Stimulated Raman Histology Interpretation by Artificial Intelligence Provides Near-Real-Time Pathologic Feedback for Unprocessed Prostate Biopsies.

PURPOSE: Stimulated Raman histology is an innovative technology that generates real-time, high-resolution microscopic images of unprocessed tissue, significantly reducing prostate biopsy interpretation time. This study aims to evaluate the ability for an artificial intelligence convolutional neural network to interpretate prostate biopsy histologic images created with stimulated Raman histology. MATERIALS AND METHODS: Unprocessed, unlabeled prostate biopsies were prospectively imaged using a stimulated Raman histology microscope. Following stimulated Raman histology creation, the cores underwent standard pathological processing and interpretation by at least 2 genitourinary pathologists to establish a ground truth assessment. A network, trained on 303 prostate biopsies from 100 participants, was used to measure the accuracy, sensitivity, and specificity of detecting prostate cancer on stimulated Raman histology relative to conventional pathology. The performance of the artificial intelligence was evaluated on an independent 113-biopsy test set. RESULTS: Prostate biopsy images obtained through stimulated Raman histology can be generated within a time frame of 2 to 2.75 minutes. The artificial intelligence system achieved a rapid classification of prostate biopsies with cancer, with a potential identification time of approximately 1 minute. The artificial intelligence demonstrated an impressive accuracy of 96.5% in detecting prostate cancer. Moreover, the artificial intelligence exhibited a sensitivity of 96.3% and a specificity of 96.6%. CONCLUSIONS: Stimulated Raman histology generates microscopic images capable of accurately identifying prostate cancer in real time, without the need for sectioning or tissue processing. These images can be interpreted by artificial intelligence, providing physicians with near-real-time pathological feedback during the diagnosis or treatment of prostate cancer.

Clinical and ultrasonographic findings of ovarian tumours in bitches: A retrospective study.

A retrospective study was carried out to investigate incidence, clinical signs and ultrasonographic findings of ovarian tumours in a population of dogs referred to the Veterinary Teaching Hospital of the University of Perugia (Italy) and Anicura Tyrus Veterinary Clinic (Terni, Italy). The period of study ranged from January 2005 to December 2021. A total of 1910 dogs were affected by neoplasia but only 35 of them (1.8%), of different breeds and ages, were found to have ovarian tumours. Ultrasound of the ovaries was performed based on clinical signs; the diagnosis was achieved after ultrasound findings prompted ovariohysterectomy and ovarian pathologic evaluation In our study, the age of bitches affected by ovarian neoplasia ranged from 3 to 20 years (mean 9.6 ± 3.8). The histopathological findings of ovarian masses identified 16 granulosa cell tumours (GCT) (46%), 7 adenomas (20%), 5 adenocarcinomas (14%), 2 teratomas (6%), 1 leiomyoma (3%), 1 luteoma (3%), 1 tecoma (3%), 1 dysgerminoma (3%), and 1 haemangiosarcoma (3%). In particular, with respect to clinical signs, 69% of bitches showed abnormalities of estrus cycle (short interestral interval, persistent estrus, prolonged interestral interval). The other main clinical signs included abdominal distention, palpable abdominal mass, vulvovaginal discharge, polyuria/polydipsia, mammary masses. When present, the laboratory abnormalities were slight anemia and leucocytosis with neutrophilia. The tumours were ultrasonographically classified as mainly solid: 12/35 (34%) (1 adenoma, 4 adenocarcinomas, 1 dysgerminoma, 1 haemangiosarcoma, 1 leyomioma, 1 luteoma, 1 GCT, 1 tecoma, 1 teratoma); solid with cystic component 13/35 (37%) (9 GCT, 2 Adenomas, 1 adenocarcinoma, 1 teratoma); and mainly cystic 10/35 (29%) (6 GCTs, 4 adenomas). In our study, the ultrasound examination allowed us to suspect ovarian neoplasia in asymptomatic subjects referred for breeding management or for preventive health check. On the basis of our data, we proposed to perform a complete periodic examination of the reproductive system once a year from 6 years. Nevertheless, the presence of ovarian neoplasms found in young subjects, during breeding management, suggest including routine ultrasound examination of the reproductive tract.

