal neutrophils regarding the cytoplasmic structures and the aspects of the phagolysosomes. It was noteworthy the presence of giant phagolysosomes enclosing bacteria in active proliferation commonly observed in CHS neutrophils after 45 min of phagocytosis, which corresponded with the impaired bactericidal activity of these leukocytes. The present results suggest that phagocytosis may also be defective in CHS, and point out to the sensitivity of the fluorochrome phagocytosis assay and its application in clinical laboratories.

[The effect of lipid emulsions on the mechanisms of organic defense in infectious injury]. / Efeito de emulsões lipídicas sobre mecanismos de defesa orgânica na agressão infecciosa.

For treatment of metabolic derangements in infective states intravenous lipidic emulsions have been used. Their use is however not harmless existing reports on fat inhibiting the function of blood polymorphonuclear leukocytes and macrophages. The purpose of the research reported herewith was to study in rats the effect of new intravenous lipidic emulsions containing medium chain triglycerides and long chain triglycerides and compare it with the effect of long chain triglycerides emulsion on function of polymorphonuclear leukocytes (chemotaxis, phagocytosis and bactericidal activity). The intraperitoneal implant of an E. coli capsule was used for the study. The transfusions of both lipidic emulsions in septic rats have not altered functions of polymorphonuclear leukocytes when compared with saline infusion. However there was found hepatic steatosis, hypertrophy and presence of fat globules in the Kupffer cells in rats infused with medium chain and long chain triglyceride emulsions. Sequential blood cultures obtained from rats infused with the emulsions showed increased bacterial growth with medium chain triglyceride emulsion. There was no significant difference between the rats that received both lipid infusions and those that received saline infusions as to the mortality. Our experimental study suggests that the use of fat emulsions in infective states be done with care and monitoring of seric triglycerides and steroids.

Functional evaluation of human neutrophils. Is the bactericidal activity correlated with nitroblue tetrazolium reduction?

The cytochemical nitroblue tetrazolium (NBT) reduction test continues to be used in clinical laboratories to detect defects in the oxidative metabolism of phagocytes. However, the specificity of the test is controversial, and it is not clear whether NBT reduction really reflects the microbicidal activity of these cells. In the present study, we evaluated the killing of Staphylococcus aureus by neutrophils from healthy adult individuals and from patients with phagocyte dysfunctions using a fluorochrome phagocytic assay, and compared the results with those obtained with a cytochemical NBT test performed simultaneously. The ability of neutrophils to reduce NBT (expressed as percent reducing neutrophils) with or without a lipopolysaccharide stimulus was not correlated with the bactericidal activity of these cells (expressed as percent killed bacteria per 100 neutrophils). The age and sex of the healthy adults did not influence the results of either assay. It seems that the superoxide anion played a small role in NBT reduction by normal neutrophils, since superoxide dismutase did not significantly inhibit this reaction. Only the absolute absence of NBT reduction reflected the low bactericidal activity of neutrophils, as seen in patients with chronic granulomatous disease (CGD). We conclude that the only clinical usefulness of the NBT test is for the screening of CGD, and that bacterial phagocytic assays are more appropriate for assessing the microbicidal function of neutrophils.

[Primary disorders of neutrophils: principal clinical and laboratory aspects]. / Disfunções primárias de neutrófilos: principais aspectos clínicos e laboratoriais.

This article presents a review of different categories of inherited neutrophil dysfunctions, such as chronic granulomatous disease, disorders of chemotaxis, defects of degranulation and the leukocyte adherence deficiency. The clinical manifestations and laboratory findings of these phagocyte abnormalities, with particular reference to various tests of neutrophil functions used for diagnostic elucidation are described.

Effect of medium- and long-chain triglycerides on human neutrophil migration.

