Comparison of molecular and serological assays on cerebrospinal fluid for the diagnosis of neurosyphilis.

BACKGROUND: Many assays are available on cerebrospinal fluid (CSF) for the diagnosis of neurosyphilis (NS) but there is no 'gold standard'. OBJECTIVES: The aim of this study was to evaluate different molecular and serological assays used in NS. METHODS: We evaluated two PCR assays and three serological techniques in parallel on CSF samples collected between 2019 and 2020 from patients suspected of NS. RESULTS: The study included 143 patients comprising 30 early NS, 7 late NS and 106 patients without a diagnosis of NS. All patients with NS were symptomatic and had either neurological (67.6%) or ophthalmological signs (54.1%). The qPCR and nPCR assays had overall sensitivities (Se) of 41% and 27%, respectively; with each an overall specificity (Sp) of 100%. VDRL had a Se of 51% and a Sp of 92%. Immunoblot had a Se of 62% and a Sp of 85%. Finally, treponemal tests (TT) had a Se of 96% and a Sp of 69%. CONCLUSIONS: Our study confirms the excellent specificity of molecular techniques allowing to avoid overdiagnosis of NS, and thus, unjustified intensive antibiotic therapy protocols. CSF TT, although not very specific, has an excellent Se confirming that there is almost never NS with negative CSF TT. VDRL and immunoblot tests have better overall diagnostic performance. However, none of these techniques has sufficient diagnostic performance to represent a 'gold standard'. Thus, the diagnosis of NS relies on a combination of clinical and biological parameters with the association of PCR with serology, associating VDRL and immunoblot, in CSF.

A New Topical Candidate in Acne Treatment: Characterization of the Meclozine Hydrochloride as an Anti-Inflammatory Compound from In Vitro to a Preliminary Clinical Study.

Acne is a chronic inflammatory multifactorial disease involving the anaerobic bacterium Cutibacterium acnes (C. acnes). Current acne treatments are associated with adverse effects, limiting treatment compliance and use. We showed that meclozine, an anti-histaminic H1 compound, has anti-inflammatory properties. In Vitro, meclozine reduced the production of CXCL8/IL-8 and IL-1ß mRNA and protein by C. acnes-stimulated human keratinocytes and monocytes. No cell toxicity was observed at the IC50. Meclozine prevented the phosphorylation of ERK and JNK. In Vivo, 1% meclozine gel significantly decreased C. acnes-mouse ear induced inflammation by 26.7% (p = 0.021). Ex vivo experiments on human skin explants showed that meclozine decreased the production of GM-CSF, IL-1ß and TNF-α at transcriptional and translational levels. In a randomized, double-blind, placebo-controlled proof-of-concept clinical trial on 60 volunteers, 2% meclozine pharmaceutical gel decreased by 20.1% (p < 0.001) the ASI score in the treated group after 12 weeks of treatment. No adverse event was reported. Together, these results indicate that meclozine is a potent topical anti-inflammatory compound of potential value for acne treatment.

Production of superoxide anions by keratinocytes initiates P. acnes-induced inflammation of the skin.

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O2(*-)), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O2(*-) was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O2(*-) was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O2(*-) abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O2(*-) with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O2(*-) production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.

Contribution of GATA6 to homeostasis of the human upper pilosebaceous unit and acne pathogenesis.

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFß signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne.

Directly Sequenced Genomes of Contemporary Strains of Syphilis Reveal Recombination-Driven Diversity in Genes Encoding Predicted Surface-Exposed Antigens.

Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.

Characterization of a Propionibacterium acnes Surface Protein as a Fibrinogen-Binding Protein.

Propionibacterium acnes (P. acnes) is a major skin-associated bacterium that was long considered commensal, until several studies revealed it to be an opportunistic pathogen. We investigated the ability of P. acnes surface proteins to recognize ECM proteins and showed that a 58 kDa P. acnes surface protein was specifically recognized by human fibrinogen (hFg). The 58 kDa protein was further characterized by two-dimensional (2-D) electrophoresis and MALDI-ToF as a P. acnes host cell-surface attachment protein, PA25957, recognizing dermatan sulfate (DsA1). This protein sequence contains 432 amino acids with the presence of three structurally different domains: an N-terminal signal peptide, a C-terminal LPXTG motif, and a PT repeat region. DsA1 is mostly produced during stationary phase. It appears to be highly glycosylated, containing GalNAc residues. Purified DsA1 strongly recognizes the Aα and Bß subunits of hFg, and specific enzymatic deglycosylation of hFg demonstrated the involvement of the protein backbone in the recognition process. The Bß subunit of hFg was cloned in four peptide fractions (Fg1-Fg4). The N-terminal Fg1 peptide of hFg was recognized by DsA1, and priming DsA1 with Fg1 inhibited DsA1/hFg recognition. We describe here for the first time, the characterization of a P. acnes surface glycoprotein recognizing human fibrinogen.

