Detection of Lactoferrin and Myeloperoxidase Release from Single Neutrophils by a Protein A Plaque Assay

A haemolytic plaque assay was adopted to detect release of lactoferrin and myeloperoxidase (MPO) from single neutrophils. Target erythrocytes coated with protein A were bound as a monolayer by poly-L-lysine to the surface of a plastic dish. Secreted lactoferrin and MPO induced plaque formation dependent on the reaction with complement and specific antiserum producing lysis of the protein-A-coated sheep red blood cells. Lactoferrin was found to be released spontaneously from a fraction of neutrophils while MPO was released only after phagocytosis, reflecting different mechanisms for degranulation of MPO-containing azurophil and lactoferrin-containing specific granules.     

Candida fermentati as a Cause of Persistent Fungemia in a Preterm Neonate Successfully Treated by Combination Therapy with Amphotericin B and Caspofungin

A case of persistent candidemia in a preterm neonate caused by Candida fermentati, identified by sequencing of the internally transcribed spacer region of ribosomal DNA (rDNA), is described. The neonate was treated for 30 days by combination therapy with amphotericin B (AmBisome) and caspofungin with a successful outcome, and no drug-related side effects were observed.     

Acremonium strictum Pulmonary Infection in a Leukemic Patient Successfully Treated with Posaconazole After Failure of Amphotericin B

A severely neutropenic patient with chronic lymphocytic leukemia developed a diffuse bilateral pulmonary infection while receiving a therapeutic daily dosage of intravenous amphotericin B for Candida glabrata esophagitis. Computed tomography of the chest showed numerous lung nodules, ground glass areas and a pleural effusion. Biopsy of one nodule demonstrated hyaline septate hyphae. Multiple sputum cultures grew Acremonium strictum. Increasing the dose of amphotericin B and the addition of itraconazole did not resolve the infection. Change of treatment to posaconazole given orally at 200 mg four times/d resulted in progressive improvement leading finally to cure after 24 weeks of therapy. Treatment with posaconazole was clinically and biologically well tolerated. Electronic Publication     

Adrenal Steroids Modulate the Immune Response during Brucella abortus Infection by a Mechanism That Depends on the Regulation of Cytokine Production

Human brucellosis is a protean disease with a diversity of clinical signs and symptoms resulting from infection with Brucella species. Recent reports suggest a cross-regulation between adrenal steroids (cortisol and dehydroepiandrosterone [DHEA]) and the immune system. Monocytes and macrophages are the main replication niche for Brucella. Therefore, we investigated the role of adrenal hormones on the modulation of the immune response mediated by macrophages in B. abortus infection. Cortisol treatment during B. abortus infection significantly inhibits cytokine, chemokine, and MMP-9 secretion. In contrast, DHEA treatment had no effect. However, DHEA treatment increases the expression of costimulatory molecules (CD40, CD86), the adhesion molecule CD54, and major histocompatibility complex class I (MHC-I) and MHC-II expression on the surface of B. abortus-infected monocytes. It is known that B. abortus infection inhibits MHC-I and MHC-II expression induced by gamma interferon (IFN-γ) treatment. DHEA reverses B. abortus downmodulation of the MHC-I and -II expression induced by IFN-γ. Taken together, our data indicate that DHEA immune intervention may positively affect monocyte activity during B. abortus infection.     

Modification of Lymphocyte and Monocyte Functional Activity by Polymorphonuclear Neutrophils in HIV Infection