Widening the Neuroimaging Features of Adenosine Deaminase 2 Deficiency.

Adenosine deaminase 2 deficiency (OMIM #615688) is an autosomal recessive disorder characterized by a wide clinical spectrum, including small- and medium-sized vessel vasculopathies, but data focusing on the associated neuroimaging features are still scarce in the literature. Here, we describe the clinical neuroimaging features of 12 patients with genetically proven adenosine deaminase 2 deficiency (6 males; median age at disease onset, 1.3 years; median age at genetic diagnosis, 15.5 years). Our findings expand the neuroimaging phenotype of this condition demonstrating, in addition to multiple, recurrent brain lacunar ischemic and/or hemorrhagic strokes, spinal infarcts, and intracranial aneurysms, also cerebral microbleeds and a peculiar, likely inflammatory, perivascular tissue in the basal and peripontine cisterns. Together with early clinical onset, positive family history, inflammatory flares and systemic abnormalities, these findings should raise the suspicion of adenosine deaminase 2 deficiency, thus prompting genetic evaluation and institution of tumor necrosis factor inhibitors, with a potential great impact on neurologic outcome.

Off-Label Use of Sirolimus and Everolimus in a Pediatric Center: A Case Series and Review of the Literature.

BACKGROUND: It has been 15 years since sirolimus, an mTOR inhibitor, received Food and Drug Administration approval to prevent acute rejection in kidney transplantation, and 8 years since its analog everolimus acquired the same status. Since then, these drugs have become more and more utilized and their immunosuppressive and antiproliferative properties have been tested in a great variety of clinical conditions, often achieving excellent results. Despite such positive evidence, the on-label indications for these rapalogs are still very restrictive, especially in children. AIMS: The aims of this study were to describe our center's experience with sirolimus and everolimus in managing rare pediatric conditions for which mTOR inhibitors have been reported as a therapeutic option, although without conclusive approval from regulatory agencies, and to evaluate safety and tolerability of the treatment at the prescribed doses. METHODS: All the subjects who received off-label sirolimus or everolimus at the Pediatric Department of the IRCCS Burlo Garofolo in the last 13 years were included. For each disease found in our case series, we reviewed the current scientific literature. RESULTS: Off-label treatment with rapalogs was prescribed in 16 children (11 males, 5 females, median age of 9.5 years, range 1-16 years). Seven had immunologic disorders: four autoimmune lymphoproliferative syndrome (ALPS), one multicentric Castleman disease (mCD), one activated PI3K delta kinase syndrome (APDS), and one immunodysregulation with polyendocrinopathy enteropathy X-linked (IPEX). Eight had proliferative disorders or vascular anomalies: one cystic lymphangioma, two Bannayan-Riley-Ruvalcaba syndrome (BRRS), one blue rubber bleb nevus syndrome (BRBNS), two tuberous sclerosis complex (TSC), and one low-flow mixed arterial and venous malformation. One case had congenital hyperinsulinism (CHI). The average dosage administered was 1 mg/m2 for sirolimus and 7 mg/m2 for everolimus. We experienced a good measurable clinical improvement in 14 patients. Nobody experienced serious adverse events (SAEs). The therapy was interrupted in two cases, for lack of efficacy and poor tolerance in one case and for occurrence of bacterial pneumonia in the other one. A review of the literature identified 101 published reports that met our inclusion criteria. CONCLUSIONS: Although use of mTOR inhibitors has been considered to be complicated, our experience shows that, using low dosages, it is possible to obtain relevant clinical improvements, with a good profile of safety and tolerability.

New molecular and cellular targets for chemoprevention and treatment of skin tumors by plant polyphenols: a critical review.

