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Biomedical subjects

Steven M Holland

Publications and source records attributed to Steven M Holland.

At least 19 recordsLinked to original sources
PubMed2003

Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion.

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

Animals↗
PubMed2003

Itraconazole to prevent fungal infections in chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease is a rare disorder in which the phagocytes fail to produce hydrogen peroxide. The patients are predisposed to bacterial and fungal infections. Prophylactic antibiotics and interferon gamma have reduced bacterial infections, but there is also the danger of life-threatening fungal infections. We assessed the efficacy of itraconazole as prophylaxis against serious fungal infections in chronic granulomatous disease. METHODS: Thirty-nine patients at least 5 years old (6 female and 33 male; mean age, 14.9 years) were enrolled in a randomized, double-blind, placebo-controlled study. After the initially assigned treatment, each patient alternated between itraconazole and placebo annually. Patients 13 years of age or older and all patients weighing at least 50 kg received a single dose of 200 mg of itraconazole per day; those less than 13 years old or weighing less than 50 kg received a single dose of 100 mg per day. The primary end point was severe fungal infection, as determined by histologic results or culture. RESULTS: One patient (who had not been compliant with the treatment) had a serious fungal infection while receiving itraconazole, as compared with seven who had a serious fungal infection while receiving placebo (P=0.10). No patient receiving itraconazole but five patients receiving placebo had a superficial fungal infection. No serious toxic effects were noted, although one patient had a rash and another had elevated results on liver-function tests; both of these effects resolved with the discontinuation of itraconazole. CONCLUSIONS: Itraconazole prophylaxis appears to be an effective and well-tolerated treatment that reduces the frequency of fungal infections in chronic granulomatous disease, but monitoring for long-term toxic effects is warranted.

Adolescent↗
PubMed2003

Persistent Mycobacterium avium infection following nonmyeloablative allogeneic peripheral blood stem cell transplantation for interferon-gamma receptor-1 deficiency.

Interferon-gamma receptor-1 (IFNgammaR1) deficiency is a rare inherited immunodeficiency. We performed a nonmyeloablative allogeneic stem cell transplantation on a boy with complete IFNgammaR1 deficiency and refractory disseminated Myco- bacterium avium infection. Despite the patient's profound immune defect, early donor stem cell engraftment was low. Full donor engraftment was accomplished only following multiple donor lymphocyte infusions. Detection of IFNgammaR1 expression on peripheral blood monocytes and neutrophils corresponded with establishment of stable, complete donor hematopoietic chimerism. However, expression of, and signaling through IFNgammaR1 disappeared shortly thereafter. Disseminated Mycobacterium avium infection persisted and the patient died. Coculture of Myco- bacterium avium with normal myeloid cells resulted in an IFNgamma signaling defect similar to that observed in vivo. Active disseminated Mycobacterium avium infection may significantly compromise normal immune reconstitution following allogeneic stem cell transplantation. Patients with IFNgammaR1 deficiency should receive transplants before developing refractory mycobacterial infections.

Child, Preschool↗
PubMed2003

Mechanical stretch enhances mRNA expression and proenzyme release of matrix metalloproteinase-2 (MMP-2) via NAD(P)H oxidase-derived reactive oxygen species.

Mechanical stretch is a hallmark of arterial hypertension and leads to vessel wall remodeling, which involves matrix metalloproteinases (MMPs). Because mechanical stretch is further capable of inducing reactive oxygen species (ROS) formation via the NAD(P)H oxidase, we assessed whether mechanical stretch enhances MMP expression and activity in a NAD(P)H oxidase-dependent manner. Therefore, vascular smooth muscle cells (VSMCs) isolated from C57BL/6 mice were exposed to cyclic mechanical stretch. The impact of ROS was assessed using VSMCs isolated from p47phox-/- mice, deficient for a NAD(P)H oxidase subunit responsible for ROS formation. Transcript levels were investigated by cDNA array and confirmed by RT-PCR. ROS formation was determined by DCF fluoroscopy and MMP-2 activity by zymography. Mechanical stretch of wild-type VSMCs resulted in a rapid ROS formation and p47phox membrane translocation that is followed by an increase in Nox-1 transcripts. ROS formation was completely abrogated in p47phox-/- VSMCs. cDNA array further revealed an increase of MMP-2 mRNA in response to mechanical stretch, which was validated by RT-PCR. Using p47phox-/- VSMCs, this increase in MMP-2 mRNA was completely blunted. mRNA expression of tissue inhibitor of MMP-2 TIMP-1 and TIMP-2 and membrane-type 1 MMP was unaffected by mechanical stretch. Gelatinolytic activity of pro-MMP-2 has been increased rapidly in wild-type VSMCs and was completely abolished in p47phox-/- VSMCs. These results indicate that mechanical stretch induces ROS formation via the NAD(P)H oxidase and thereby enhances MMP-2 mRNA expression and pro-MMP-2 release. These results are consistent with the notion that in arterial hypertension, reactive oxygen species are involved in vascular remodeling via MMP activation. The full text of this article is available online at http://www.circresaha.org.