The aim of this study was to determine whether potymorphonuclear neutrophils (PMN) can modify the immune response in HIV cases. Supernatants of PMN (PMNS) from 33 HIV-infected patients (16 with lymphoadenopathy syndrome, 17 with AIDS-related complex) were tested for their influence on the functional activity oflymphocytes and monocytes from 6 healthy donors. PMNS from another 6 healthy donors comprised a control group. It was found that PMNS from HIV-infected patients, but not from healthy donors, induced suppression oflymphocyte proliferative response and down-regulation of CD8 receptor expression on lymphocytes. Decrease of NK-cell cytotoxicity in the presence of PMNS from HIV-infecled patients was the same as that from healthy donors. PMNS did not influence the production of anti-HIV antibody by lymphocytes from HIV-infected patients, as well as non-specific IgG by lymphocytes from healthy donors. PMNS effect on functional activity of lymphocytes was blocked completely after treatment of PMN by catalase and superoxidedismutase. At the same time PMNS from HIV-infected patients but not from healthy donors induced increased production of TNF-a by monocytes and up-regulation of monoeyte phagoeytosis. These effeets were independent of catalase and superoxide dismutase and were not abrogated by antibody against IL-1, IL-8, TNF-γ, IFN-γ or IFN-γ.     

Regulation of fungal infection by a combination of amphotericin B and peptide 2, a lactoferrin peptide that activates neutrophils

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 x 10(8) cells) or Aspergillus fumigatus (1 x 10(8) cells) was prolonged 3 to 5 days by the injection of 10 microg of peptide 2 (a lactoferrin peptide) and 10 microg of alpha-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 microg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 microg of amphotericin B. When mice received a combined injection of peptide 2 (10 microg/day) with amphotericin B (0.5 microg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as alpha-defensin 1, beta-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O2-) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O2- -generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47phox as well as p67phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.     

Activity of MGCD290, a Hos2 Histone Deacetylase Inhibitor,in Combination with Azole Antifungals against Opportunistic Fungal Pathogens