Looking for the target cell of Kaposi's sarcoma-associated herpesvirus.

The level of Kaposi's sarcoma-associated herpesvirus (KSHV) cellular viremia in patients with Kaposi's sarcoma could be related to both the tumor burden and its progression. The origin of the spindle cell, the hallmark cell of Kaposi's sarcoma, is still debated. Two hypotheses may be formulated, favoring a lymphatic endothelial cell or an endothelial-cell precursor evolving into a lymphatic phenotype, both preferentially targeted by KSHV.

Superoxide anions produced by Streptococcus pyogenes group A-stimulated keratinocytes are responsible for cellular necrosis and bacterial growth inhibition.

Gram-positive Streptococcus pyogenes (group A Streptococcus or GAS) is a major skin pathogen and interacts with keratinocytes in cutaneous tissues. GAS can cause diverse suppurative and inflammatory infections, such as cellulitis, a common acute bacterial dermo-hypodermitis with a high morbidity. Bacterial isolation yields from the lesions are low despite the strong local inflammation observed, raising numerous questions about the pathogenesis of the infection. Using an in vitro model of GAS-infected keratinocytes, we show that the major ROS produced is the superoxide anion ([Formula: see text]), and that its production is time- and dose-dependent. Using specific modulators of ROS production, we show that [Formula: see text] is mainly synthesized by the cytoplasmic NADPH oxidase. Superoxide anion production leads to keratinocyte necrosis but incomplete inhibition of GAS growth, suggesting that GAS may be partially resistant to the oxidative burst. In conclusion, GAS-stimulated keratinocytes are able to develop an innate immune response based on the production of ROS. This local immune response limits GAS development and induces keratinocyte cell death, resulting in the skin lesions observed in patients with cellulitis.

Salivary lactoferrin is recognized by the human herpesvirus-8.

Human herpesvirus-8 (HHV-8) is commonly detected in all epidemiologic forms of Kaposi's sarcoma. Despite the broad cellular tropism of HHV-8, studies on mucosal shedding of HHV-8 have shown that infectious particles are restricted to saliva isolated from the oropharynx. We used biotinylated purified HHV-8 particles in a direct binding assay to whole clarified human salivary samples isolated from HHV-8-infected and uninfected individuals. We found that the major binding activity was carried out by a protein of 78-kDa size, which was further characterized as human lactoferrin (hLf) using 2-D electrophoresis and MALDI-ToF analysis. Preliminary comparison of HHV-8 binding activity of 76 salivary samples from HHV-8-infected and uninfected individuals showed that 7.8% of the uninfected population exhibited a form of Lf not recognized by HHV-8. Deglycosylation of hLf by PNGase F did not reduce HHV-8 binding activity, whereas endoproteinase cleavage of native hLf generated a non-glycosylated 8-kDa peptide recognized by HHV-8 particles and was located at the position Ala606-Tyr679 in the native hLf amino acid sequence, corresponding to the C-terminal region of the glycoprotein. This work identify the lactoferrin in saliva as a ligand for HHV-8 and suggests that this glycoprotein could be used as a carrier for the viral particles.

Oxidative stress markers are increased since early stages of infection in syphilitic patients.

Clinical symptoms of syphilis are the consequence of the spirochete propensity to induce persistent chronic inflammation, which could participate to oxidative stress increase. The present study was designed to evaluate the level of oxidative stress biomarkers and antioxidant defences in a cohort of syphilitic patients. Serum oxidative status was explored in 63 patients diagnosed with early syphilis, 34 consulting patients negative for syphilis and 19 healthy controls. Total plasma thioredoxin (Trx) and thiols were determined as antioxidant capacity markers, °NO, advanced oxidation protein products (AOPP) and protein carbonyl levels as oxidative stress status biomarkers, and CRP as marker of inflammation. Mean serum levels of Trx, AOPP, carbonyls, and nitrates/nitrites were significantly higher, whereas thiols level was lower in syphilitic patients compared to non-syphilitic patients and healthy controls (respectively, p < 0.05/p < 0.01 for Trx, p < 0.005/p < 0.0001 for AOPP, p < 0.05/p < 0.005 for carbonyls, p < 0.005/p < 0.05 for nitrates/nitrites and p < 0.01/p < 0.0001 for thiols). According to the stage of the disease, results highlighted a marked and sustained oxidative stress imbalance from the first stage to the latent period of the disease. Moreover, syphilitic patients presented a low inflammation status reflected by median of CRP level (1.7 mg/L, range 5th-95th percentile from <0.1 to 33.7 mg/L), correlated with antioxidant capacity decrease (thiols) at stage 1 (r = -0.725; p < 0.0001) and nitrosative stress increase (nitrates/nitrites) at stage 2 and latent (respectively, r = 0.285, p < 0.05 and r = 0.650, p < 0.05). These findings indicate that at all stages of the disease, despite a low-grade inflammatory state, syphilis infection generates a major oxidative and nitrosative stress which may be involved in the pathophysiology of the disease.

