Performance measures for guidelines on preventing opportunistic infections in patients infected with human immunodeficiency virus.

This article serves as a complement to the 1999 US Public Health Service/Infectious Diseases Society of America guidelines on the prevention of opportunistic infections in persons infected with HIV, published in this issue of Clinical Infectious Diseases [1]. A number of performance measures to assess compliance with the guidelines and to aid in their implementation are proposed.

Successful treatment of systemic Exophiala dermatitidis infection in a patient with chronic granulomatous disease.

Exophiala dermatitidis, one of the saprophytic dematiaceous fungi, is a rare cause of human infection that, when invasive, is nearly always fatal. Besides the more common subcutaneous infection usually caused by traumatic inoculation, infection can also spread hematogenously, in which case the organism has a distinct neurotropism. A patient with autosomal recessive chronic granulomatous disease of childhood who was found to have a progressive pulmonary and central nervous system infection with E. dermatitidis responded to an aggressive, multifaceted therapeutic approach. Scanning electron microscopy of the cultured conidiogenous cells confirmed that the manner of conidiogenesis is typical of the genus Exophiala. We report the first successful treatment of an infection involving the lungs and central nervous system by a combination of surgical resection of the pulmonary source and medical therapy with amphotericin B, flucytosine or ketoconazole, and transfused white cells, followed by a prolonged course of fluconazole.

In vivo interferon-gamma therapy augments the in vitro ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae.

During the recently completed double-blind, placebo-controlled, randomized trial of recombinant interferon-gamma (rIFN-gamma) therapy in chronic granulomatous disease (CGD), a metabolic assay of neutrophil damage to Aspergillus fumigatus hyphae was used to monitor neutrophil function before and during therapy. In this assay, 5 x 10(4) conidia that had germinated into hyphae were exposed to 5 x 10(5), 15 x 10(5), or 50 x 10(5) CGD neutrophils. By analysis of variance, neutrophils from patients on rIFN-gamma were found to produce significantly more damage to hyphae than those from the placebo group (P less than .01). In subgroup analysis, this effect was best seen in the hyphae exposed to 50 x 10(5) CGD neutrophils, where neutrophils from patients receiving rIFN-gamma produced significantly more damage to the hyphae than those from the placebo group (P less than .05). In vivo rIFN-gamma therapy improves the ability of CGD neutrophils to damage Aspergillus fumigatus hyphae in an in vitro assay.

Invasive infection with Sarcinosporon inkin in a patient with chronic granulomatous disease.

Sarcinosporon inkin, a rare skin fungus, was found to have caused progressive pneumonia in a young male with chronic granulomatous disease. Histologic sections of right upper lobe lung tissue showed clusters of globose to oblong hyalin-walled, septate, sporangia throughout the necrotic areas within the pyogranulomas. Pure cultures of S. inkin were recovered from the surgical specimen of the lung. Current status of the taxonomy of S. inkin is reviewed and clarified. Treatment of the patient with Amphotericin B and white blood cell transfusions led to clinical and radiographic response. This is the first documented case of systemic infection caused by S. inkin.

Trimethoprim-sulfamethoxazole prophylaxis in the management of chronic granulomatous disease.

Long-term oral antimicrobial prophylaxis is accepted practice in the management of patients with chronic granulomatous disease (CGD). Reports of adverse outcome with trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis in other patient groups, and the recent occurrence of several severe fungal infections in patients followed at the National Institutes of Health (NIH), prompted a review of the NIH experience to examine the incidence of nonfungal and fungal infections in CGD patients with and without TMP-SMX prophylaxis. Prophylaxis decreased the incidence of nonfungal infections from 7.1 to 2.4 per 100 patient-months in patients with autosomal CGD (P less than .01) and from 15.8 to 6.9 infections per 100 patient-months (P = .06) in X-linked CGD patients. There was no significant change in the incidence of fungal infection in CGD patients on TMP-SMX (1.5-0.3 fungal infections/100 patient-months in autosomal CGD and 1.7-0.2 fungal infections/100 patient-months in X-linked CGD patients). TMP-SMX prophylaxis is indicated for the management of patients with CGD and decreases the incidence of non-fungal infections without increasing the incidence of fungal infections.

Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae.

