Thyroid hormone action in epidermal development and homeostasis and its implications in the pathophysiology of the skin.

Thyroid hormones (THs) are key endocrine regulators of tissue development and homeostasis. They are constantly released into the bloodstream and help to regulate many cell functions. The principal products released by the follicular epithelial cells are T3 and T4. T4, which is the less active form of TH, is produced in greater amounts than T3, which is the most active form of TH. This mechanism highlights the importance of the peripheral regulation of TH levels that goes beyond the central axis. Skin, muscle, liver, bone and heart are finely regulated by TH. In particular, skin is among the target organs most influenced by TH, which is essential for skin homeostasis. Accordingly, skin diseases are associated with an altered thyroid status. Alopecia, dermatitis and vitiligo are associated with thyroiditis and alopecia and eczema are frequently correlated with the Graves' disease. However, only in recent decades have studies started to clarify the molecular mechanisms underlying the effects of TH in epidermal homeostasis. Herein, we summarize the most frequent clinical epidermal alterations linked to thyroid diseases and review the principal mechanisms involved in TH control of keratinocyte proliferation and functional differentiation. Our aim is to define the open questions in this field that are beginning to be elucidated thanks to the advent of mouse models of altered TH metabolism and to obtain novel insights into the physiopathological consequences of TH metabolism on the skin.

Characterization of the immune response of human cord-blood derived gamma/delta T cells to stimulation with aminobisphosphonate compounds.

Vγ9Vδ2 T lymphocytes have been shown to respond to a variety of non-peptide antigens including alkylamines and phosphoantigens. Recently, aminobisphosphonates have also been shown to stimulate this subset of γδ+ T cells. In this study we analyzed the proliferative responses of freshly isolated γδ T lymphocytes obtained from human cord blood when challenged with pyrophosphomonoesters or aminobisphosphonates. Nitrogen-containing aminobisphopsphonates, in contrast to phoshoantigens, readily stimulated expansion of Vδ2Vγ9 cells in human cord blood. Expanded cells displayed an activated mature phenotype, and were capable of producing TNFalpha and IFNgamma but not perforin following secondary stimulation, consistent with the development of a regulatory, as opposed to cytotoxic, phenotype. This approach may provide a useful strategy for a new approach to the treatment of neonatal pathologies.

High serum levels of TNF-α and IL-6 predict the clinical outcome of treatment with human recombinant erythropoietin in anaemic cancer patients.

BACKGROUND: A number of anaemic cancer patients are not responsive to treatment with recombinant human erythropoietin (rHuEPO). The aim of the present study is to investigate whether serum levels of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6 and additional laboratory parameters, together with clinical variables, can predict the clinical outcome of treatment with rHuEPO in anaemic cancer patients. PATIENTS AND METHODS: Thirty-five cancer patients and 25 healthy controls were enrolled in this study. Patients were treated with epoetin alfa at the dose of 150 IU/kg s.c. three times a week for 12 weeks. If the haemoglobin (Hb) level failed to improve at least 2 g/dl above baseline by week 6 of treatment, dose was increased to 300 IU/kg s.c. for the remainder of the treatment period. All patients filled out the Brief Fatigue Inventory (BFI), a questionnaire for the self-evaluation of cancer-related fatigue. Serum samples from patients and control groups were frozen at -80 degrees C and TNF-alpha, IL-1beta and IL-6 were later examined by enzyme-linked immunosorbent assay. RESULTS: Fatigued cancer patients had significant higher levels of circulating TNF-alpha, IL-1beta and IL-6 than healthy controls. Responders (Rs) to erythropoietin had significant lower medium levels of TNF-alpha and IL-6 than nonresponders (NRs). Fatigued patients with a general BFI score > or =6 presented higher medium level of cytokines than nonfatigued patients (general BFI score <6), but each group responded similarly to treatment with rHuEPO. CONCLUSIONS: High serum levels of TNF-alpha and IL-6 at the baseline are significantly correlated with a negative response to administration with rHuEPO. Thus, pretreatment evaluation of TNF-alpha and IL-6 serum levels can help to select those patients who are most likely to benefit from treatment with rHuEPO. On the contrary, Hb level, red blood cell count, lactate dehydrogenase and BFI score do not predict the outcome of treatment with rHuEPO.

