Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst

The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N- ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N- ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N- ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.     

Bone marrow transplantation in dogs after radio-ablation with a new Ho- 166 amino phosphonic acid bone-seeking agent (DOTMP)

beta-emitting 166Ho (t1/2 = 26.78 hours, E(beta)max = 1.8 MeV) complexed with the phosphonic acid chelator, 1,4,7,10 tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid) (DOTMP) at a ligand-to-metal ratio of 1.5:1 binds to bone. This radioactive complex is a marrow-ablating radiopharmaceutical that appears useful for preparation of bone marrow (BM) transplant recipients without the morbidity usually associated with total body irradiation preparatory regimens. We have found with seven splenectomized young adult beagle dogs that a 166Ho radiopharmaceutical dosage of 370 MBq/kg body weight provides an initial skeletal radioactivity burden of at least 1.5 GBq/kg skeleton and results in complete ablation of hematopoietic marrow cell populations within 7 days. The beta particle flux distribution in BM-forming skeletal tissue is not uniform. Red marrow radiation doses varied from 30 to 115 Gy as estimated by direct radioassay and autoradiographic analyses of both bone biopsies and postmortem samples; the median value of 61 Gy agreed with our theoretical expectations. In vivo radioactivity distribution was evaluated with nuclear imaging methods. Apparently, normal hematopoiesis was restored in three dogs with autologous BM transplants performed 5 to 6 days after administration of the marrow ablative radiopharmaceutical, 166Ho-DOTMP. BM biopsies at 7 to 10 months posttransplantation indicate continued normal hematopoietic activity.     

Mycobacterium tuberculosis dysregulates MMP/TIMP balance to drive rapid cavitation and unrestrained bacterial proliferation

Active tuberculosis (TB) often presents with advanced pulmonary disease, including irreversible lung damage and cavities. Cavitary pathology contributes to antibiotic failure, transmission, morbidity and mortality. Matrix metalloproteinases (MMPs), in particular MMP‐1, are implicated in TB pathogenesis. We explored the mechanisms relating MMP/TIMP imbalance to cavity formation in a modified rabbit model of cavitary TB. Our model resulted in consistent progression of consolidation to human‐like cavities (100% by day 28), with resultant bacillary burdens (>10⁷ CFU/g) far greater than those found in matched granulomatous tissue (10⁵ CFU/g). Using a novel, breath‐hold computed tomography (CT) scanning and image analysis protocol, we showed that cavities developed rapidly from areas of densely consolidated tissue. Radiological change correlated with a decrease in functional lung tissue, as estimated by changes in lung density during controlled pulmonary expansion (R² = 0.6356, p < 0.0001). We demonstrated that the expression of interstitial collagenase (MMP‐1) was specifically greater in cavitary compared to granulomatous lesions (p < 0.01), and that TIMP‐3 significantly decreased at the cavity surface. Our findings demonstrated that an MMP‐1/TIMP imbalance is associated with the progression of consolidated regions to cavities containing very high bacterial burdens. Our model provided mechanistic insight, correlating with human disease at the pathological, microbiological and molecular levels. It also provided a strategy to investigate therapeutics in the context of complex TB pathology. We used these findings to predict a MMP/TIMP balance in active TB and confirmed this in human plasma, revealing the potential of MMP/TIMP levels as key components of a diagnostic matrix aimed at distinguishing active from latent TB (PPV = 92.9%, 95% CI 66.1–99.8%, NPV = 85.6%; 95% CI 77.0–91.9%). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Distinct mutation patterns of breast cancer-associated alleles of the HRAS1 minisatellite locus

DNA sequence analysis of 130 alleles of the HRAS1 minisatellite has demonstrated that breast cancer-associated variants arise as a consequence of both replication errors and gene conversions. Unlike mutations at other variable number of tandem repeats (VNTRs), high-risk variants of the HRAS1 minisatellite do not demonstrate positional polarity. Instead, most mutations occur at three hotspots, with replication errors confined to one hotspot, gene conversions to a second and a mixed pattern of mutation at the third. DNA sequence analysis of 66 low-risk a1 alleles revealed no evidence for hypermutation. Therefore, while the HRAS1 minisatellite may serve as a reporter for a broad-based group of mutational mechanisms, these results are consistent with a direct pathogenetic contribution by high- risk alleles as the biological basis underlying cancer association of this VNTR.      

《亚洲男性学杂志》“21世纪精液分析特刊”前言

精液分析成为不育研究的重要部分经历了一个极其漫长的历史变迁过程，并声名狼藉，曾经被排除在常规的病理学检查之外，直到近些年才引起学术界的重视。精液分析起源于19世纪，那时对精子的认识仅仅局限于检测性交后宫颈黏液中是否存在精子，曾认为是“有失体面的，不自重的^[1]。”