The value of five blood markers in differentiating mycosis fungoides and Sézary syndrome: a validation cohort.

BACKGROUND: Clinical and histological diagnosis of Sézary syndrome (SS) and mycosis fungoides (MF) is challenging in clinical routine. OBJECTIVES: We investigated five blood markers previously described for SS (T-plastin, Twist, KIR3DL2, NKp46 and Tox) in a prospective validation cohort of patients. METHODS: We included 447 patients in this study and 107 patients were followed up for prognosis. The markers were analysed by reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) on peripheral blood leucocytes and CD4+ T cells in a cohort of consecutive patients with early MF, erythrodermic MF and SS and compared with patients presenting with benign inflammatory dermatoses (BID) and erythrodermic BID. The markers were assessed in parallel to gold standard values such as CD4/CD8 ratio, loss of CD7 and CD26 membrane expression and CD4 absolute values. Sensitivity and specificity were analysed by receiver operator characteristic curves. The prognostic value of selected markers was analysed on a subset of patients. This study was conducted in one centre. RESULTS: We defined cut-off values for each marker. T-plastin, Twist and KIR3DL2 had the best validity. SS may be overrepresented. The combination of T-plastin and Twist was able to differentiate between erythrodermic MF or BID and SS. The additional analysis of KIR3DL2 may be useful to predict the prognosis. CONCLUSIONS: We propose T-plastin, Twist and KIR3DL2 measured by RT-qPCR as new diagnostic markers for Sézary syndrome.

Systematic screening for tuberculosis among hospital outpatients in Cameroon: The role of screening and testing algorithms to improve case detection.

BACKGROUND: Better screening and testing approaches are needed to improve TB case finding, particularly in health facilities where many people with TB seek care but are not diagnosed using the existing approaches. OBJECTIVE: We aimed to evaluate the performance of various TB screening and testing approaches among hospital outpatients in a setting with a high prevalence of HIV/TB. METHODS: We screened outpatients at a large hospital in Cameroon using both chest X-ray and a symptom questionnaire including current cough, fever, night sweats and/or weight loss. Participants with a positive screen were tested for TB using smear microscopy, the Xpert MTB/RIF assay, and culture. RESULTS: Among 2051 people screened, 1137 (55%) reported one or more TB symptom and 389 (19%) had an abnormal chest X-ray. In total, 1255 people (61%) had a positive screen and 31 of those screened (1.5%) had bacteriologically confirmed TB. To detect TB, screening with cough >2 weeks had a sensitivity of 61% (95% CI, 44-78%). Screening for a combination of cough >2 -weeks and/or abnormal chest X-ray had a sensitivity of 81% (95% CI, 67-95%) and specificity of 71% (95% CI, 69-73%), while screening for a combination of cough >2 weeks or any of 2 or more symptoms had a similar performance. Smear microscopy and Xpert MTB/RIF detected 32% (10/31) and 55% (17/31), respectively, of people who had bacteriologically-confirmed TB. CONCLUSIONS: Screening hospital outpatients for cough >2 weeks or for at least 2 of current cough, fever, night sweats or weight loss is a feasible strategy that had a high relative yield to detect bacteriologically-confirmed TB in this population. Clinical diagnosis of TB is still an important need, even where Xpert MTB/RIF testing is available.

Study of gene expression alteration in male androgenetic alopecia: evidence of predominant molecular signalling pathways.

BACKGROUND: Male androgenetic alopecia (AGA) is the most common form of hair loss in men. It is characterized by a distinct pattern of progressive hair loss starting from the frontal area and the vertex of the scalp. Although several genetic risk loci have been identified, relevant genes for AGA remain to be defined. OBJECTIVES: To identify biomarkers associated with AGA. METHODS: Molecular biomarkers associated with premature AGA were identified through gene expression analysis using cDNA generated from scalp vertex biopsies of hairless or bald men with premature AGA, and healthy volunteers. RESULTS: This monocentric study reveals that genes encoding mast cell granule enzymes, inflammatory mediators and immunoglobulin-associated immune mediators were significantly overexpressed in AGA. In contrast, underexpressed genes appear to be associated with the Wnt/ß-catenin and bone morphogenic protein/transforming growth factor-ß signalling pathways. Although involvement of these pathways in hair follicle regeneration is well described, functional interpretation of the transcriptomic data highlights different events that account for their inhibition. In particular, one of these events depends on the dysregulated expression of proopiomelanocortin, as confirmed by polymerase chain reaction and immunohistochemistry. In addition, lower expression of CYP27B1 in patients with AGA supports the notion that changes in vitamin D metabolism contributes to hair loss. CONCLUSIONS: This study provides compelling evidence for distinct molecular events contributing to alopecia that may pave the way for new therapeutic approaches.