As the incidence of skin tumors has been steadily growing, there is an urgent need for the preventive measures as well as the improved therapeutic approaches. In the last two decades, natural plant derived polyphenols (PPs, resveratrol, silibinin, green tea polyphenols, flavonoids, anthocyanins, etc.) have been drawing particular interest as emerging active substances in dermatological/cosmeceutical compositions for the prevention, slowing, or reversion of skin tumorigenesis (chemoprevention). When chronically applied to the skin, they supposedly would not damage normal skin cells or negatively affect their functions while they would suppress tumorigenic cell transformation, inhibit tumor cell proliferation, and activate tumor cell apoptosis. PPs are also reported to synergize with conventional anti-cancer therapies. The major aim of this critical review is to provide recent updates on the molecular and cellular targets for the prevention and therapy of skin tumors with a special focus on the crossroad between inflammation and carcinogenesis as the most promising approach to chemoprevention. Novel therapeutic targets as different as epidermal stem cells, cellular senescence, epigenetic enzymes involved in carcinogenesis, epidermal growth factor and aryl hydrocarbon receptors, and metabolic CYP1 subfamily enzymes are highlighted. The mechanisms of PPs interaction with these molecular and cellular targets are reviewed. The feasibility of PPs to prevent/ cure specific cutaneous toxicity connected to anti-EGFR therapy and to reduce multidrug resistance of skin tumors is also discussed.

Modulation of human keratinocyte responses to solar UV by plant polyphenols as a basis for chemoprevention of non-melanoma skin cancers.

Excessive exposure to solar UVA and UVB radiation is widely considered to cause skin cancers such as squamous cell carcinoma and basalioma. Direct UVB damage to skin cell DNA as well as UV-induced chronic skin inflammation, accelerated keratinocyte proliferation, inhibited apoptosis, and immunosuppression seem to underlie the UV-induced carcinogenesis. Also, UVB induces cytochrome P450 subfamilies (CYP1A1 and CYP1B1) involved in metabolic activation of organic pro-carcinogens and their conversion to ultimate carcinogens. Here, the effects of several glycosylated and non-glycosylated plant polyphenols (verbascoside, resveratrol, polydatin, rutin, and quercetin) on the inflammatory, apoptotic, metabolic, and proliferative responses of cultured human epidermal keratinocytes (HEK) to non-cytotoxic doses of solar-simulated UVA+UVB and chemical mediators of UV signalling in HEK, 6-formylindolo[3,2-b]carbazole and squalene isolated from photo-oxidized skin surface lipids (SSL), were evaluated. We showed that the stilbenes and quercetin being exposed to UV were photo-destroyed within a short period of time, while verbascoside and rutin were photo-stable. When SSL were exposed to UV, the stilbenes and quercetin remarkably accelerated photo-oxidation of alpha-tocopherol, squalene, and cholesterol fractions, whilst verbascoside protected them. Verbascoside invariably inhibited molecular pathways in HEK leading to inflammatory cytokine expression (NFkappaB and EGFR/ERK phosphorylation), and cell proliferation (EGFR nuclear translocation), and displayed a stimulus-specific effect on the metabolic axis aryl hydrocarbon receptor (AhR)-CYP1A1/CYP1B1. By contrast, the stilbenes inhibited UV-connected inflammatory cytokines excluding IL-8, but they prevalently stimulated NFkappaB, EGFR nuclear translocation and the AhR-CYP pathway. We conclude that, among the PPs investigated, verbascoside does interfere with multiple UV-sensitive signalling in HEK in a way that it could have a major impact on skin cancer chemoprevention.

Plant phenylpropanoids as emerging anti-inflammatory agents.