Animals↗
PubMed2003

Reinfection, rather than persistent infection, in patients with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by severe recurrent infections with Staphylococcus aureus, certain gram-negative rods, Nocardia species, and fungi. When infections with the same species recur, they may represent relapses or new infections. We collected organisms from infections that occurred between 1992 and 2000 in patients with CGD and determined the biochemical phenotypes, in vitro antibiotic susceptibility patterns, and pulsed-field gel electrophoresis (PFGE) patterns of the organisms causing the initial and recurrent infections. Recurrence of infection with Burkholderia cepacia or Serratia marcescens was caused by a new strain in 9 of 10 cases (P=.001). Recurrent S. aureus infections were caused by new strains in 7 of 8 cases (P=.006). In patients with CGD, recurrence of infection with the same bacterial species after appropriate antibiotic therapy usually represents new infection.

Adolescent↗
PubMed2003

Deficiency of NADPH oxidase components p47phox and gp91phox caused granulomatous synovitis and increased connective tissue destruction in experimental arthritis models.

Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-L-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox(-/-) and gp91(-/-) mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-alpha, interleukin-1alpha, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-kappaB ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage.

Animals↗
PubMed2003

Phagocyte NADPH oxidase, but not inducible nitric oxide synthase, is essential for early control of Burkholderia cepacia and chromobacterium violaceum infection in mice.

Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens. Using mice deficient in generating phagocyte superoxide (p47(phox)(-/-)) and mice deficient in generating inducible nitric oxide synthase (iNOS(-/-)), we examined the roles of these reactive species in host defense against Burkholderia cepacia and Chromobacterium violaceum, organisms known to have unusual virulence in chronic granulomatous disease. Intraperitoneal B. cepacia challenge (4.0 x 10(3) to 4.0 x 10(5) organisms/mouse) resulted in mortality in all p47(phox)(-/-) mice, with the survival interval being inversely proportionate to the amount of inoculum. Pretreatment with gamma interferon did not affect survival. C. violaceum was strikingly virulent in p47(phox)(-/-) mice (the 50% lethal dose [LD(50)] was <13 organisms). iNOS(-/-) and wild-type mice were resistant to B. cepacia challenges of at least 10(6) organisms per mouse, and the LD(50) of C. violaceum was between 10(6) and 10(7) organisms per mouse. Consistent with the survival data, numbers of organisms in cultures of B. cepacia from multiple sites were higher for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice at day 4 after challenge, but numbers of organisms for different B. cepacia strains varied. The recovery of C. violaceum was strikingly greater at 18 h after challenge for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice, in which the organism burdens were virtually nil. In vitro, both B. cepacia and C. violaceum were sensitive to H(2)O(2) and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly. Host defense against B. cepacia and C. violaceum is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.

Animals↗
PubMed2003

Nocardia veterana as a pathogen in North American patients.

The molecular methodologies used in our laboratories have allowed us to define a group of Nocardia isolates from clinical samples which resemble the type strain of Nocardia veterana. Three patient isolates and the type strain of N. veterana gave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the N. veterana type strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the Nocardia nova type strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the N. veterana type strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the N. veterana type strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was N. veterana. Biochemical and susceptibility testing showed no significant differences among the patient isolates and the N. veterana type strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for N. nova, indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that N. veterana is a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of N. veterana from the related species Nocardia africana and N. nova because of the very high degree of 16S rRNA gene similarity among them.

