Sanford R. Simon

Hypocomplementemic urticarial vasculitis syndrome: Clinical and serologic findings in 18 patients

We identify and describe clinical findings in hypocomplementemic urticarial vasculitis syndrome (HUVS), an uncommon to rare illness related to systemic lupus erythematosus (SLE). A patient with recurrent, idiopathic urticaria-like lesions was diagnosed as having HUVS if a lesional biopsy showed leukocytoclastic vasculitis, the serum C1q was markedly decreased, and antibody to C1q was detected in the patients serum. The clinical characteristics, serologic findings, and outcome of patients who met these criteria were determined from prospective and retrospective data, including hospital and office records, patient interviews, previously banked serum samples, and freshly drawn sera. Eighteen patients with HUVS were identified, and high incidences of angioedema, ocular inflammation, glomerulonephritis, and obstructive pulmonary disease were found. Renal and lung biopsies showed mesangial or membranoproliferative glomerulonephritis and severe pulmonary emphysema without vasculitis. Pulmonary function was measured in 17 patients, 11 of whom had dyspnea. All dyspneic patients had moderate to severe airflow obstruction, which progressed in all 11 and subsequently improved in only 1. Six of these 11 patients died of respiratory failure, 1 underwent lung transplantation, and 3 of the remaining 4 have moderately severe to life-threatening respiratory insufficiency. Treatment did not appear to alter the progression of obstructive lung disease. In contrast, renal insufficiency improved with treatment in 2 of 2 patients. Angioedema, ocular inflammation, obstructive lung disease, and glomerulonephritis appear to be common in HUVS, and lung disease causes substantial morbidity and mortality. The pathogenesis of HUVS may involve humoral autoimmunity, although it is not clear how autoimmunity would participate in development of obstructive lung disease. Cigarette smoking appears to be a risk factor for fatal lung disease in HUVS. All patients with HUVS should be made aware of this possibility and should be advised, encouraged, and helped to avoid tobacco smoke.

Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass.

BACKGROUND Acute lung injury (ALI) after cardiopulmonary bypass (CPB) results from sequential priming and activation of neutrophils. Activated neutrophils release neutral serine, elastase, and matrix metalloproteinases (MMPs) and oxygen radical species, which damage alveolar-capillary basement membranes and the extracellular matrix, resulting in an ALI clinically defined as adult respiratory distress syndrome (ARDS). We hypothesized that treatment with a potent MMP and elastase inhibitor, a chemically modified tetracycline (CMT-3), would prevent ALI in our sequential insult model of ALI after CPB. METHODS AND RESULTS Anesthetized Yorkshire pigs were randomized to 1 of 5 groups: control (n=3); CPB (n=5), femoral-femoral hypothermic bypass for 1 hour; LPS (n=7), sham bypass followed by infusion of low-dose Escherichia coli lipopolysaccharide (LPS; 1 microgram/kg); CPB+LPS (n=6), both insults; and CPB+LPS+CMT-3 (n=5), both insults plus intravenous CMT-3 dosed to obtain a 25-micromol/L blood concentration. CPB+LPS caused severe lung injury, as demonstrated by a significant fall in PaO(2) and an increase in intrapulmonary shunt compared with all groups (P<0.05). These changes were associated with significant pulmonary infiltration of neutrophils and an increase in elastase and MMP-9 activity. CONCLUSIONS All pathological changes typical of ALI after CPB were prevented by CMT-3. Prevention of lung dysfunction followed an attenuation of both elastase and MMP-2 activity. This study suggests that strategies to combat ARDS should target terminal neutrophil effectors.

Comparison of fluorescence-based techniques for the quantification of particle-induced hydroxyl radicals

BackgroundReactive oxygen species including hydroxyl radicals can cause oxidative stress and mutations. Inhaled particulate matter can trigger formation of hydroxyl radicals, which have been implicated as one of the causes of particulate-induced lung disease. The extreme reactivity of hydroxyl radicals presents challenges to their detection and quantification. Here, three fluorescein derivatives [aminophenyl fluorescamine (APF), amplex ultrared, and dichlorofluorescein (DCFH)] and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.ResultsAPF, amplex ultrared, and DCFH react similarly to the presence of hydroxyl radicals. Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity. Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.ConclusionThe most useful probe evaluated here for hydroxyl radicals formed from cell-free particle suspensions is APF due to its sensitivity and selectivity.