We report on the in vitro activity of the Hos2 fungal histone deacetylase (HDAC) inhibitor MGCD290 (MethylGene, Inc.) in combination with azoles against azole-resistant yeasts and molds. Susceptibility testing was performed by the CLSI M27-A3 and M38-A2 broth microdilution methods. Testing of the combinations (MGCD290 in combination with fluconazole, posaconazole, or voriconazole) was performed by the checkerboard method. The fractional inhibitory concentrations were determined and were defined as <0.5 for synergy, ≥0.5 but <4 for indifference, and ≥4 for antagonism. Ninety-one isolates were tested, as follows: 30 Candida isolates, 10 Aspergillus isolates, 15 isolates of the Zygomycetes order, 10 Cryptococcus neoformans isolates, 8 Rhodotorula isolates, 8 Fusarium isolates, 5 Trichosporon isolates, and 5 Scedosporium isolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 μg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 μg/ml). MGCD290 was synergistic with fluconazole against 55 (60%) of the 91 isolates, with posaconazole against 46 (51%) of the 91 isolates, and with voriconazole against 48 (53%) of the 91 isolates. Synergy between fluconazole and MGCD290 was observed against 26/30 (87%) Candida isolates. All 23 of the 91 Candida isolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, ≥64 μg/ml] to susceptible [MIC, ≤8 μg/ml]) when fluconazole was combined with MGCD290 at 0.12 to 4 μg/ml. The activity of fluconazole plus MGCD290 was also synergistic against 6/10 Aspergillus isolates. Posaconazole plus MGCD290 demonstrated synergy against 14/15 Zygomycetes (9 Rhizopus isolates and 5 Mucor isolates). Voriconazole plus MGCD290 demonstrated synergy against six of eight Fusarium isolates. Thus, MGCD290 demonstrated in vitro synergy with azoles against the majority of clinical isolates tested, including many azole-resistant isolates and genera inherently resistant to azoles (e.g., Mucor and Fusarium). Further evaluation of fungal HDAC inhibitor-azole combinations is indicated.At present, the azole class of antifungal agents constitutes one of the cornerstones of therapy for opportunistic mycoses due to many yeasts and molds (3, 16, 20, 24, 28, 30, 31, 33). Unfortunately, the clinical efficacy of this class of agents may be compromised by intrinsic or acquired resistance (11, 25, 27, 30). Resistance to azoles has been studied most extensively in Candida spp., in which the upregulation of genes encoding the lanosterol demethylase target enzyme (ERG11) and the Candida drug resistance (CDR) efflux pumps may occur upon exposure of the organism to azole antifungal agents. These alterations in gene regulation can result in increases in MICs and compromised clinical efficacy (25, 27, 30, 34). Given the proven safety, efficacy, and ease of use of these agents, the availability of strategies that may be used to avoid the emergence of resistance is important. Combination antifungal therapy with agents of different mechanistic classes could promote fungal killing and clinical efficacy and provide an alternative to monotherapy for patients with infections caused by multiresistant species and for patients who fail to respond to standard treatments.Histone deacetylases (HDACs) are a family of enzymes which deacetylate lysines on core histones and other cellular proteins (9, 10, 32). They play an important role in gene regulation and also in the control of other cellular functions, such as proliferation, cell death, and motility (9, 10, 22, 32). Inhibitors of HDACs belong to several chemical classes that act by binding to the catalytic site of the enzyme, causing cell cycle arrest, apoptosis, and terminal differentiation (9, 22). HDAC enzymes have been explored as potential targets in the treatment of cancer cells and infections caused by several eukaryotic microorganisms (1a, 7, 9, 22, 26, 29). Modulation of fungal gene expression through fungal HDAC inhibition may be an alternative approach to the treatment of fungal infections (17, 29). Smith and Edlind (29) have shown in Candida albicans and two other Candida spp. that trichostatin A, a nonselective HDAC inhibitor with cytoxic properties in mammalian cells, markedly decreased the upregulation of the ERG11 and CDR genes following exposure to sterol biosynthesis inhibitors, such as fluconazole and terbinafine.We previously examined the potential chemosensitizing interaction between a novel Hos2 inhibitor, MGCD290, developed by MethylGene, Inc. (Montreal, Quebec, Canada), and three triazole antifungal agents (fluconazole, itraconazole, and voriconazole) against a panel of 45 clinical isolates of Candida spp. (16 of which were fluconazole resistant) and 16 clinical isolates of Aspergillus spp. In the previous study, MGCD290 displayed synergy with fluconazole against 76% of the Candida isolates tested and with voriconazole and itraconazole against 69% of the Aspergillus isolates tested (8a). Our results suggest a potential clinical use for the combination of HDAC inhibitors and azoles in the treatment of fungal infections.In the present study, we expand upon our initial findings by examining the interaction between MGCD290 and three triazoles (fluconazole, voriconazole, and posaconazole) against a larger and more diverse collection of yeasts and molds, most of which were azole resistant.     

In Vivo Susceptibility of Cryptococcus neoformans to Hamycin, Amphotericin B, and 5-Fluorocytosine

Previous investigations have shown that hamycin is 5 to 10 times more active in vitro than amphotericin B against Cryptococcus neoformans, whereas 5-fluorocytosine is approximately 15 times less active. Present studies are concerned with a comparison of these drugs in vivo. Three strains of C. neoformans which varied in both their virulence for mice and their susceptibilities to the antifungal agents were studied. Acute experimental infections were established in mice by using inocula containing approximately 4 × 10⁶ cells. The mice were treated by gastric intubation for 28 days; daily dosages of the three drugs ranged from 12.5 to 250 mg/kg. The polyenes were suspended in 5% dimethyl sulfoxide, whereas 5-fluorocytosine was suspended in saline. Amphotericin B was the most effective drug, with almost complete absence of toxic deaths in control mice and increased survival in mice infected with two of the three strains and treated with 25 mg/kg per day or more. 5-Fluorocytosine was nontoxic but provided protection against only one strain. Hamycin was both the least effective and the most toxic compound, providing only slight protection at doses of 12.5 or 25 mg/kg per day and causing toxic deaths in over 50% of uninfected mice at doses above 25 mg/kg per day.     

