Biomarkers of mitochondrial dysfunction and inflammaging in older adults and blood pressure variability

GeroScience - Most physiopathological mechanisms underlying blood pressure variability (BPV) are implicated in aging. Vascular aging is associated with chronic low-grade inflammation occurring in...     

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.     

Mapping myasthenia gravis-associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice

Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8xDQ6 and DQ8xDR3 F(1) transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (alpha320-337, alpha304-322, and alpha419-437), of which the response to alpha320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR- and alpha320-337 peptide-specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG(2b), and IgG(2c) Ab's and reduced IL-2 and IFN-gamma secretion by H-AChR- and peptide alpha320-337-specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence alpha320-337 as a dominant trait.     

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells

We previously reported that Cd3e‐deficient mice adoptively transferred with CD4⁺ T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B‐cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation‐induced cytidine deaminase. Furthermore, GC B cells from Cd3e^–/– mice accumulate fewer somatic mutations as compared with GC B cells from wild‐type mice, and exhibit impaired affinity maturation and reduced differentiation into long‐lived plasma cells. Reconstitution of Cd3e^–/– mice with regulatory T (Treg) cells restored Tfh‐cell numbers, GC B‐cell numbers and B‐cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh‐cell numbers and GC B‐cell numbers and dynamics were also restored by pre‐reconstitution of Cd3e^–/– mice with Cxcr5^–/– Treg cells or non‐regulatory, memory CD4⁺ T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh‐cell response for an efficient and long‐lasting serological response.     

Multiplex ligation-dependent probe amplification for the detection of 1p and 19q chromosomal loss in oligodendroglial tumors

Multiplex ligation-dependent probe amplification (MLPA) is a new assay for the detection of multiple chromosomal deletions in tumor tissue in a single experiment. Since genotyping of gliomas with oligodendroglial features by the detection of 1p/19q chromosomal deletions became essential for treatment decisions, we developed and validated an MLPA-based assay to determine these losses in formalin fixed and paraffin embedded oligodendroglial tumors (OG). Nineteen OGs, and 10 control samples were analyzed by MLPA and the results were correlated with those obtained by fluorescent in situ hybridization (FISH). The MLPA results were reproducible in all samples in which repeated experiments were performed. In 18 of 19 OGs, MLPA and FISH were concordant for presence or absence of 1p deletion. In 3 OGs, MLPA detected a 19q deletion not shown by FISH. For the other 15 OGs, MLPA and FISH were concordant. In one sample with 50% to 75% of tumor, MLPA failed to detect the 1p/19q deletions revealed by FISH (though with borderline values of significance). We conclude that MLPA is a valid and reproducible method for the detection of 1p/19q chromosomal deletions in OGs stored on formalin fixed, paraffin embedded tissue.     

Genetic control of anti-idiotypic vaccination against coronavirus infection

The idiotypic network can be experimentally altered to induce protective immune responses against microbial pathogens. Both internal image and noninternal image anti-idiotypic (anti-Id) antibodies have been shown to trigger antigen (Ag)-specific immune responses. Therefore, mechanisms of anti-Id vaccination appear to go beyond structural mimicry of Ag, but remain undefined. Using the neurotropic murine coronavirus animal model, we have previously shown that a polyclonal noninternal image anti-Id (Ab2) could vaccinate BALB/c mice. To characterize its mode of action, we have examined the immune modulating capability of this Ab2 in vivo in strains of mice with different H-2 haplotypes. Even though only internal image anti-Id are expected to induce non-genetically restricted immunity, this noninternal image Ab2 induced protective immunity in four of eight genetically different strains of mice susceptible to coronavirus infection. These were BALB/c (H-2^d), DBA/1 (H-2^d), DBA/1 (H-2^q), and SWR (H-2^q) mice. Protection was generally correlated with the induction of specific antiviral Ab (Ab3) that showed biological properties, such as virus neutralization in vitro, similar to the initial Ab1. To evaluate the genetic implication of the H-2 haplotypes in protection, congenic mice were also tested. Vaccination profiles suggest that cooperation between background gene(s) of the BALB/c mouse with H-2^d and H-2^q loci is necessary for an optimal protective immune response, although the main genetic element(s) regulating the antiviral response to Ab2 inoculation appeared to be located outside the major histocompatibility complex. These results are consistent with the ability of Ab2 to induce protective antiviral antibodies in genetically different animals by biological mimicry.     

Diagnostic utility of a p63/alpha-methyl-CoA-racemase (p504s) cocktail in atypical foci in the prostate.