Down-regulation of trypsinogen-2 expression by chemically modified tetracyclines: association with reduced cancer cell migration.

Many types of human tumor express trypsinogen‐2, which may be a significant factor in the activation of pro‐MMPs and the invasiveness of tumors. Prevention of trypsinogen‐2 expression in cancer cells might be of benefit in cancer therapy. We describe here chemicals capable of down‐regulating the expression of trypsinogen‐2. Doxycycline (DOXY) and chemically modified tetracyclines ( CMTs ), previously known as inhibitors of the matrix metalloproteinase (MMP)–dependent proteinase cascade, down‐regulated the mRNA and protein expression of trypsinogen‐2 by COLO‐205 human colon adenocarcinoma cells at therapeutically attainable concentrations (0.1 to 1.0 μM). DOXY specifically inhibited the activation of pro‐MMP‐9 and cell migration induced by enteropeptidase, a specific activator of trypsinogen. Pro‐MMP‐9 activation and cell migration were also inhibited by tumor‐associated trypsin inhibitor (TATI), which is a highly specific inhibitor of trypsin. CMT‐3 as well as CMT‐5 also inhibited cell migration, but an effect on the enteropeptidase‐enhanced activation of pro‐MMP‐9 was not observed. Our results indicate that CMTs, DOXY and TATI inhibit cancer cell migration by down‐regulating trypsinogen‐2 expression or activity. Inhibition of trypsinogen‐2 expression may represent a mechanism contributing to the ability of CMTs to suppress the pericellular proteolytic activity of some tumors. Int. J. Cancer 86:577–581, 2000.

Polymerized liposomes as stable oxygen‐carriers

We have produced a surrogate erythrocyte (‘hemosomes’) by encapsulating human hemoglobin in polymerized vesicles composed of diacetylenic phospholipids plus or minus cholesterol. Hemoglobin (in the presence or absence of allosteric effectors) was encapsulated by a freeze‐thaw method in large, unilamellar vesicles composed of monomeric lipids. Entrapment was demonstrated by molecular‐sieve chromatography. Brief irradiation with ultraviolet light produced polymeric hemosomes with polymerization kinetics and conversions similar to liposomes in the absence of protein. Photo‐induced oxidation of the heme was eliminated or severly limited by a combination of prior ligation with CO and the maintenance of high intravesicular hemoglobin concentrations (5–10 mM internal hemoglobin). The inclusion of allosteric effectors within polymerized hemosomes facilitated near‐quantitative conversion to the oxy‐HbA form. Gas permeability of monomeric and polymeric hemosomes was demonstrated by spectroscopic methods. Reversible spectral shifts, corresponding to oxygenation‐deoxygenation, were obtained after brief evacuation and exposure to oxygen or nitrogen. The gas permeability of polymerized hemosomes appears sufficient for the vesicles to act as oxygen carriers in vivo, a notion that is strengthened by their apparent hemocompatibility.

Tumor necrosis factor release from lipopolysaccharide-stimulated human monocytes: Lipopolysaccharide tolerance in vitro

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.

Attenuation of oxidative damage and inflammatory responses by apigenin given to mice after irradiation