Does inflammatory acne result from imbalance in the keratinocyte innate immune response?

Acne is a multifactorial chronic disease affecting around 80% of teenage population. The pathogenesis of acne involves inflammatory reactions and colonization by the Propionibacterium acnes (P. acnes) strain. P. acnes stimulates the keratinocytes involved in the innate immune response, the intensity of which could be influenced either by bacterial intrinsic factors or by endogenous factors of the host.

Nicotinamide inhibits Propionibacterium acnes-induced IL-8 production in keratinocytes through the NF-kappaB and MAPK pathways.

BACKGROUND: Propionibacterium acnes (P. acnes) has been implicated in the inflammatory phase of acne vulgaris. It has been shown to activate interleukin-8 (IL-8) secretion by interacting with Toll-like receptor 2 (TLR-2) on the surface of keratinocytes. Nicotinamide has been shown to be an effective treatment for skin inflammation in various conditions, including acne vulgaris. OBJECTIVE: To investigate the molecular mechanisms underlying the anti-inflammatory properties of nicotinamide in keratinocytes stimulated by P. acnes. METHODS: HaCaT cells and primary keratinocyte cell lines were stimulated by P. acnes in the presence of nicotinamide. IL-8 production was monitored by ELISA on the cell culture supernatant and by qRT-PCR on total RNA extract. A luciferase reporter system assay was used to assess nicotinamide activity with the IL-8 promoter in transfected keratinocytes. We used western blotting to analyze the effect of nicotinamide on activation of the NF-kappaB and MAPK pathways. RESULTS: Nicotinamide significantly decreased IL-8 production in a dose-dependent manner, decreasing both mRNA and protein levels for this chemokine in immortalized HaCaT cells and primary keratinocytes. P. acnes-induced IL-8 promoter activation seemed to be downregulated by nicotinamide, which inhibited IkappaB degradation and the phosphorylation of ERK and JNK MAP kinases. CONCLUSION: Our results indicate that nicotinamide inhibits IL-8 production through the NF-kappaB and MAPK pathways in an in vitro keratinocytes/P. acnes model of inflammation. Keratinocytes involved in the innate immune response may be a suitable target for treatment during the early phase of inflammation.

Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry.

Epidemiological aspects of syphilis in Western countries have undergone a significant change with respect to the number of cases. Detection of Treponema pallidum is difficult, and the correct diagnosis of secondary syphilis can be critical. In this study, biopsy samples from skin lesions of 12 patients with secondary syphilis were used. Diagnosis of syphilis was based on clinical presentation, dark-field microscope analysis, and serological tests. Using a polyclonal antibody directed against T. pallidum, we show the presence of T. pallidum in 90% of the samples studied with the bacteria located in the epidermis and the upper dermis. The T. pallidum 47-kDa surface protein gene could be amplified by PCR in 75% of the skin lesions. When combining both techniques, T. pallidum was detected in 92% of the samples from patients with secondary syphilis but not in the control samples. Our work suggests that both immunohistochemistry and PCR could be useful for the diagnosis of secondary syphilis and may be helpful in some rare cases when serological assays failed to detect T. pallidum antibodies.

Evaluation of receptor binding specificity of Escherichia coli K88 (F4) fimbrial adhesin variants using porcine serum transferrin and glycosphingolipids as model receptors.

Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets. K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells. There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities. We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants. We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 microM), while neither K88ac nor K88ad adhesin recognizes pSTf. Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf. Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizes N-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf. All three K88 variants were found to bind preferentially to GSL containing a beta-linked N-acetylhexosamine (HexNAc), either GlcNAc or N-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position. Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a beta-linked HexNAc. In addition, the presence of a terminal galactose beta-linked to this HexNAc residue enhances K88 adhesin binding.

Recruitment of murine neutrophils in vivo through endogenous sialidase activity.

Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface. This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis. We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo. A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activities in vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8. We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitment in vivo. The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes. Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response.