Regulation of CFTR by protein phosphatase 2B and protein kinase C

 The activity of the CFTR Cl^– channel is dependent on its phosphorylation status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regulators of the cystic fibrosis conductance regulator (CFTR). Treating CFTR-expressing 3T3 cells with either of the two specific PP2B blockers cyclosporin A (CsA, 1 μM) or deltamethrin (DM, 30 nM) caused rapid activation of CFTR in cell-attached patches. As determined by noise analysis of multi channel patches, DM- or CsA-activated CFTR displayed gating kinetics comparable to those of forskolin-activated CFTR. After activation of CFTR by blocking PP2B, CFTR still inactivated. CFTR-mediated currents were, on average, 6.1 times larger when cells were stimulated by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphorylation site of CFTR is regulated by cellular PKA, PP2B and another phosphatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, CsA and DM had no effect on CFTR activity in both cell-attached patch-clamp and transepithelial experiments. In contrast, when exogenous PP2B was added to patches excised from 3T3 or Calu-3 cells, PKA-activated CFTR currents were quickly inactivated. This indicates that free exogenous PP2B can inactivate CFTR in patches from both cell types. We propose that in order to regulate CFTR in an intact cell, PP2B may require a selective subcellular localization to become active. When excised patches were PKC-phosphorylated, the gating kinetics of CFTR were significantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indiscriminate dephosphorylation of different phosphorylation sites by PP2B. Received: 29 October 1997 / Received after revision: 13 February 1998 / Accepted: 2 March 1998     

Regulation of B Cell Function by Bucillamine,a Novel Disease-Modifying Antirheumatic Drug

Bucillamine [N-(2-mercapto-2-methylpropionyl)-L-cysteine] (BUC) is a thiol compound that differs from D-penicillamine (DPC) in that it contains two free sulfhydryl groups. Clinical trials have demonstrated that its efficacy in rheumatoid arthritis is superior to that of DPC, but its mechanism of action remains unclear. We therefore examined the effects of BUC on the in vitro function of human B cells in comparison to those of DPC. IgM production was induced from highly purified B cells from healthy donors by stimulation with Staphylococcus aureus Cowan I (SA) plus factors generated from mitogen-activated T cells (TF) or interleukin-2 (IL-2) or with immobilized anti-CD3-activated CD4⁺ T cells. BUC suppressed the production of IgM at concentrations of 0.3-100 μg/ml irrespective of the presence of CUSO⁴. Whereas BUC suppressed the production of IL-2 and interferon-γ by immobilized anti-CD3-activated CD4⁺ T cells, its suppressive effects on the production of IgM in anti-CD3-stimulated cultures were not overcome by addition of TF or IL-2, indicating that the action of BUC involves direct inhibition of B cell function. BUC suppressed the initial stages of B cell activation, but not the maturation of previously activated B cells. In contrast to DPC, the suppressive activities of BUC did not require the presence of copper and were not overcome by the addition of monocytes or catalase. The effects of SA981, a metabolite of BUC with an intramolecular disulfide, on B cell function were more marked than those of BUC, whereas the effects of SA679, another metabolite of BUC with one of the two sulfhydryl bonds methylated, were similar to those of DPC. SA672, a metabolite of BUC with both of the two sulfhydryl bonds methylated, did not suppress B cell function. These results indicate that BUC as well as some of its metabolites inhibit cytokine production by T cells and also suppress the production of IgM at least in part by directly inhibiting B cells. These compounds exert immunosuppressive effects that are similar to those of DPC, but also unique inhibitory effects that depend upon the capacity of BUC to form an intramolecular disulfide between its two sulfhydryl groups.     