Using a metabolic test of hyphal viability, the interaction between neutrophils and Aspergillus hyphae was investigated over a broad range of hyphae-to-neutrophil ratios. Normal neutrophils were found to damage hyphae whereas neutrophils from patients with both chronic granulomatous disease (CGD) and myeloperoxidase (MPO) deficiency did not. Further, both azide and catalase + superoxide dismutase inhibited the ability of normal neutrophils to damage hyphae, suggesting that this damage is mediated by products of the respiratory burst and by the MPO-halide system. Also, mixtures of small numbers of normal neutrophils with larger numbers of CGD neutrophils (range, 1:5 to 1:15) damaged hyphae more efficiently than either population of cells alone. Further, mixtures of CGD and MPO-deficient neutrophils, neither of which alone could efficiently damage hyphae, were able to damage the hyphae almost as well as a comparable number of normal neutrophils. These data demonstrate that intact neutrophils can cooperate to synergistically damage Aspergillus hyphae, possibly by extracellular mixing of hydrogen peroxide and MPO.

An elevation in the concentration of free cytosolic calcium is sufficient to activate the oxidative burst of granulocytes primed with recombinant human granulocyte-macrophage colony-stimulating factor.

In granulocytes harvested from human blood, an elevation of the cytosolic concentration of Ca2+ ions is by itself insufficient to activate the cell's respiratory burst. We report herein that, when granulocytes are "primed" by a 90-min preincubation with the recombinant human hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSFrh), elevation of the concentration of cytosolic Ca2+ ions ([Ca2+]i) becomes a more effective transduction signal capable of triggering the generation of substantial quantities of superoxide (O2-) anions by the cell. In these studies, we used four separate and independent maneuvers to induce elevation of [Ca2+]i: 1) depolarization of the cell's electrical potential through obliteration of the transmembrane Na+ and K+ gradients; 2) acidification of the cytoplasm using propionic acid; 3) addition of the calcium ionophore ionomycin; and 4) treatment of the cells with the monoclonal antibody to the C3bi receptor, PMN7C3. In all cases, elevation of [Ca2+]i through these manipulations resulted in the release of substantially greater quantities of O2- by GM-CSFrh-primed granulocytes than by unprimed, control cells. The generation of O2- was in all cases markedly reduced by chelation of either intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or extracellular Ca2+ with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid. We conclude that during the process of GM-CSFrh priming, the metabolic assembly responsible for O2- anion production in the granulocyte becomes altered in such a way that a subsequent elevation in [Ca2+]i provides a potent signal for its activation.

Induction of signal transduction in human neutrophils by Candida albicans hyphae: the role of pertussis toxin-sensitive guanosine triphosphate-binding proteins.

Nonopsonized Candida hyphae elicit from human neutrophils a transient rise in cytosolic calcium concentrations and an oxidative burst without a detectable change in membrane potential. To determine if the signal-transduction pathway used by these organisms is mediated by guanine nucleotide-binding proteins (GNPs), we examined the functional responsiveness of neutrophils pretreated with pertussis toxin (PT). In response to serum-opsonized hyphae or zymosan, the rise in cytosolic calcium, membrane depolarization, and the respiratory burst were only partially abrogated. The transient rise in calcium induced by unopsonized hyphae was, however, completely eliminated in PT-treated neutrophils. Despite total abrogation of the calcium response, PT-treated cells could still mount a respiratory burst in response to these nonopsonized hyphae. Thus, neutrophil signaling by both serum-opsonized particles and nonopsonized hyphae is only partially mediated by PT-sensitive GNPs. Furthermore, the ability of unopsonized hyphae to elicit a respiratory burst without a calcium response suggests these events are separable and confirms the versatility of these organisms as probes for investigating neutrophil activation.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes.

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh-primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh-untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).

Clostridium difficile toxin A stimulates intracellular calcium release and chemotactic response in human granulocytes.

Clostridium difficile, a common enteric pathogen, mediates tissue damage and intestinal fluid secretion by release of two protein exotoxins: toxin A, an enterotoxin, and toxin B, a cytotoxin. Because toxin A elicits an intense inflammatory reaction in vivo, we studied the effects of highly purified C. difficile toxins on activation of human granulocytes. Toxin A at concentrations of 10(-7) to 10(-6) M, but not toxin B, elicited a significant chemotactic and chemokinetic response by granulocytes that was comparable with that induced by the chemotactic factor N-FMLP (10(-7) M). Neither toxin stimulated release of superoxide anion from granulocytes. Toxin A produced a rapid, transient rise in cytosolic [Ca2+]i, as measured by quin 2 fluorescence. Pertussis toxin and depletion of intra- and extracellular calcium blocked the toxin A effect on cytosolic [Ca2+]i. These findings suggest that the inflammatory effects of C. difficile toxin A in the intestine may be related to its ability to mobilize intracellular Ca2+ and elicit a chemotactic response by granulocytes.