Plant-derived phenylpropanoids (PPPs) compose the largest group of secondary metabolites produced by higher plants, mainly, for the protection against biotic or abiotic stresses such as infections, wounding, UV irradiation, exposure to ozone, pollutants, and herbivores. PPPs are parent molecules for biosynthesis of numerous structurally and functionally diverse plant polyphenols (simple phenolic acids and esters, glycosylated derivatives of primary PPPs, flavonoids, isoflavonoids, stilbenes, coumarins, curcuminoids, lignans, etc.), which play multiple essential roles in plant physiology. During the last few decades, extensive research has been dedicated to natural and biotechnologically produced PPPs for medicinal use as antioxidants, UV screens, anticancer, antiviral, anti-inflammatory, wound healing, and antibacterial agents. In the present review, the metabolic pathways of phenylpropanoid biosynthesis in plants and their re-construction in biotechnologically engineered systems are described. Chemical physical peculiarities of PPPs defining their antioxidant, metal chelating, and UV-protecting effects as a molecular basis for their anti-inflammatory properties are discussed as well. We focused also on the discovery of PPPs-based anti-inflammatory agents since distinct PPPs were found to modulate molecular pathways underlying inflammatory responses in human cells triggered by different pro-inflammatory stimuli in vitro and to inhibit inflammation in various tissues in vivo. The problem of low bioavailability, fast metabolism, and potential toxicity/sensitization as limiting factors for the development of PPPs-based anti-inflammatory drugs is also highlighted.

Efficacy and safety of biologicals against immune-mediated diseases: do benefits outweigh risks?

The success of molecular biology in identifying molecular pathways underlying chronic immune-mediated diseases and the rapid development of gene/cell engineering biotechnologies has resulted in the development of a number of targeted biological drugs, which have revolutionized the therapy of these diseases. Numerous data published over the last 10-15 years demonstrate a dramatic improvement in the clinical efficacy of biologics compared with conventional drugs. However, professional and public concern about serious biological drug-associated adverse events has also been growing steadily. We critically analyze recent literature on the efficacy and safety of biologics in the management of rheumatoid arthritis, psoriasis, psoriatic arthritis and immune thrombocytopenia. Our analysis of benefits, resistance to the therapy, risk of infections, tumors and other serious complications related to chronic administration of biologics is based on the molecular/cellular mechanisms of their interaction with the immune system. We also address whether it is feasible to attenuate the risks associated with biologics without limiting their benefits.

Anti-transglutaminase antibodies in non-coeliac children suffering from infectious diseases.

Anti-transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti-transglutaminase antibodies. The aim was to measure positive anti-transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti-transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty-two children suffering from infectious diseases were enrolled prospectively along with seven biopsy-proven coeliacs. Serum samples were tested for anti-transglutaminase antibodies and anti-endomysium antibodies; positive samples were tested for coeliac-related human leucocyte antigen (HLA)-DQ2/8 and anti-viral antibodies. Purified anti-transglutaminase antibodies from the two study groups were tested for urea-dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell-cycle in Caco-2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti-transglutaminase, one of whom also tested positive for anti-endomysium antibodies. This patient was positive for HLA-DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA-DQ8. Levels of anti-transglutaminase returned to normal in all subjects, despite a gluten-containing diet. Purified anti-transglutaminase of the two study groups induced actin rearrangements and cell-cycle progression. During an infectious disease, anti-transglutaminase antibodies can be produced temporarily and independently of gluten. The infection-triggered anti-transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in-vivo potential for damage.

alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes.

BACKGROUND AND PURPOSE: The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. EXPERIMENTAL APPROACH: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods. KEY RESULTS: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. CONCLUSIONS AND IMPLICATIONS: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Plant polyphenols and tumors: from mechanisms to therapies, prevention, and protection against toxicity of anti-cancer treatments.

Polyphenolic molecules produced by higher plants in response to biotic and abiotic stresses exert numerous effects on tumorigenic cell transformation, and on tumor cells in vitro and in vivo, and may interact with conventional anti-tumor therapies. In the present review, we collected and critically discussed data on: (i) redox-dependent and redox-independent mechanisms underlying cytotoxic/cytostatic effects of PPs and their metabolites towards tumor cells and cytoprotection of normal cells; (ii) mechanisms of anti-angiogenic and anti-metastatic action of PPs; (iii) PPs-associated phototoxicity against tumor cells and photoprotection of non-tumor cells; (iv) PPs effects on drug-metabolizing enzymes as a basis for their synergism or antagonism with chemotherapy; (v) molecular pathways leading to tumor chemoprevention by PPs; and (vi) PPs as protectors against toxic effects of chemo-, radio-, and photodynamic therapies.

