Characterization of an 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi that contains a conserved surface-exposed epitope.

Identification of antigenically conserved surface components of Haemophilus ducreyi may facilitate the development of reagents to diagnose and prevent chancroid. A hybridoma derived from a mouse immunized with nontypeable Haemophilus influenzae produced a monoclonal antibody (MAb), designated 3B9, that bound to 35 of 35 H. ducreyi strains isolated from diverse geographic regions. The MAb 3B9 bound to a non-heat-modifiable H. ducreyi outer membrane protein (OMP) whose apparent molecular weight was 18,000 (the 18K OMP), and the 3B9 epitope did not phase vary at a rate of greater than 10(-3) in H. ducreyi. In immunoelectron microscopy, the 3B9 epitope was surface exposed, and there was intrastrain and interstrain variability in the amount of 3B9 labelling of whole cells. The MAb 3B9 cross-reacted with many species of the family Pasteurellaceae and bound to the 16.6K peptidoglycan-associated lipoprotein (P6 or PAL) of H. influenzae. Unlike P6, the 18K OMP did not copurify with peptidoglycan. In Western blots (immunoblots), five of seven serum samples obtained from patients with chancroid and four of five serum samples obtained from patients with other genital ulcer diseases at the time of presentation contained antibodies that bound to the 18K OMP. In a competition enzyme-linked immunosorbent assay, four of these serum samples inhibited the binding of 3B9 to H. ducreyi by more than 50%. We conclude that members of Pasteurellaceae expressed a conserved epitope on OMPs that sometimes had different physical characteristics. Patients with chancroid usually have antibodies to the 18K OMP and the 3B9 epitope that may have resulted from infection with H. ducreyi or previous exposure to other Haemophilus or Actinobacillus sp. strains.     

Effect of a single, open-sea, air scuba dive on human micro- and macrovascular function

Purpose

Previous studies have shown that bubble formation induced endothelial damage on conduit arteries. We aim to evaluate the effect of diving on microvascular and macrovascular function.

Methods

Nine divers took part in a SCUBA dive at 30 msw (400 kPa), for 30 min of bottom time. Pre- and post-dive, they underwent an assessment of endothelial-dependent (acetylcholine) and endothelial-independent (sodium nitroprusside) microvascular function (laser Doppler flowmetry), as well as endothelial-dependent (flow-mediated dilation) and endothelial-independent (nitroglycerin-mediated dilation) function. Bubble grades were monitored with Doppler according to the Spencer grade.

Results

The mean KISS bubble score ranged from 21.10 ± 4.7 at rest to 55.03 ± 8.8 after knee flexion. The increase in cutaneous vascular conductance elicited by either acetylcholine (25.34 ± 6.71 to 7.63 ± 1.25 %, p = 0.021) or sodium nitroprusside (35.24 ± 8.75 to 7.61 ± 1.86 %, p = 0.017) was significantly reduced after diving. Similarly, both flow-mediated dilation (10.8 ± 0.9 to 5.4 ± 1.5 %, p = 0.002) and nitroglycerin-mediated dilation (15 ± 1.1 to 6.5 ± 1.6 %, p = 0.002) were also significantly decreased. There were no correlations between vascular parameters and bubble formation.

Conclusions

There appears to be a reduction in endothelium-dependent and endothelium-independent, macro- and microvascular function associated with diving. Our results suggest that in the process of vascular dysfunction during diving, functional changes in the vessel wall may not be limited to the endothelium and may be mediated by alterations in vascular smooth muscle.     

Percutaneous vertebroplasty in the treatment of diseases causing vertebral bone loss

Background. Percutaneous vertebroplasty involves the injection of acrylic surgical cement into the vertebral body. The basic principles of vertebroplasty and the authors' own clinical experiences are described. Material and methods. Between November 1999 and January 2005 the authors performed percutaneous vertebroplasty on 75 patients: 45 with osteoporotic compression fractures, 15 with angiomas of the vertebral bodies, and 12 patients with spinal neoplasms. There were also 3 patients with coexisting spinal angiomas and osteoporotic compression fractures. All these patients were treated under local anesthesia. Cement injections were realized by the transpedicular approach under fluoroscopic guidance; in certain cases a CT-guided approach was used. The clinical outcome was assessed based on follow-up examinations, the Oswestry questionnaire, and the Visual Analog Pain Scale. Plain x-rays or CT scans were made for purposes of radiological evaluation. Results. Follow-up examinations revealed pain relief or significant reduction of pain in 89% of the patients. In 2 cases (3%) vertebroplasty was complicated by intracanal leakage of cement. Conclusions. Percutaneous vertebroplasty is well tolerated by patients. Filling with cement is effective in the treatment of osteoporotic compression fractures and of vertebral angiomas.     