Activation of Human Neutrophils with Chemotactic Peptide, Opsonized Zymosan and the Calcium Ionophore A23187, But Not with a Phorbol Ester, is Accompanied by Efflux and Store-Operated Influx of Calcium

The objective of this study was to monitor alterations in cellular Ca²⁺ metabolism following activation of neutrophils with receptor(chemotactic peptide, FMLP, 1 M; opsonized zymosan, OZ, 0.5 mg/ml) and non-receptor (calcium ionophore, A23187, 1 M; phorbol 12-myristate 13-acetate, PMA, 25 ng/ml)-mediated stimuli of the pro-inflammatory functions of these cells. Ca²⁺ fluxes in activated neutrophils were measured using a fura-2-based spectrofluorimetric method in combination with radiometric (⁴⁵Ca) procedures which facilitate distinction between net efflux and net influx of the cation. Exposure of neutrophils to receptor-mediated stimuli and to A23187 was associated with an abrupt increase in cytosolic Ca²⁺ coincident with a rapid efflux of the cation which terminated at around 30 s. In the case of FMLP and OZ, this was followed by a delayed (30–60 s), store-operated influx of Ca²⁺, which was complete at around 5 min after addition of the stimulus. With A23187, however, influx of Ca²⁺ occurred immediately following activation of the cells. There were no detectable alterations in cytosolic Ca²⁺ or measurable net efflux or influx of the cation above control levels in PMA-activated neutrophils. These data demonstrate that FMLP, OZand A23187-mediated alterations in neutrophil cytosolic Ca²⁺ are due to mobilization of both intracellular and extracellular cation, while activation of neutrophils by PMA is independent of alterations in cytosolic Ca²⁺.     

Regulation of immunity and autoimmunity by B cells

Compelling evidence has demonstrated that IL-10-producing regulatory B cells (B(regs)) are specialized to suppress immune responses and control various immunopathologies. Several reports have provided information on the phenotype, generation, and mechanism of action of B(regs). Recent work has also identified B(regs) in humans and has begun to unravel their phenotype and mode of suppression. A complete analysis of their function in immune-mediated diseases is required for possible future use of B(regs) as a part of new clinical strategies for treating autoimmune diseases and in the induction of transplant tolerance.     

In Vitro Amphotericin B Resistance in Clinical Isolates of Aspergillus terreus, with a Head-to-Head Comparison to Voriconazole

Amphotericin B therapy continues to be the "gold standard" in the treatment of invasive aspergillosis in the immunocompromised host. Although Aspergillus fumigatus and Aspergillus flavus constitute the major species, several reports have described invasive pulmonary or disseminated disease due to the less common Aspergillus terreus and dismal clinical outcomes with high-dose amphotericin B. We therefore evaluated 101 clinical isolates of A. terreus for their susceptibility to amphotericin B and the investigational triazole voriconazole by using the National Committee for Clinical Laboratory Standards M27-A method modified for mould testing. Forty-eight-hour MICs indicated 98 and 0% resistance to amphotericin B and voriconazole, respectively. We conclude that A. terreus should be added to the list of etiologic agents refractory to conventional amphotericin B therapy and suggest the potential clinical utility of voriconazole in aspergillosis due to this species.     

Regulation of IgG antibody synthesis by a B cell component.

An immunoglobulin-G recruiting component (GRC) prepared from splenic B cells of antigen-primed mice was shown to be effective in recruiting more IgG plaque forming cells than normally appear among splenic cells experiencing a primary immune response. GRC caused increases in all classes of IgG PFC except perhaps IgG3, and the largest improvements were in IgG1 and IgG2a. GRC is synthesized by IgG2a-bearing cells and is effective at 96-120 h after spleen cells have been exposed to antigen. It is incapable of substituting for allogeneic effect factor, and the latter apparently must have its input on antibody producing cells before GRC can act. Together the data suggest that during a primary immune response a definite number of splenic B cells become poised for synthesizing IgG antibody, but only a portion of them are able to secrete. Apparently, the quiescent cells among them can be activated to secrete by exposure to GRC, A B cell product.     

Unexpected Blastomyces dermatitidis Etiology of Fungal Sinusitis and Erosive Palatal Infection in a Diabetic Patient

We present what is believed to be the initial report of hard-palate infection caused by Blastomyces dermatitidis. The organism was cultivated from biopsy material obtained from a diabetic patient presenting with complaints of headache and malaise. Radiologic findings revealed a malignant-appearing soft-tissue mass with paranasal sinus base destruction.