KIR3DL2 (CD158k) is a potential therapeutic target in primary cutaneous anaplastic large-cell lymphoma.

BACKGROUND: KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. OBJECTIVES: To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. METHODS: Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by in vitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. RESULTS: KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8 ± 14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. CONCLUSIONS: pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.

Peripheral blood regulatory T cells in patients with diffuse systemic sclerosis (SSc) before and after autologous hematopoietic SCT: a pilot study.

The present pilot study aims to evaluate the frequency and the function of regulatory T (Treg) cells in patients with diffuse cutaneous SSc (dcSSc) before and after autologous hematopoietic SCT (aHSCT). Peripheral blood lymphocytes from seven dcSSc patients were analyzed before and 24 months after aHSCT and were compared with those from seven healthy donors (controls). Immunophenotyping of CD4(+)CD25(high)FoxP3(+) natural Treg (nTreg), CD4(+)CD25(+)TGF-ß(+) and CD4(+)CD25(+)IL-10(+) adaptive Treg (aTreg) cell subsets was performed using four-color flow cytometry. Treg-suppressive capability was measured after coculture with autologous T effector cells by evaluation of T-cell proliferation using (3)H-thymidine incorporation. Peripheral CD4(+)CD25(high)FoxP3(+) (2±0.5 vs 4.2±1.1, P<0.01), CD4(+)CD25(+)TGF-ß(+) (6.9±1.8 vs 14.6±5.0, P<0.05) and CD4(+)CD25(+)IL-10(+) (10.7±0.5 vs 16.1±3.2, P<0.01) Tregs as well as CD4(+)CD25(high)CD127(low) Tregs suppressive capacity (P<0.05) were decreased in dcSSc patients vs controls. After aHSCT (n=7), the percentages of CD4(+)CD25(high)FoxP3(+) (4.1±1.8) and CD4(+)CD25(+)IL-10(+) (15.7±2.2) Treg cells and the suppressive activity of CD4(+)CD25(high)CD127(low) were restored to the levels in controls. The decreased frequency and the functional defect of peripheral Treg cells from patients with dcSSc are reversed following aHSCT to reach those observed in controls. This pilot study brings evidence of an effective restoration of nTreg and aTreg subsets, and recovery of nTreg suppressive function following aHSCT.

[Cytokines in systemic sclerosis]. / Les cytokines dans la sclérodermie systémique.

The balance in the production and release of cytokines "Th1/Th2" or "Th17" or "regulatory T" is one of the key events in the pathogenesis of systemic sclerosis (SSc). Specifically, the Th2 cytokine response, characterized by the production of IL-4, IL-10 and TGF-ß, leads to tissue fibrosis in patients with SSc. Many studies have shown the importance of analyzing the levels of cytokines as diagnostic or prognostic markers in the blood or in situ in patients with SSc. The restoration of the Th1/Th2/Th17/Treg balance will contribute to the effectiveness of treatment and the use of cytokine modulators may therefore be considered in developing new therapeutic approaches.

Imperforate hymen: congenital or acquired from sexual abuse?

Imperforate hymen diagnosed beyond the newborn period may present a dilemma for the physician. Three case studies are reviewed in which children with the diagnoses of imperforate hymen presented for evaluation of suspected sexual abuse. Clear descriptions of genital anatomy documented at well-child visits may be critical to later interpretations of findings encountered during examinations for suspected sexual abuse.

Humoral and cellular responses to histamine and pollen allergen in a skin chamber model: effect of mizolastine.