We determined the in vivo efficacy of apigenin, as an anti-oxidant and anti-inflammatory agent, given to mice after irradiation. Various concentrations of apigenin (0, 10, 20, and 40mg/kg body weight) were administered to mice by a single intraperitoneal injection 3hr after receiving 0 or 3Gy of (137)Cs gamma rays. Mice receiving vehicle only (no radiation and no apigenin) served as sham controls. We assessed the anti-oxidative activity of apigenin in vivo by measuring levels of 8-hydroxy-2-deoxy guanosine (8-OH-dG) in bone marrow (BM) cells, collected at days 3 and 10 after irradiation, from groups of mice (5 mice per treatment group) with or without apigenin treatment. Simultaneously, we evaluated the ability of apigenin to diminish radiation-induced inflammatory responses in bone-marrow-derived macrophages (BMDMs) from the same individual mice used for measuring the level of 8-OH-dG. To do this, the levels of activated nuclear factor-kappa B (NF-kappa B) and NF-kappa B-regulated pro-inflammatory cytokines [i.e. interleukin 1-beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha)] were measured in BMDMs. Our results indicated significant reductions (p<0.01 or <0.05) in the levels of 8-OH-dG in BM cells collected at both harvest times from irradiated mice receiving apigenin treatment, at all apigenin concentrations tested. Likewise, activation of NF-kappa B in BMDMs collected from gamma-irradiated mice that received apigenin was suppressed at both harvest times. Further, the levels of pro-inflammatory cytokines in gamma-irradiated mice treated with 20 or 40mg/kg body weight apigenin were significantly lower than those in mice receiving radiation only (p<0.01 or <0.05) even at day 10 post-irradiation. Additionally, the ratio of neutrophils to lymphocytes indicated that apigenin ameliorated radiation-induced hematological toxicity. Our study is the first to demonstrate the mitigative/therapeutic effects of apigenin given to mice after irradiation.

Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat Block Bacillus anthracis Lethal Factor Activity in Viable Cells

ABSTRACT Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin of B. anthracis.

Interaction of Matrix Metalloproteinases with Serine Protease Inhibitors

Proteolytic activity is responsible for inflammatory damage to connective tissues in a number of acute and chronic processes involving such diverse organs as the lungs, heart, joints, skin, and periodontiurn. Regardless of the nature of the inflammatory stimulus, a significant contribution to the tissue damage associated with inflammatory injury appears to come from unregulated degradation by the proteases from activated leukocytes. Neutrophil elastase (NE) is an especially potent protease which can degrade multiple components of the interstitial matrix in vitro and which has been implicated in inflammatory tissue damage in vivo. In the plasma and the interstitial stroma, the major endogenous defense against excessive NE activity comes from al-antitrypsin (aI-protease inhibitor, q-PI), a plasma serine protease inhibitor, or serpin, which is present in interstitial fluid at about half its normal plasma concentration of 1-2 mg/mL. We recently demonstrated that al-PI can be bound tightly to a complete interstitial extracellular matrix (ECM) in vitro with retention of its antielastase activity.’ Such ECM-bound antiprotease activity could contribute, along with fluid phase inhibitor, to the overall defenses against proteolytic damage to stromal tissues in vivo. Several laboratories have shown that fluid phase aI-PI can be completely inactivated by proteolytic cleavage of a constrained loop in the native molecule. A number of matrix metalloproteinases (MMPs), such as neutrophil collagenase (MMP-8), can proteolyze the loop in fluid phase al-PI at specific sites” in addition to cleaving matrix proteins. In this communication, we have extended the earlier fluid phase studies to ECM-bound aI-PI to determine whether matrix binding affects the sensitivity of this serpin to inactivation by MMPs. It is our hypothesis that inhibition of the MMP-8 activity of activated neutrophils could be of therapeutic value in helping to sustain the normal protective serpin levels within the interstitium while also blocking collagenolytic activity. One such class of inhibitors of MMP activity that could be useful for this application is the tetracy-

Improved pulmonary distribution of recombinant human Cu/Zn superoxide dismutase, using a modified ultrasonic nebulizer

Prophylactic, intratracheal instillation of recombinant human Cu/Zn superoxide dismutase (rhSOD) has been shown to lessen lung injury produced by 48 h of hyperoxia and mechanical ventilation in neonatal piglets. However, instillation of small volumes of rhSOD intratracheally would not be expected to result in uniform pulmonary distribution. Aerosolization is a technique that may improve pulmonary distribution of drugs, but is limited by the poor efficiency of most nebulizers. A newly modified ultrasonic nebulizer was tested to assess pulmonary distribution of rhSOD compared to that achieved by intratracheal instillation. rhSOD was dual‐labeled with technetium‐99m (99mTc) and a fluorescent analog (permitting quantitative and qualitative assessments of pulmonary distribution), and administered to neonatal piglets by intratracheal instillation or by aerosolization.