Adult↗
PubMed2003

Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension.

Tetrahydrobiopterin is a critical cofactor for the NO synthases, and in its absence these enzymes become "uncoupled," producing reactive oxygen species (ROSs) rather than NO. In aortas of mice with deoxycorticosterone acetate-salt (DOCA-salt) hypertension, ROS production from NO synthase is markedly increased, and tetrahydrobiopterin oxidation is evident. Using mice deficient in the NADPH oxidase subunit p47(phox) and mice lacking either the endothelial or neuronal NO synthase, we obtained evidence that hypertension produces a cascade involving production of ROSs from the NADPH oxidase leading to oxidation of tetrahydrobiopterin and uncoupling of endothelial NO synthase (eNOS). This decreases NO production and increases ROS production from eNOS. Treatment of mice with oral tetrahydrobiopterin reduces vascular ROS production, increases NO production as determined by electron spin resonance measurements of nitrosyl hemoglobin, and blunts the increase in blood pressure due to DOCA-salt hypertension. Endothelium-dependent vasodilation is only minimally altered in vessels of mice with DOCA-salt hypertension but seems to be mediated by hydrogen peroxide released from uncoupled eNOS, since it is inhibited by catalase. Tetrahydrobiopterin oxidation may represent an important abnormality in hypertension. Treatment strategies that increase tetrahydrobiopterin or prevent its oxidation may prove useful in preventing vascular complications of this common disease.

Animals↗
PubMed2003

Disseminated trichosporonosis in a murine model of chronic granulomatous disease.

Over a period of ten months, five mice submitted to our service (the Pathology Section of the Veterinary Resources Program, Office of Research Services at the National Institutes of Health, Bethesda, Md.) were diagnosed with disseminated trichosporonosis. These mice had pyogranulomatous inflammation in multiple organs, including lung, liver, lymph nodes, salivary gland, and skin. Fungal elements in many of the lesions were identified, using special histochemical stains, and Trichosporon beigelii was obtained by use of culture of specimens at affected sites. This saprophytic fungus has caused disseminated disease in immunosuppressed humans. However, despite widespread use of immunosuppressed rodents in research, to the authors' knowledge, this organism had not previously been reported to cause spontaneous disseminated disease in laboratory mice. All affected mice had a genetically engineered defect in p47(phox), a critical component of the nicotinamide dinucleotide phosphate (NADPH) oxidase, the enzyme responsible for generating the phagocyte oxidative burst. These animals are used as a murine model of human chronic granulomatous disease. We discuss the lesions, differential diagnosis, identification of the organism, and the role of NADPH oxidase in protecting against disseminated trichosporonosis.

Animals↗
PubMed2002

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II.

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.

Angiotensin II↗
PubMed2002

Nocardia infection in chronic granulomatous disease.

To determine the clinical characteristics and outcome of Nocardia infection in patients with chronic granulomatous disease (CGD), we reviewed data on 28 episodes of Nocardia infection in 23 patients with CGD. All episodes involved pulmonary infection. The frequency of disseminated nocardiosis was 25% for the case series overall, but it was 56% among episodes in patients receiving neither interferon-gamma (IFN-gamma) nor sulfonamide prophylaxis. Patients receiving prophylaxis with IFN-gamma and/or a sulfonamide were significantly less likely to have disseminated nocardiosis than were patients not receiving these medications, and no patient receiving both medications developed disseminated nocardiosis. One-third of the patients had concomitant fungal infections, and 2 patients had concomitant Legionella infections. The majority of patients were successfully treated with a sulfonamide-containing regimen, even though some patients had developed Nocardia infection while receiving sulfonamide prophylaxis. Nocardia infections in patients with CGD are not usually fatal if treated properly, and prophylaxis with IFN-gamma and a sulfonamide may protect against dissemination.

Adolescent↗
PubMed2002

The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression.