BACKGROUND: Mizolastine is a new non-sedative antihistamine and antiallergic drug proven to be effective and safe in the treatment of allergic rhinitis and urticaria. OBJECTIVE: To quantitatively explore the time course of mediator release and cell recruitment during allergen challenge and the effects of mizolastine on the event, using the skin chamber model. METHODS: Twelve pollen-sensitive patients (23+/-6 years) were included in a double-blind crossover study. Patients received 10 mg mizolastine or placebo once daily in the first 4-day period and, after a 3-week washout period, vice-versa in the crossover period. On day 4 of each period, a non-invasive in vivo skin chamber technique was used to determine the alteration of vascular permeability, mast cell mediator release, the release of soluble intercellular adhesion molecule -1(sICAM-1) in skin sites challenged with exogenous histamine or grass pollen allergen extract, over an 8-hour period. RESULTS: Challenge with allergen-induced significant mast cell activation, as indicated by the release of histamine, tryptase and LTC4, in chamber fluids 2 hours after initiation of the allergic reaction and during the following 6 hours. Both exogenous histamine and allergen induced significant vasodilatation, which was sustained during the 8-hour challenge, as indicated by the accumulation of protein in the chamber fluids. Likewise, both histamine and allergen induced the release of significant amounts of ICAM-1 throughout the 8-hour period. Mizolastine significantly inhibited the histamine- and allergen-induced extravasation (after 2 hours, P = .003; after 8 hours, P = .009; after 2 hours, P = .044; after 8 hours, P = .003 respectively) and the histamine- and allergen-induced--ICAM-1 release (after 2 hours, P = .004; after 8 hours, P = .05; after 2 hours, P = .03 respectively). CONCLUSION: Mizolastine strongly inhibited the local response to histamine in this skin chamber model with, of interest, inhibition of the release of the soluble adhesion-molecule ICAM-1.

Sudden infant death syndrome, child sexual abuse, and child development.

Since the introduction of the Back to Sleep Campaigns, there has been a dramatic reduction in sudden infant death syndrome in this country. Steven Blatt and Victoria Meguid review the literature surrounding sleep position. Investigators have continued efforts to find other modifiable risk factors of sudden infant death syndrome. A prospective study of more than 33,000 neonates found a link between a prolonged QT electrocardiogram interval and sudden infant death syndrome. Also discussed are investigations seeking to explain the relationship between smoking and sudden infant death syndrome. Ann Botash, Florence Jean-Louis and Mongkae Ploy Siripornsawan review the latest thinking on genital warts and their relation to specific viral etiologies and child sexual abuse. Other symptoms and signs of sexual abuse are the focus of a number of articles that can help the practitioner care for these unfortunate children. Catherine Church reviews medication options for children diagnosed with pervasive developmental disorders or autism spectrum disorders. Finally, in this article, risperidone, fluoxetine and naltrexone are reviewed.

Ophthalmomyiasis and nasal myiasis in New Zealand: a case series.

We report three cases of ophthalmomyiasis in New Zealand, due to the larvae of Oestrus ovis. All three patients reported eye injury caused by a fly. The larvae were removed from the conjunctival sac without difficulty under local anaesthesia. Presenting ocular symptoms of foreign body sensation, irritation, redness and photophobia all resolved swiftly. Topical antibiotic and steroid eye drops were administered. All three patients also developed nasal symptoms such as sneezing, nasal discharge and epistaxis. Otolaryngology follow-up demonstrated nasal myiasis in two patients which was treated with nasal decongestants. In addition, all three patients were treated with ivermectin (Mectizan).

Production, metabolism and effect of platelet-activating factor on the growth of the human K562 erythroid cell line.

The human immature K562 erythroid cell line was studied for its capacity to produce and to metabolize the phospholipid molecule platelet-activating factor (PAF). K562 cells produced PAF under calcium ionophore stimulation. Lyso PAF and acetyl-CoA (the acetate donor molecule for the acetylation of lyso PAF into PAF) had no effect on the amounts of PAF produced by ionophore-stimulated cells. The metabolism of PAF and lyso PAF by K562 cells was compared to that of freshly-isolated human bone marrow erythroblasts and blood erythrocytes. K562 cells rapidly metabolized [3H]PAF and [3H]lyso PAF with 1-alkyl analogue of phosphatidylcholine as the major metabolic product. In contrast, blood erythrocytes did not. PAF acetylhydrolase activity levels in K562 cells and bone marrow erythroblasts were similar and higher than in blood erythrocytes. PAF (1-100 nM) stimulated [3H]thymidine incorporation in K562 cells grown in low serum concentration, a non-metabolizable PAF agonist being more potent than PAF to stimulate thymidine incorporation. PAF receptor mRNA was detected in K562 cells by polymerase chain reaction on reverse transcripts. The present study demonstrates that K562 cells produce and metabolize PAF and underlines the putative role of erythroid precursors in the modulation of bone marrow PAF concentrations. The effect of PAF on the growth of K562 cells might be mediated through PAF receptors suggesting a potential role of PAF on the proliferation and functions of human erythroid marrow precursors.

Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.

In vertebrate tissues, cell integrity is maintained by at least three mechanisms. During an immune response, injured cells are eliminated by cytotoxic lymphoid cells that produce perforin, granzyme B, and Fas ligand (FasL). Second, epithelial cells can produce FasL as an immunosuppressive protein, probably to protect the tissue against immune-mediated damage. Third, locally secreted antimicrobial peptides can be operative in the protection of animal and human epithelia. In this work, as another contribution to local mechanisms of host defense, the ability of human epidermal keratinocytes to produce cytotoxic proteins was investigated. To address this question, freshly isolated human epidermal cells and keratinocytes grown in vitro were studied. Freshly isolated epidermal cells did not express the cytolytic proteins. In contrast, keratinocyte growth to confluence was associated with granzyme B, perforin, and FasL mRNA and protein synthesis. These proteins were secreted in the culture medium. Further analysis showed that they were identical with the ones used by cytotoxic lymphocytes. Their function was then investigated with a view to a potential role in epidermal cell integrity. The data showed that activated human keratinocytes were able to protect against invading pathogens through granzyme B expression. This was demonstrated by the ability of granzyme B to greatly decrease the bacterial growth of Staphylococcus epidermidis. In addition, keratinocytes expressing FasL were found to prevent immune epidermal cell damage. Apoptosis of Fas-sensitive T cells occurred during coculture with confluent epidermal keratinocytes and was largely reduced by the addition of a FasL inhibitor. The data favor keratinocyte involvement in the regulation of dermal inflammatory responses.

Heparin induces fibroblast proliferation, cell-matrix interaction and epidermal growth inhibition.

We have examined the effect of heparin on fibroblasts cultivated in monolayer or in a 3-dimensional culture system: the so-called collagen lattices. Thereafter, we have investigated the effect of heparin on the kinetics of epidermal growth on the collagen lattices. In monolayer culture, heparin stimulated the fibroblast growth with an optimal response at 0.01 mg/ml. The volume of treated fibroblasts was smaller than that of untreated controls. In the collagen lattices, heparin stimulated the fibroblast growth with an optimal response at 0.1 mg/ml. The volume of treated fibroblasts was greater than that of untreated controls, the opposite to the result observed in monolayer culture. The beginning of the contraction of the collagen lattices was inhibited by heparin. Heparin inhibited epidermal growth on the immersion as well as on the emersion collagen lattices. These effects of heparin should be the consequences of heparin-induced modifications of cell-matrix interactions.

Effect of human basic fibroblast growth factor on fibroblast proliferation, cell volume, collagen lattice contraction: in comparison with acidic type.

Human recombinant basic fibroblast growth factor (bFGF) is a potent mitogen for normal human dermal fibroblasts in the presence of heparin which binds to and stabilizes it. The optimal mitotic response is obtained with a concentration of 1 ng/ml of bFGF in monolayer cultures (non-differentiated fibroblasts) as well as in the better-differentiated fibroblasts obtained through the 'collagen lattices' culture system (fibroblasts embedded in a 3-dimensional collagen gel) achieving a doubling of the cell number in 8 days. Despite increasing the number of cells, bFGF decreases the ability of fibroblasts to contract collagen fibers. This inhibition is concentration-dependent and reaches a plateau at a dose of about 1 ng/ml. This effect is associated with a bFGF-induced decrease of fibroblast volume. Various dosing regimens indicate that although the highest response was obtained by daily dosing nearly optimal response was obtained either by early daily dosing or short intermittent treatment. Interestingly, the fibroblast mitotic response to bFGF decreases steadily when fibroblasts mature in collagen gels. The mitogenic properties of bFGF associated to its ability to inhibit fibroblasts contraction, if demonstrated in vivo, may be of interest in the management of wound healing.

Production of paf-acether by human epidermal cells.