Rolling Circle Amplification : A New Approach to Increase Sensitivity for Immunohistochemistry and Flow Cytometry

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.     

Evaluation of borinic acids as new,fast hydrogen peroxide–responsive triggers

Hydrogen peroxide (H₂O₂) is responsible for numerous damages when overproduced, and its detection is crucial for a better understanding of H₂O₂-mediated signaling in physiological and pathological processes. For this purpose, various “off–on” small fluorescent probes relying on a boronate trigger have been prepared, and this design has also been involved in the development of H₂O₂-activated prodrugs or theranostic tools. However, this design suffers from slow kinetics, preventing activation by H₂O₂ with a short response time. Therefore, faster H₂O₂-reactive groups are awaited. To address this issue, we have successfully developed and characterized a prototypic borinic-based fluorescent probe containing a coumarin scaffold. We determined its in vitro kinetic constants toward H₂O₂-promoted oxidation. We measured 1.9 × 10⁴ m⁻¹⋅s⁻¹ as a second-order rate constant, which is 10,000-fold faster than its well-established boronic counterpart (1.8 m⁻¹⋅s⁻¹). This improved reactivity was also effective in a cellular context, rendering borinic acids an advantageous trigger for H₂O₂-mediated release of effectors such as fluorescent moieties.

Reactive oxygen species (ROS) are involved in various physiological processes. In particular, hydrogen peroxide (H₂O₂) plays a critical role in the regulation of numerous biological activities as a signaling molecule (1, 2). However, aberrant production or accumulation of H₂O₂ leads to oxidative stress conditions, which can cause lesions associated with aging, cancer (3), and several neurodegenerative diseases such as Alzheimer’s or Parkinson’s (4, 5). Differentiation of physiological or abnormal conditions is closely connected with slight changes in H₂O₂ levels. However, the generation and degradation of H₂O₂ are variable within different cellular compartments, and this small molecule is highly diffusive, rendering the capture of small H₂O₂ fluctuations and the study of its spatial and temporal dynamics difficult. Therefore, the development of selective and sensitive H₂O₂-reactive tools for applications in a biological context represents a challenge for a better understanding of H₂O₂-mediated signaling in physiological and pathological processes or the use of H₂O₂ activation for the release of biological effectors (6).Numerous strategies have been developed to implement H₂O₂-reactive molecular triggers, as exemplified by “off–on” small fluorescent probes. Such probes have attracted particular attention due to their easy implementation, high expected signal-to-noise ratio, and compatibility with standard equipment present in cellular biology research environments (7–9). Activation in such a context is triggered or modified by H₂O₂-mediated transformation of a suitable chemical species. Several approaches have been explored including probes based on arylsulfonyl ester perhydrolysis (10), oxidation of arylboronates (11), Baeyer–Villiger oxidation of diketones (12), Tamao oxidation of silanes (13), a tandem Payne–Dakin reaction (14) or a seleno-Mislow–Evans rearrangement (15). Among them, designs based on the boronate esters oxidation pioneered by Chang are the most explored, due to their remarkable stability, low toxicity profile, ease of preparation, and specificity toward H₂O₂, as illustrated in recent reviews (16–18). Upon reaction with H₂O₂, these compounds undergo an oxidative conversion into aryl borate esters that further hydrolyze into the corresponding phenols along with borate esters or boric acid (Scheme 1A). This conversion turns on probe fluorescence or activates drug release either directly or via the degradation of a self-immolative spacer. This chemospecific and biologically compatible reaction allowed, for instance, developing highly selective fluorescent probes for H₂O₂ imaging in cells (19–23). However, H₂O₂-triggered conversion of boronic acids to phenols is still not completely satisfactory in a biological context (24) since most of these probes have second-order reaction rate constants of 0.1 to 1.0 m⁻¹⋅s⁻¹ (14). In cells, H₂O₂ is present in the 1 to 100 nm concentration range in physiological conditions and could reach up to 100 μm under oxidative stress conditions (25). Therefore, most of the boronate-based systems need an incubation time longer than 30 min for activation at an H₂O₂ concentration of 100 μm. At such a time scale, H₂O₂ typically diffuses over a distance of 2 mm (evaluated as (D_H2O2τ)^0.5 with D_H2O2 = 1.7 × 10⁻⁹ m²⋅s⁻¹ from ref. 26 and τ = 30 min). Hence, to improve spatial resolution for H₂O₂ imaging, alternative H₂O₂ triggers with rapid reaction rates allowing real-time activation by H₂O₂ are still required.Open in a separate windowScheme 1.