An NADPH oxidase is thought to be a main source of vascular superoxide (O(2)(-)) production. The functional role of this oxidase, however, and the contribution of the different subunits of the enzyme to cellular signaling are still incompletely understood. We determined the role of the p47phox subunit of the oxidase in O(2)(-) generation and signaling in aortic rings and cultured smooth muscle cells (SMC) from wild-type (WT) and p47phox-deficient (p47phox -/-) mice. Basal O(2)(-) levels in aortae of p47phox -/- mice were lower than those in WT aortae. Infusion of [val(5)]-angiotensin II increased O(2)(-) levels in aortae from WT more than in aortae from p47phox -/- mice. O(2)(-) generation was similar in quiescent SMC from WT and p47phox -/- mice. However, exposure to thrombin selectively increased O(2)(-) generation in VSMC from WT, but not from p47phox -/- mice. Thrombin-activated redox-mediated signal transduction and gene expression was attenuated in VSMC from p47phox -/- compared to cells from WT mice as determined by p38 MAP kinase activation and VEGF gene expression. We conclude that p47phox is important for vascular ROS production and redox-modulated signaling and gene expression in VSMC.

Angiotensin II↗
PubMed2002

Role of NADPH oxidase in the mechanism of lung neutrophil sequestration and microvessel injury induced by Gram-negative sepsis: studies in p47phox-/- and gp91phox-/- mice.

We addressed the role of O(2) generated by the NADPH oxidase complex in the mechanism of polymorphonuclear leukocyte (PMN) accumulation and transalveolar migration and lung microvascular injury. Studies were made in mice lacking the p47(phox) and gp91(phox) subunits of NADPH oxidase (p47(phox-/-) and gp91(phox-/-)) in which PMN are incapable of the respiratory burst. The mice were challenged i.p. with live Escherichia coli to induce sepsis. We observed time-dependent increases in PMN sequestration and migration from 1 to 6 h after challenge with 2 x 10(8) E. coli. The responses in knockout mice were greater post-E. coli challenge compared with control mice; i.e., transalveolar PMN migration post-E. coli challenge increased by approximately 50% in the null mice above values in wild type. The increased PMN infiltration was associated with decreased lung bacterial clearance. The generation of the chemoattractant macrophage-inflammatory protein-2 in lung tissue was greater in NADPH oxidase-defective mice after E. coli challenge than control mice; moreover, macrophage-inflammatory protein-2 Ab pretreatment prevented the PMN infiltration. We also observed that E. coli failed to increase lung microvascular permeability in p47(phox-/-) and gp91(phox-/-) mice despite the greater lung PMN sequestration. Thus, O(2) production is required for the induction of sepsis-induced lung microvascular injury. We conclude that NADPH oxidase-derived O(2) generation has an important bactericidal role, such that an impairment in bacterial clearance in NADPH oxidase-defective mice results in increased chemokine generation and lung tissue PMN infiltration.

Animals↗
PubMed2002

White blood cell defects: molecular discoveries and clinical management.

In this review, we present the most recent discoveries at the molecular level in white blood cell defects, and explain how their identification helped us to understand the underlying pathophysiology and directed our approach in clinical management. These lately discovered genes, relevant to immune disorders of mononuclear phagocytes and neutrophils, include defects in the interferon gamma (IFNg)/interleukin 12 (IL-12) pathway, such as IFNg receptor (IFNgR) defects, IL-12 defect, IL-12 receptor (IL-12R) defect, and signal transducer and activator of transcription 1 (STAT-1) defect. We have also included NF-kappaB essential modifier (NEMO) defects, which lead to X-linked ectodermal dysplasia, with or without lymphedema and osteopetrosis, and a wide range of involvement of the immune system, which can mimic the hyper-IgM phenotype. Neutrophil-specific granule deficiency and neutrophil elastase deficiency are discussed, the latter being the molecular defect in both cyclic neutropenia and in some sporadic cases of severe congenital neutropenia.

Hematologic Diseases↗
PubMed2002

Host susceptibility factors in mycobacterial infection. Genetics and body morphotype.

Through identification and evaluation of mutations and polymorphisms in components of the IFN gamma response pathways, a better understanding of the mechanisms and risk factors influencing the development of mycobacterial disease is gained. This may lead the way for development of therapeutic and preventative strategies. Although conventional science has focused on identifying discrete mutations, greater awareness of the impact of subtle changes, both at the genetic (polymorphisms) and physical levels (body morphotype), may prove critical in the investigative process. There has been extraordinary progress in the understanding of mycobacterial susceptibility factors over the last few years. The recognition of characteristic phenotypes will lead to the identification of new genetic bases for disease.

Genetic Predisposition to Disease↗