A novel natural inhibitor from Candida albicans hyphae causing dissociation of the neutrophil respiratory burst response to chemotactic peptides from other post-activation events.

Previous work established that Candida albicans hyphae release several inhibitors of human neutrophil function. We now report that the crude hyphal inhibitory product (CHIP) inhibits superoxide anion (O2-) production stimulated by FMLP in a dose-related manner with an EC50 of approximately 2 micrograms/ml. CHIP also inhibited O2- production stimulated by A23187 and by opsonized zymosan, although this effect could be overcome by increasing the concentration of agonist. No inhibition of the PMA-stimulated burst was seen at any concentration of PMA tested, indicating that CHIP neither affected polymorphonuclear neutrophil viability nor quenched superoxide anion detection. A saturating dose of inhibitor had no effect on chemotaxis stimulated either by 0.1 to 100 nM FMLP or by zymosan-activated serum. Peak inositol trisphosphate levels stimulated by FMLP were not inhibited by a dose of CHIP producing maximum inhibition of FMLP-induced superoxide production. Peak changes in cytosolic free calcium levels (as measured by indo-1 fluorescence) stimulated by 50 nM or greater FMLP were unaffected by CHIP, although for subsaturating doses of FMLP a more rapid decline from peak calcium levels was seen in CHIP-exposed cells. Taken together, these data suggest that the common fungal pathogen C. albicans releases a substance that selectively impairs the neutrophil respiratory burst. It appears to do so without inhibiting the fully assembled NADPH oxidase and with minimal or no effect on events tightly coupled to FMLP-R/G protein activation, suggesting that these events may be uncoupled from activation of the burst. In addition, the absence of effect of CHIP on chemotaxis despite profound inhibition of the respiratory burst suggests these neutrophil functions may be mediated by divergent transduction pathways.

Temporal association of calcium mobilization, inositol trisphosphate generation, and superoxide anion release by human neutrophils activated by serum opsonized and nonopsonized particulate stimuli.

We have investigated the involvement of phospholipase C mediated polyphosphoinositide turnover in activation of polymorphonuclear leukocytes by particulate stimuli. Results showed that stimulation of leukocytes with serum opsonized zymosan (ingestible particle), serum opsonized Candida albicans hyphae (noningestible particle), or nonopsonized hyphae was followed by a transient rise in cellular inositol phosphates as has been described for neutrophil activation via the formyl peptide receptor. The kinetics of inositol trisphosphate generation paralleled both the time course of changes in cytosolic calcium concentration and the onset of superoxide anion generation, suggesting that these may be related events.

Unopsonized Candida albicans hyphae stimulate a neutrophil respiratory burst and a cytosolic calcium flux without membrane depolarization.

We have defined major differences between events elicited during activation of human neutrophils by opsonized Candida hyphae (noningestible) or zymosan (ingestible) and by unopsonized Candida hyphae (also noningestible). In response to opsonized particles, a transient increase in cytosolic free calcium was initially observed, followed by depolarization in the transmembrane potential and a respiratory burst. Without opsonins, zymosan did not elicit a response from neutrophils. In contrast, unopsonized Candida hyphae elicited a transient increase in cytosolic calcium, as well as triggered a respiratory burst; they did not, however, elicit a depolarization of the neutrophil membrane. Furthermore, the rate of superoxide generation decreased after a prolonged lag period. The calcium flux was also delayed and coincided with the onset of superoxide generation. Thus, our data demonstrate that Candida albicans hyphae activate neutrophils in the absence of added serum components and initiate a neutrophil respiratory burst without an antecedent membrane depolarization.

Effects of recombinant human granulocyte and macrophage colony-stimulating factors on signal transduction pathways in human granulocytes.