Mechanisms of immune-mediated skin diseases: an overview.

The skin is a frequent site of pathological immune responses that can take place in the dermal and/or the epidermal compartments.These immunopathological reactions often occur towards innocuous antigens and may be the result of T cell-dependent and/or autoantibody dependent mechanisms. Defective immune regulation is increasingly recognized as very relevant in many skin and systemic immune-mediated disorders. In some instances (e.g., psoriasis and atopic dermatitis) genetic predisposition can affect also the capacity of keratinocytes to initiate or perpetuate inflammatory responses. A more precise understanding of the molecular and cellular mechanisms underlying each disorder may allow the identification of novel targets for more effective therapeutic strategies.

Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokine production profile in response to T cell-derived cytokines.

BACKGROUND: Atopic dermatitis (AD) and psoriasis are genetically determined inflammatory skin disorders. Keratinocytes actively participate in cutaneous inflammatory responses by elaborating various chemokines. OBJECTIVE: We investigated the capacity of IL-4, IFN-gamma, and TNF-alpha to modulate the expression of CCL and CXCL chemokines in cultured keratinocytes from patients and healthy individuals, as well as chemokine expression in situ. METHODS: Keratinocyte cultures were established from normal-looking skin of adult patients with AD or psoriasis vulgaris and from healthy subjects. Monocyte chemoattractant protein 1 (MCP-1)/CCL2, RANTES/CCL5, IL-8/CXCL8, and IFN-gamma-induced protein of 10 kd (IP-10)/CXCL10 production was evaluated at the mRNA and protein levels by using RNase protection assay and ELISA, respectively. The expression of the same chemokines was studied in chronic lesional skin by means of immunohistochemistry or in situ hybridization. RESULTS: Only IL-8 mRNA was detected in unstimulated ke-ratinocyte cultures. MCP-1 and IP-10 were potently induced by IFN-gamma, whereas IL-8 and RANTES were preferentially upregulated by TNF-alpha and, to a lesser extent, by IFN-gamma. IL-4 weakly induced IP-10, RANTES, and IL-8 but not MCP-1. Keratinocytes of patients with AD invariably responded with significantly earlier and higher RANTES expression. By contrast, keratinocytes of patients with psoriasis displayed much higher levels of both constitutive and induced IL-8 and a stronger induction of MCP-1 and IP-10. RANTES and MCP-1 mRNA(+) keratinocytes were detected in the basal layer of lesions of patients with AD and psoriasis. IP-10 and IL-8 were consistently upregulated in the epidermis of patients with psoriasis but not in lesions of patients with AD. CONCLUSIONS: Keratinocytes of patients with AD and psoriasis show an intrinsically abnormal and different chemokine production profile and may thus favor the recruitment of distinct leukocyte subsets into the skin.

Regulatory activity of autocrine IL-10 on dendritic cell functions.

IL-10 is a critical cytokine that blocks the maturation of dendritic cells (DCs), but the relevance of autocrine IL-10 on DC functions has not been investigated. In this study, we found that immature monocyte-derived DCs released low but sizeable amounts of IL-10. After stimulation with bacteria, LPS, lipoteichoic acid, or soluble CD40 ligand, DCs secreted high levels of IL-10. Addition of an anti-IL-10-neutralizing Ab to immature DCs as well as to soluble CD40 ligand- or LPS-maturing DCs led to enhanced expression of surface CD83, CD80, CD86, and MHC molecules and markedly augmented release of TNF-alpha and IL-12, but diminished IL-10 mRNA expression. Moreover, DCs treated with anti-IL-10 Ab showed an increased capacity to activate allogeneic T cells and primed naive T cells to a more prominent Th1 polarization. DC maturation and IL-10 neutralization were associated with enhanced accumulation of the IL-10 receptor binding chain (IL-10R1) mRNA and intracellular IL-10R1 protein. In contrast, surface IL-10R1 and IL-10 binding activity diminished in mature DCs. These results indicate that autocrine IL-10 prevents spontaneous maturation of DCs in vitro, limits LPS- and CD40-mediated maturation, and increases IL-10 production by DCs. Moreover, IL-10R expression appears to be regulated by both transcriptional and posttranscriptional mechanisms. Endogenous IL-10 and IL-10R can be relevant targets for the manipulation of DC functions.