The production of the inflammatory mediator paf-acether (paf) from human epidermal cells was investigated in vitro. Human epidermal cells, freshly isolated from normal skin or in culture, were incubated in Tyrode's buffer containing 0.25% lipid-free bovine serum albumin in the presence of 2 microM calcium ionophore A23187, at 37 degrees C, for 1 to 60 min. Paf production slightly began at the first min of stimulation, was significant after 10 min, reached a maximum at 20 min (251 +/- 25 pg/l X 10(6) cells, mean +/- 1 SD), and decreased thereafter. About 50% of the paf amount produced by epidermal cells was recovered in supernatants. Addition of the non-acetylated paf precursor 1-O-octadecyl-sn-glycero-3-phosphocholine, i.e., lyso-paf, at 0.1 microM to epidermal cells during A23187-stimulation did not alter this production. In contrast, addition of acetyl-coenzyme A at 0.1 mM enhanced paf production by 5 times. The material produced by epidermal cells was identical to synthetic paf because: 1) the aggregation of aspirin-treated and ADP-insensitive washed rabbit platelets it induced was inhibited by BN 52021, an antagonist of the paf putative receptor; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse-phase (RP) high-pressure liquid chromatography (HPLC) elution. The paf precursors, i.e., lyso-paf and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, were also detected in epidermal cells, stimulated with A23187 or not. As determined by RP-HPLC analysis and confirmed by gas chromatography analysis, these precursors and the paf produced by epidermal cells exhibited more than 90% of a hexadecyl chain at the sn-1 position of the molecule. The present results demonstrate the synthesis and release of paf by normal human epidermal cells. Paf production within the epidermis might account for the development of cutaneous inflammation and the pathogenesis of many skin disorders.

Biological and cellular responses to grass pollen in sensitive patients.

Using a noninvasive skin chamber technique, we studied the in vivo development of anaphylactic reactions in 8 pollen-sensitive patients suffering from seasonal allergic rhinitis/conjunctivitis/asthma and showing positive cutaneous reactions after intradermal allergen challenge. As agonists, histamine and pollen were introduced into the skin chambers and left in contact with superficial dermis during 6 h. The release of mediators (histamine and prostaglandin [PG] D2) and the modifications in protein diffusion occurring during the immediate (30 min) and the late (6 and 24 h) cutaneous reaction phases were quantitatively analyzed. 24 h after agonist introduction, the recruitment of inflammatory cells on the superficial dermis was studied by use of Rebuck's windows. Histamine release in pollen-containing skin chambers was immediate and persisted until the 24th h despite replacement of the agonists by control medium at the 6th h. An intense PGD2 release occurred as soon as the first 30 min in chambers containing either exogenous histamine or pollen and was maintained until the 24th h. Protein diffusion induced by histamine and pollen was similar to the control one at 30 min but was intensely enhanced at the 6th h. At the 24th h, pollen-induced protein diffusion was still intense whereas that induced by histamine was analogous to the control one. 24 h after pollen challenge, numerous eosinophils were recruited on the superficial dermis but almost none were observed after control medium or histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of paf-acether factor-acether by human skin fibroblasts in vitro.

The synthesis and release of paf-acether by fibroblasts from normal human skin was investigated in vitro. When fibroblasts in suspension (1 X 10(6) cells) were stimulated with 2 microM Ca1+ ionophore A23187 (Io), they synthesized a material that aggregated aspirin-treated washed rabbit platelets and was identified as paf because 1) the platelet aggregation it induced was inhibited by BN 52021, an antagonist of paf putative receptors; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse phase HPLC elution. Paf production by fibroblasts occurred as soon as the first min of Io stimulation (287 +/- 92 pg/1 X 10(6) cells), reached a maximum at 5 min (369 +/- 85 pg/1 X 10(6) cells) and decreased thereafter. Half of the fibroblast-produced paf was recovered in supernatants. Addition of exogenous 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-paf) at 0.1 microM and/or acetyl-coenzyme A at 0.1 mM to fibroblasts during Io stimulation enhanced paf production by two- and three-fold, respectively. The paf precursors, i.e., 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC) and lyso-paf, were detected in fibroblasts either stimulated with Io or not. These precursors exhibited 80% hexadecyl and 20% octadecyl chains at the sn-1 position of the molecules, as determined by reverse phase HPLC and gas chromatography analysis. The present results are the first to demonstrate the synthesis and release of paf by fibroblasts from normal human skin. Such production within the dermis might account for the development of cutaneous inflammation and for the pathogenesis of many skin disorders.