We studied the ability of the recombinant human-active hemopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSFrh) and granulocyte colony-stimulating factor (G-CSFrh) to activate receptor-mediated transduction pathways which have been implicated in the stimulation of cytotoxic functions in granulocytes. With the use of a panel of fluorescent probes, we found that these two growth factors exerted no detectable immediate effect on the resting transmembrane electrical potential, the intracellular concentration of free calcium ions, or the cytosolic pH of isolated, mature granulocytes. However, when granulocytes were "primed" by preincubation for 90 min with GM-CSFrh or G-CSFrh, the rate of membrane depolarization induced by 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, but not the rate of rise in free calcium ions, was greatly accelerated. In examining potential mechanisms to account for the priming effect of these growth factors, we found that although they did not induce translocation of protein kinase C or stimulate significant degranulation, they each directly caused prompt release of arachidonic acid from plasma membrane phospholipids. Our data indicate that although GM-CSFrh and G-CSFrh do not activate the transduction signals that have most clearly been implicated in receptor-mediated activation of cytotoxic functions in granulocytes--namely, those coupled to membrane depolarization or release of intracellular calcium ions--they appear directly to induce the release of arachidonic acid esterified to membrane phospholipids, an event which may represent the receptor-mediated activation of membrane phospholipases and which may contribute to the "priming" of the cells for enhancement of their functional responsiveness.

The effects of phorbol myristate acetate and chemotactic peptide on transmembrane potentials and cytosolic free calcium in mature granulocytes evolve sequentially as the cells differentiate.

We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor.

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.

Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst.

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.

Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate.

We examined the biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA). Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu-[125I]iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on d-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product (Mr, 31,000 for neutrophils; Mr, 29,000 for d-HL-60) as receptor on the surface of unstimulated cells. Formyl peptide receptor detected by affinity labeling in neutrophil specific granule-enriched subcellular fractions is identical to receptor found on the surface of unstimulated cells appearing as equal amounts of two isoelectric forms (isoelectric points, 5.8 and 6.2) at Mr 55,000 to 70,000. There is twice as much receptor present in the specific granule-enriched fraction per cell equivalent compared with plasma membrane. Azurophil granules contain trace amounts of receptor. Similar analysis of neutrophils treated with papain before subcellular fractionation shows that papain cleaved receptor fragment is detectable almost exclusively in the plasma membrane-enriched fraction. Most of the affinity-labeled formyl peptide receptor present in specific granule enriched fraction is present in membranes other than plasma membrane or Golgi membrane, because specific granule-enriched fraction contains only a small amount of plasma membrane marker and an amount of Golgi membrane marker equal to that found in plasma membrane-enriched fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

An antibody binding to human neutrophils demonstrates antigenic heterogeneity detected early in myeloid maturation which correlates with functional heterogeneity of mature neutrophils.

An IgG1 mouse monoclonal antibody (31D8) labels a subpopulation of human peripheral neutrophils. Heterogeneous binding to granulocyte precursors is seen in the bone marrow as early as the myelocyte stage of maturation. 31D8 binding is not affected by the in vitro stimulation or incubation of neutrophils. We demonstrate that the cells which bind 31D8 strongly (31D8-bright) are the same cells which depolarize, reduce nitroblue tetrazolium, and migrate chemotactically when stimulated with the chemoattractant N-formylmethionylleucylphenylalanine. Treatment with cytochalasin B modulates functional activity so that all cells depolarize or reduce nitroblue tetrazolium in response to this chemoattractant, but the heterogeneous binding of 31D8 does not change. The stable binding of 31D8 at 37 degrees C and during activation permitted characterization of the functional capacity of the two populations. These results demonstrate neutrophil heterogeneity early in myeloid maturation and correlate antigenic with functional heterogeneity. 31D8 will make it possible to study the significance of neutrophil heterogeneity in a variety of clinical situations.

A transient rise in intracellular free calcium is not a sufficient stimulus for respiratory burst activation in human polymorphonuclear leukocytes.

Binding of murine monoclonal antibody PMN7C3 to human neutrophils results in a large rapid, dose dependent transient increase in intracellular free calcium as measured by QUIN-2 fluorescence. Unlike other calcium mobilizing agents PMN7C3 does not induce any increase in respiratory burst activity over basal level. The PMN7C3 effect requires multivalent binding. Chelation of extracellular calcium does not significantly decrease the fluorescence transient generated by exposure to PMN7C3. Lowering of basal levels of intracellular free calcium concentration by maintaining QUIN-2-loaded PMN in calcium free medium eliminates the fluorescence transient. The observations demonstrate that a cell surface receptor mediated intracellular free calcium transient may be generated without any associated respiratory burst activation.