Eczema and food allergy in an Italian pediatric cohort: no association with TLR-2 and TLR-4 polymorphisms.

Recent studies have indicated that Toll-like receptor polymorphisms or their impaired signalling, specifically TLR-2 and TLR-4, were correlated with a higher risk for allergy. The purpose of this study is to evaluate the associations of TRL-2 and TRL-4 single nucleotide polymorphisms (SNP) and atopic traits in a cohort of 159 Italian allergic children (102 affected by eczema and 57 by IgE-mediated food allergy) and 147 healthy controls recruited in Rome, Italy. DNA was isolated from the peripheral blood and TLR-2 R753Q/TLR-4 D299G polymorphisms were determined by TaqMan MGB probes using Real-Time PCR technique. In the control group, the TLR-2 polymorphism R753Q had a prevalence of 2.5% while the frequency of the TLR-4 D299G was 12%. None of the 159 allergic patients showed the R753Q SNP. By contrast, 7/57 patients with food allergy (12%) and 6/102 subjects with eczema (6%) carried the TLR-4 mutation. In our cohort, no evidence of correlation between TLR-2 or TLR-4 polymorphism and eczema and food allergy incidence and/or severity was found. Further studies are needed to clarify the possible role of TLR-2 and TLR-4 polymorphism in allergic disease, in Italian children.

Nuclear factor kappaB activity is increased in peripheral blood mononuclear cells of children affected by atopic and non-atopic eczema.

Atopic and non-atopic eczema is an inflammatory cutaneous disease which is common in childhood and is associated with a dysregulation of the immune system. Many genes encoding immune receptors, cytokines, chemokines, chemokine receptors, and adhesion molecules involved in the development of the disease are under the control of transcription factors belonging to the nuclear factor (NF)-kappaB family. To investigate the role of NF-kappaB in the development of eczema, 20 children, affected by relapsing chronic eczema, were enrolled in this study. Eleven of the 20 children showed IgE immunoreactivity and had a positive prick test. The DNA binding activity of NF-kappaB in nuclear extracts of the patients' peripheral blood mononuclear cells (PBMC) was examined by electrophoretic mobility shift assay. We found that basal NF-kappaB-DNA binding activity in PBMC was significantly higher in the eczema patient group in comparison with the same parameter in the healthy age-matched control group. Moreover, we observed a significant correlation between NF-kappaB-DNA binding activity and patients clinical score (SCORAD). Based on these observations we speculate that NF-kappaB can play an important role in the immunopathogenesis of eczema and therefore could be considered as a potential therapeutic target.

The role of macrophage cell death in tuberculosis.

Studies of host responses to infection have traditionally focused on the direct antimicrobial activity of effector molecules (antibodies, complement, defensins, reactive oxygen and nitrogen intermediates) and immunocytes (macrophages, lymphocytes, and neutrophils among others). The discovery of the systems for programmed cell death of eukaryotic cells has revealed a unique role for this process in the complex interplay between microorganisms and their cellular targets or responding immunocytes. In particular, cells of the monocyte/macrophage lineage have been demonstrated to undergo apoptosis following intracellular infection with certain pathogens that are otherwise capable of surviving within the hostile environment of the phagosome or which can escape the phagosome. Mycobacterium tuberculosis is a prototypical 'intracellular parasite' of macrophages, and the direct induction of macrophage apoptosis by this organism has recently been reported from several laboratories. This paper reviews the current understanding of the mechanism and regulation of macrophage apoptosis in response to M. tuberculosis and examines the role this process plays in protective immunity and microbial virulence.