Dysregulated activation of activator protein 1 in keratinocytes of atopic dermatitis patients with enhanced expression of granulocyte/macrophage-colony stimulating factor.

Keratinocytes of patients with atopic dermatitis produce high amounts of granulocyte/macrophage colony-stimulating factor, a factor essential for dendritic cell function and thus for the development of skin immune responses. In contrast to keratinocytes cultured from nonatopic, healthy individuals, granulocyte/macrophage colony-stimulating factor mRNA could be detected in unstimulated cultures of atopic dermatitis keratinocytes, and phorbol myristate acetate induced much greater granulocyte/macrophage colony-stimulating factor mRNA levels in these cells, although the decay kinetics were not altered. Using reporter gene (chloramphenicol acetyl transferase) analysis, a minimal granulocyte/macrophage colony-stimulating factor promoter was shown to confer constitutive and phorbol-myristate-acetate-induced regulation of transcriptional activity in keratinocytes, and significantly higher levels of chloramphenicol acetyl transferase activity were measured in lysates of unstimulated and phorbol-myristate-acetate-treated atopic dermatitis keratinocytes than in control keratinocyte cultures. Electrophoretic mobility shift assays showed that low levels of NF-kappa B binding activity could be induced by phorbol myristate acetate in both normal and atopic dermatitis keratinocytes. By contrast, activator protein 1 complexes were efficiently induced, and they were invariably present at higher levels in nuclear lysates of atopic dermatitis keratinocytes. Atopic dermatitis keratinocyte nuclear lysates had higher constitutive levels of c-Jun, and phorbol myristate acetate promoted an earlier and stronger expression of c-Jun, JunB, and of the phosphorylated forms of c-Fos. A dysregulated activation of activator protein 1 may be implicated in the molecular mechanisms leading to increased granulocyte/macrophage colony-stimulating factor expression in atopic dermatitis keratinocytes. J Invest Dermatol 115:1134-1143 2000

Interferon-gamma promotes exaggerated cytokine production in keratinocytes cultured from patients with atopic dermatitis.

Recent studies suggest that skin keratinocytes from patients with atopic dermatitis (AD) and nonatopic subjects differ in their intrinsic ability to respond to proinflammatory stimuli. In this study keratinocyte cultures established from the normal-looking skin of six adult patients with AD and six healthy, nonatopic control subjects were compared in their response to interferon (IFN)-gamma, a potent proinflammatory lymphokine whose expression is increased in chronic AD lesions. Basal expression of IFN-gamma receptor as well as IFN-gamma-induced membrane expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 were evaluated by flow cytometry. Keratinocyte release of IL-1alpha, IL-1 receptor antagonist (IL-1ra), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha were measured by ELISA on culture supernatants after treatment with IFN-gamma or medium alone. Expression of membrane IFN-gamma receptor was similar in keratinocytes cultured from nonatopic subjects and subjects with AD. IFN-gamma (10 to 500 U/ml) induced comparable levels of membrane HLA-DR and ICAM-1 in both groups of keratinocytes. In contrast, spontaneous release of IL-1alpha, IL-1ra, GM-CSF, and TNF-alpha was increased in the supernatants of unstimulated keratinocytes from patients with AD compared with keratinocytes from control subjects, with IL-1ra and GM-CSF reaching statistically significant difference. Moreover, IFN-gamma-induced release of all the cytokines tested was much higher for keratinocytes from patients with AD, but the IL-1ra/IL-1alpha ratio for the two groups of keratinocytes was not substantially different, either basally or after IFN-gamma stimulation. The results indicate that keratinocytes from patients with AD are hyperresponsive to IFN-gamma in terms of cytokine release.