Prostatic needle biopsy is the preferred method for diagnosing early prostate cancer, providing specific information. In cases of histological cancer mimics, a diagnosis of atypical small acinar proliferation suspected of but not diagnosed as malignancy can be made. In such cases, and in small focus carcinomas, pathologists use 34betaE12, cytokeratin (CK) 5/6 or p63 immunostaining to label basal cells, and alpha-methylacyl-CoA racemase (AMACR/p504s) immunostaining as a positive prostate cancer marker on two distinct slides. However, in cases of small foci, ambiguous lesions might disappear. The purpose of our study was to improve the sensitivity of a cocktail of two antibodies (p63/p504s) with a sample incubation on 260 prostatic specimens, in order to help make a decision in conjunction with standard histology and CK 5/6 immunostaining. We tested 101 small focus prostatic cancers, 104 atypical small acinar proliferation, 19 high-grade prostatic intraepithelial neoplasia, two atypical adenomatous hyperplasia and 34 benign mimics of cancer. After p63/p504s immunostaining, the final diagnoses retained were as follows: 154 prostatic cancers, 14 atypical small acinar proliferation, 30 high-grade prostatic intraepithelial neoplasia, three atypical adenomatous hyperplasia and 62 benign mimics of cancer. To differentiate malignant from benign lesions, we used the criteria of greater sensitivity to p504s/p63 (95%) than to CK 5/6 (57%) or p63 (86%), and higher specificity for p504s/p63 (95%) than for CK 5/6 (88%) or p63 (81%). With the p504s/p63 cocktail, 89% of the ambiguous lesions were classified vs 53% for CK 5/6. Combined use of the two antibodies, one (p504s) as a positive marker and the other (p63) as a negative marker, with a simple immunostaining procedure, may improve diagnostic performance, sensitivity and specificity, leading to a reduction in the risk of false negatives; this technique in cases of atypical small acinar proliferation should reduce the percentage of residual ambiguous lesions and the need for additional biopsies.     

The arterial supply of the coracoid process

Objective

The aim of this study was to describe the arterial supply of the coracoid process and to define its possible involvement in complications of Latarjet procedure.

Method

Five shoulder dissections were performed to highlight the extraosseous blood supply of the coracoid process. Postmortem arteriographies of the upper limb were performed. Diaphanization of a scapula enabled to view its intraosseous blood supply.

Results

The vertical part of coracoid process was supplied by supra-scapular artery, and the horizontal part by branches of the axillary artery.

Discussion and conclusion

This anatomical study has shown that the coracoid process had its own blood supply. During the Latarjet procedure, vascular sacrifices are mandatory to allow coracoid process transfer to the scapular neck. Such sacrifices could explain lysis or non-union of the coracoid process after Latarjet procedure. Preservation of axillary artery branches supplying horizontal part of the coracoid process could be a possible solution to prevent non-union and lysis of the bone transfer.     

PCR-based identification of eight Lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of South African women at risk of HIV and BV

Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant fixative BoonFix of one hundred women attending a health centre for HIV testing in South Africa were available for this study. In the Ndlovu Medical Centre in Elandsdoorn, South Africa, identification of 18 hr-HPV genotypes was done using the INNO-LiPA method. An inventory of lactobacilli organisms was performed using microarray technology. On the basis of the Lactobacillus and Lactobacillus biofilm scoring, the cases were identified as Leiden bacterial vaginosis (BV) negative (BV-; n = 41), Leiden BV intermediate (BV±; n = 25), and Leiden BV positive (BV+; n = 34). Fifty-one women were HIV positive and 49 HIV negative. Out of the 51 HIV positive women, 35 were HPV infected. These 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples. Lactobacillus salivarius and Lactobacillus iners were the most prevalent lactobacilli as established by the microarray technique. In women with HPV infection, the prevalence of Lactobacillus crispatus was significantly reduced. In both HIV and HPV infection, a similar (but not identical) shift in the composition of the lactobacillus flora was observed. We conclude that there is a shift in the composition of vaginal lactobacilli in HIV-infected women. Because of the prominence of HPV35, HPV52, HPV33, and HPV66, vaccination for exclusively HPV16 and HPV18 might be insufficient in South African HIV+ women.     

Metalloproteinases and their Tissue Inhibitors in Multiple Sclerosis

Matrix metalloproteinases (MMPs) comprise a family of proteolytic enzymes. MMPs are capable of disrupting the blood-brain barrier (BBB), mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in the processing of a variety of cell surface molecules, including the proinflammatory cytokine TNF-alpha. Each of these mechanisms are thought to be important in the pathogenesis of multiple sclerosis (MS). We investigated mRNA expression of MMP-3, MMP-9 and two tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in parallel in blood mononuclear cells (MNC) from patients with MS and controls, using in situ hybridization. Numbers of MMP-9 mRNA-expressing cells in blood were higher in patients with MS compared to other neurological diseases (OND), other inflammatory neurological diseases (OIND) and healthy subjects (P<0.0001 for all comparisons). Patients with MS had also higher levels of MMP-3 and TIMP-1 mRNA expressing blood MNC compared to patients with OND and healthy subjects. A positive correlation was observed for MMP-9 and TIMP-1 mRNA expression in MS. These results demonstrate that MMPs and TIMPs are upregulated in MS and may contribute to the pathogenesis of the disease.