(A) Current boronic acid (R = H) or boronate (R,R = tetramethylethylene) as H₂O₂-responsive group releasing a hydroxyaryl as effector and a boric acid or a borate ester respectively. (B) This study: a borinic acid as H₂O₂-responsive group releasing a hydroxyaryl as effector and a boronic acid.To address this issue, we envisioned the use of borinic acids, structures in which one of the boron–oxygen bonds of the boronic acid is replaced by a boron–carbon bond. Due to these electronic modifications, borinic acids exhibit more electrophilic properties (27–30) compared to their boronic acid counterparts and could be more prone to rapid oxidation. These structures have been mainly exploited as catalysts in various reactions such as epoxide ring opening (31), hydrosilylation (32), transamidation (33), aldol reaction (34, 35), C–H activation (36, 37), selective monoalkylation, acylation and sulfonation of diols (38, 39), or regioselective glycosylation reactions (40–42). Surprisingly, the reactivity of these borinic species remains underexplored (43–45), probably due to their limited synthetic access (46–49). They were usually obtained through the addition of strong organometallic reagents (RLi/RMgBr) onto boron-based electrophiles such as trialkylborates, boron halides, diborane, or boronate esters. To date, a detailed study of the reactivity of borinic acids toward oxidation including reaction with H₂O₂ has not been reported and their use as triggers for the direct release of a probe or an effector has not been considered.Herein, we report the design, synthesis, and evaluation of a borinic-triggered prototypic probe prone to direct and rapid activation by the H₂O₂ molecule (Scheme 1B). We establish a detailed kinetic analysis of the H₂O₂-promoted oxidation of this borinic acid as well as a comparative study with its corresponding boronic analog. Furthermore, we demonstrate the shorter response time of the borinic trigger compared to the boronic trigger against H₂O₂-mediated oxidation in a cellular environment.     

Expression of inducible nitric oxide synthase in skin lesions of patients with american cutaneous leishmaniasis

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of Leishmania major or Leishmania donovani infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human Leishmania infections in vivo.     

Interaction of Dr adhesin with collagen type IV is a critical step in Escherichia coli renal persistence

The pathogenic mechanism of recurrent or chronic urinary tract infection is poorly understood. Escherichia coli cells bearing Dr fimbriae display unique tropism to the basement membrane (BM)-renal interstitium that enables the bacteria to cause chronic pyelonephritis in experimental mice. The renal receptors for Dr-fimbriated E. coli are type IV collagen and decay-accelerating factor (DAF). We hypothesized that type IV collagen receptor-mediated BM-interstitial tropism is essential for E. coli to cause chronic pyelonephritis. To test the role of the type IV collagen tropism of Dr-fimbriated E. coli in renal persistence, we constructed an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to DAF and examined its virulence in the mouse model. The collagen-binding mutant DrI113T was eliminated from the mouse renal tissues in 6 to 8 weeks, while the parent strain caused persistent renal infection that lasted at least 14 weeks (P < or = 0.02). Transcomplementation with the intact Dr operon restored collagen-binding activity, BM-interstitial tropism, and the ability to cause persistent renal infection. We conclude that type IV collagen binding mediated by DraE adhesin is a critical step for the development of persistent renal infection in a murine model of E. coli pyelonephritis.