M. T. Van Den Barselaar

Antibacterial Activity of Human Neutrophil Defensins in Experimental Infections in Mice Is Accompanied by Increased Leukocyte Accumulation

Neutrophil defensins (or human neutrophil peptides-HNP) are major constituents of the azurophilic granules of human neutrophils and have been shown to display broad-spectrum antimicrobial activity. Other activities of these defensins, which are released from stimulated neutrophils, include cytotoxic, stimulatory, and chemotactic activities toward a variety of target cells. We studied the potential use of HNP-1 for antibacterial therapy of experimental bacterial infections in mice. In experimental peritoneal Klebsiella pneumoniae infections in mice, HNP-1 injection was shown to markedly reduce bacterial numbers in the infected peritoneal cavity 24 h after infection. This antibacterial effect was found to be associated with an increased influx of macrophages, granulocytes, and lymphocytes into the peritoneal cavity. These leukocytes appeared to be a requirement for the antibacterial effect, since in leukocytopenic mice administration of HNP-1 did not display antibacterial activity. HNP-1 treatment also reduced bacterial numbers in experimental K. pneumoniae or Staphylococcus aureus thigh muscle infections. In this model, radiolabeled HNP-1 was found to accumulate at the site of infection, whereas most of the injected HNP-1 was rapidly removed from the circulation via renal excretion. These results demonstrate that neutrophil defensins display marked in vivo antibacterial activity in experimental infections in mice and that this activity appears to be mediated, at least in part, by local leukocyte accumulation.

Requirement of extracellular complement and immunoglobulin for intracellular killing of micro-organisms by human monocytes.

The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells. Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity. Intracellular killing of Staphylococcus aureaus, S. epidermidis, and Escherichia coli by human monocytes does not occur or is low in the absence of serum, and maximal killing is only reached when fresh serum is present; intermediate values are obtained in the presence of heat-inactivated serum. These findings indicate that complement stimulates intracellular killing. Isolated heterogeneous immunoglobulin (Ig)G, pFc fragments of heterogeneous IgG, and both IgG1 and IgG3 stimulate intracellular killing of S. aureaus by monocytes to the same degree as heat-inactivated serum. Sphingomyelinase, which decreases the number of Fc receptors, and neuraminidase, which increases these receptors, respectively, decreased and increased the intracellular killing, whereas anti-monocyte serum completely abolished the stimulation of intracellular killing by inactivated serum. These results prove that interaction of the Fc receptor with the Fc part of IgG is required for the intracellular killing. Inhibition of the activation of complement components via the alternative pathway gave a considerable reduction in the intracellular killing of S. aureaus; impairment of the activation via the classical pathway had no effect. The addition of complement components to heat-inactivated serum showed that intracellular killing is maximal only when C3b is generated. Reduction of the number of C3b receptors in the membrane by trypsin or pronase decreased intracellular killing in the presence of fresh serum; anti-monocyte serum completely abolished the stimulation of intracellular killing by fresh serum. These results lead to the conclusion that intracellular killing is also dependent on the interaction between C3b and its receptor in the membrane.

Complete and partial deficiencies of complement factor D in a Dutch family.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patients serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patients serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patients serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patients serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.

Arachidonic acid, but not its metabolites, is essential for FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcγ receptor (FcγR)‐mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcγR‐stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo‐oxygenase and lipoxygenase, markedly suppressed the FcγR‐mediated killing process. The production of O−2 by monocytes upon FcγR cross‐linking was inhibited by 4‐bromophenacyl bromide in a dose‐dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen‐dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcγR cross‐linking. Furthermore, FcγR cross‐linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcγR‐mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcγR‐stimulated intracellular killing of S. aureus by monocytes.

Influence of human monocytes on the antibacterial activity of kanamycin and gentamicin for Staphylococcus aureus.

The present study was performed to compare the antibacterial activities of kanamycin and gentamicin on Staphylococcus aureus phagocytosed by human monocytes and on nonphagocytosed S. aureus. The method used permitted the measurement of the effect of antibiotics on intracellular bacteria independent of phagocytosis and intracellular killing by the monocytes. A morphological assay with lysostaphin established the intracellular localization of about 70% of the cell-associated S. aureus in the monocyte-bacterium suspension. After 1 h of incubation, the antibacterial activity of both aminoglycosides was greater against intracellular than against nonphagocytosed S. aureus, but after 3 h, the reverse was true. The maximal effect on phagocytosed S. aureus, i.e., killing of about 98% of the bacteria, was reached in the first hour of incubation at kanamycin and gentamicin concentrations of 5 and 1 microgram/ml, respectively. A cell-free medium in which monocytes had been incubated increased the antibacterial activity of kanamycin, indicating that monocytes secrete a factor that enhances the antibacterial activity of aminoglycosides.

Functions of granulocytes after allogeneic bone marrow transplantation

SummaryThe phagocytosis and intracellular killing by granulocytes as well as the opsonizing capacity of the serum were studied in 13 patients who had undergone allogeneic bone marrow transplantation. Phagocytosis was normal in all patients. A moderately impaired opsonic activity of the serum was found in two patients, who were investigated within 30 days after the transplantation. The intracellular killing was less than control values in two patients. In one patient this was probably due to the existence of a split chimerism.

Defective intracellular killing of micro-organisms by murine alveolar macrophages

Peritoneal and alveolar macrophages differ in phenotype, endocytic activities, and oxidative metabolism.

Molecular Characterization and Determination of Antibiotic Resistance of Acinetobacter Baumanii Isolates from a Bulgarian Hospital

The recent literature is replete with reports on the occurrence of multiresistant epi-demic Acinetobacter baumannii in hospi-tals. Most reports are from Western Euro-pean countries, but publications from other parts of the world including South East Asia and South America are on the in-crease. Relatively little is known about the occurrence of multiresistant

Characteristics and Functions of Monocytes and Promonocytes in Monocytic Leukemia

The characteristics of mononuclear phagocytes (promonocytes and bone-marrow and blood monocytes) of 27 patients with acute or chronic monocytic leukemia were studied. The percentages of promonocytes with peroxidase and esterase activity and of cells with Fcgamma and C3b receptors showed some divergence from those found in normal individuals. The phagocytic activity of monocytes and their ability to kill ingested Staphylococcus aureus were not decreased in this group of patients. The 3H-thymidine-labeling index of the promonocytes did not differ greatly from normal values, which indicates that the percentage of dividing promonocytes had not increased. The very low labeling index of the circulating monocytes means that these cells do not divide and therefore cannot be blast cells or monoblasts.