Cetirizine and hydrocortisone differentially regulate ICAM-1 expression and chemokine release in cultured human keratinocytes.

BACKGROUND: Cetirizine is a H1 histamine antagonist which possesses anti-inflammatory properties through inhibition of leucocyte recruitment and activation, and reduction of ICAM-1 expression on mucosal epithelial cells. No studies have addressed the potential anti-inflammatory activities of cetirizine on skin keratinocytes. OBJECTIVES: Cetirizine and hydrocortisone were compared in their capacity to counteract human keratinocytes activation by IFNgamma. In particular, expression of immuno-modulatory membrane molecules and chemokine release have been examined. METHODS: Keratinocyte cultures established from normal skin of healthy donors were activated by IFNgamma (100-500 U/mL) in the absence or presence of cetirizine (10(-3)-10(3) microM) or hydrocortisone (10(-3)-10(2) microM), and tested for expression of ICAM-1, HLA-DR, MHC class I and CD40 as well as for release of RANTES, IL-8, macrophage chemotactic protein-1 (MCP-1) and granulocyte macrophage-colony stimulating factor (GM-CSF). RESULTS: Cetirizine at high concentrations (10(2)-10(3) microM) markedly inhibited IFNgamma-induced expression of membrane ICAM-1, HLA-DR and up-regulation of MHC class I, but had no effect on CD40 expression. In contrast, hydrocortisone (10(2) microM) enhanced IFNgamma-induced membrane ICAM-1, reduced expression of HLA-DR and did not alter expression of MHC class I and CD40. Consistently, high doses of cetirizine decreased, whereas hydrocortisone increased, soluble ICAM-1 levels in the supernatants of IFNgamma-treated keratinocytes. The inhibiting and stimulating effects of cetirizine and hydrocortisone, respectively, on ICAM-1 expression were confirmed at the mRNA level by Northern blot analysis. Finally, cetirizine, but not hydrocortisone, inhibited the release of MCP-1 and RANTES from IFNgamma-stimulated keratinocytes. In contrast, hydrocortisone, but not cetirizine, reduced GM-CSF and IL-8 release. CONCLUSIONS: The results indicate that cetirizine has the capacity to block the IFNgamma-induced activation of keratinocytes, and thus can exert important regulatory effects on TH1 cell-mediated immune responses in the skin. The high doses required for evidencing these activities suggest the potential benefits of a topical use of cetirizine.

Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.

Lesional skin of atopic dermatitis (AD) harbors high numbers of dendritic cells with enhanced stimulatory capacity for T lymphocytes. In this study, lesional AD skin was shown to stain heavily in both epidermal and dermal compartments for GM-CSF, a cytokine crucial to dendritic cell functions. Keratinocyte cultures established from uninvolved skin of AD patients exhibited markedly increased spontaneous and PMA-stimulated release of GM-CSF compared with keratinocytes from nonatopic controls. Correspondingly, keratinocytes from AD patients showed higher constitutive as well as PMA-induced GM-CSF gene expression. Larger amounts of GM-CSF were produced by AD keratinocytes, also in response to IL-1alpha, but not after stimulation with LPS, lipoteichoic acid, or staphylococcal enterotoxin B. Hydrocortisone reduced GM-CSF gene expression and protein release in both atopic and control keratinocytes. Supernatants from atopic keratinocytes were able to strongly stimulate PBMC proliferation in a GM-CSF-dependent manner. Moreover, conditioned medium from PMA-treated AD keratinocytes, together with exogenous IL-4, could support phenotypical and functional maturation of peripheral blood precursors into dendritic cells. Enhanced production of GM-CSF by keratinocytes may contribute relevantly to the establishment and chronicity of AD lesions, in particular to the increased number, sustained activation, and enhanced antigen-presenting functions of dendritic cells.