There is some controversy concerning the effect of intravenous long-chain triglyceride (LCT) emulsions on the phagocytic system and little is known about the effect of medium-chain triglyceride (MCT)-containing emulsions. We evaluated the chemotaxis and random migration of human neutrophils from 18 healthy adults after preincubation with the following fat emulsions: LCT, MCT and a mixture of 50% MCT and 50% LCT (MCT/LCT). Leukocyte-rich plasma (4 x 10(6) cells/ml) was diluted 4:1 (v/v) with commercial fat emulsions (LCT, MCT, or MCT/LCT, 1:1) or saline and tumbled at 20 cycles/min for 30 min at 37 degrees C. The final composition of the emulsion was 20 mg/ml fat, 0.24% egg yolk lecithin, and 0.5% glycerol and the dispersion was made isotonic by adding NaCl. In a second set of experiments, the LCT and MCT concentrations were adjusted to be equimolar. Leukocyte viability was > or = 95% after exposure to the treatment with fat emulsions. For emulsions with the same weight of each fat, random migration and chemotaxis of neutrophils were unaffected by the LCT emulsion but there was a significant decrease in both chemotaxis and random migration in MCT-(79 and 74%) or MCT/LCT-treated (60 and 56%) neutrophils. Similar results were obtained when LCT and MCT were equimolar. These results demonstrate an inhibitory effect of MCT on two human neutrophil functions which may be dose dependent.

Effect of medium and long chain triglycerides on human neutrophil migration

There is some controversy concerning the effect of intravenous long-chain triglyceride (LCT) emulsions on the phagocytic system and little is known about the effect of medium-chain triglyceride (MCT) containing emulsions. We evaluated the chemotaxis and random migration of human neutrophils from 18 healthy adult after preincubation with the following fat emulsions: LCT, MCT and a mixture of 50% MCT and 50% LCT (MCT/LCT). Leukocyte-rich plasma (4 x 10 6 cells/ml) was diluted 4:1 (v/v) with commercial fat emulsions (LTC, MCT, or MCT?LCT, 1:1) or saline and tumbled at 20 cycles?min for 30 min at 37 grade C. The final composition or the emulsion was 20 mg/ml fat, 0.24% egg yolk lecithin, and 0.5% glycerol and the dispersion was made isotonic by adding NaCl. In a second set of experiments, the LCT and MCT concentrations were adjusted to be equimolar. Leukocyte viability was * 95% after exposure to the treatment with fat emulsions. For emulsions with the same weight of each fat, random migration and chemotaxis of neutrophils were unaffected by the LCT emulsion but there was a significant decrease in both chemotaxis and random migration in MCT- (79 and 74%) or MCT/LCT-treated (60 and 56%) neutrophils. Similar results were obtained when LCT and MCT were equimolar. These results demonstrate an inhibitory effect of MCT on two human neutrophil functions which may be dose dependent

Evaluation of a fluorochrome assay for assessing the bactericidal activity of neutrophils in human phagocyte dysfunctions.

We evaluated a method for the assessment of the phagocytic and bactericidal activity of human peripheral neutrophils against Staphylococcus aureus Cowan I, which is a modified version of the acridine orange staining technique originally described by Smith and Rommel (1977). The modification consisted of the use of free leukocyte suspensions rather than coverglass adhered leukocytes in order to avoid two main problems: the inefficient neutrophil adherence to glass that can be observed in specimens from patients with certain functional phagocyte defects, and the risk of selecting among neutrophils. An additional advantage of the modified procedure is that it permits a uniform bacteria: phagocyte ratio in different cell samples. The method was tested on 25 healthy adults and on four children with functional phagocytic defects (chronic granulomatous disease of infancy, Chediak-Higashi syndrome, and Rothmund-Thomson syndrome associated to persistent neutropenia and low chemotactic response). The neutrophils of all four patients showed a low bactericidal activity, with percent values of intracellular killed bacteria below the mean +/- 2 SD range observed in the healthy population at all incubation times tested (5, 15 and 30 min). A significant reduction in phagocytosis index and in % killed unopsonized S. aureus was observed in relation to bacteria treated with a pool of normal human serum. These results demonstrate the high sensitivity of the method, which could be used to determine intrinsic and extrinsic functional alterations in human neutrophils.