Expression of TAFII70 RNA and protein during oogenesis and development of the amphibian Pleurodeles waltl.

TAFs are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. TAFII60/70 was studied in yeast, Drosophila and humans: in the present work, we analyzed the homologue PwTAFII70 in Pleurodeles. The gene is expressed in ovarian oocytes and throughout development, and the level of expression decreases in late embryos. The transcripts are localized in the animal hemisphere of the fertilized eggs and in the animal blastomeres of embryos at cleavage; later PwTAFII70 mRNA is expressed in the neural plate and folds. TAFII70 protein, which is present in fertilized eggs and throughout development, progressively shows a lower level of expression starting from the neurula stage.

Role of mycobacteria-induced monocyte/macrophage apoptosis in the pathogenesis of human tuberculosis.

Apoptosis is a physiological programmed cell death process whose dysregulation plays an important role in different human infectious diseases. An increasing number of intracellular pathogens are known to induce target cell apoptosis, while some other parasites inhibit it. Unlike necrosis, apoptosis is a silent immunological event occurring without inflammation. Infection-induced target cell apoptosis may be a successful strategy to eliminate pathogens and assure host survival. Conversely, apoptosis inhibition could represent an adaptive mechanism for pathogen survival, while it may be beneficial for the host to initiate an effective immune response. The worldwide increase in tuberculosis has stimulated more research aimed at defining the interaction between Mycobacterium tuberculosis and the immune system. M. tuberculosis possesses sophisticated strategies to circumvent its fate within target monocytic cells. Apoptosis of alveolar macrophages and monocytes has been described as a consequence of M. tuberculosis infection. Moreover, the observation that mycobacterial lipoproteins activate macrophages through Toll-like receptor (TLR) 2 suggests that innate immune receptors contribute to defence against M. tuberculosis. There is evidence that TLR-induced apoptosis modulates inflammation and immune activation during M. tuberculosis infection. Finally, the role of apoptotic-infected cells as a source of microbial antigens for cross-priming of effector T-cells is also discussed.

Atopic dermatitis: molecular mechanisms, clinical aspects and new therapeutical approaches.

Atopic dermatitis (AD) is a genetically determinated, chronic inflammatory skin disorder associated with cutaneous erythema and severe pruritus, affecting 10-15% of children with increasing incidence and socio-economical relevance. Frequently, AD is associated with development of allergic rhinitis and/or asthma later in childhood. In most of patients AD is associated with a sensitization to food and/or environmental allergens and increased serum-IgE, while only a fewer percentage missed links to the classical atopic diathesis. Currently investigated pathogenetic aspects of AD include imbalanced Th1/Th2 responses, altered prostaglandin metabolism, intrinsic defects in the keratinocyte function, delayed eosinophil apoptosis, and IgE-mediated facilitated antigen presentation by epidermal dendritic cells. An inflammatory response of the two-phase-type and the effects of staphylococcal superantigens (SAgs) are also reported. At present a standardized cure of AD and a consensus on therapeutical approach of the severe form of the disease have not been established. Current management of AD is directed to the reduction of cutaneous inflammation and infection, mainly by S. aureus, and to the elimination of exacerbating factors (irritants, allergens, emotional stresses). Since patient with AD show abnormalities in immunoregulation, therapy directed to adjustment of their immune function could represent an alternative approach, particularly in the severe form of the disease. In this review, we analyse the clinical and genetic aspects of AD, the related molecular mechanisms, and the immunobiology of the disease, focusing our attention on current treatments and future perspectives on this topic.

Identification of an amphibian oocyte nuclear protein as a candidate for a role in embryonic DNA replication.

Monoclonal antibody B24/3 recognizes a nuclear protein of 104 kD in germinal vesicles of newt oocytes. Immunohistostaining of oocytes at different stages of growth shows an accumulation of B24 protein throughout oogenesis. During development B24 protein is located inside embryonic cell nuclei from the onset of cleavage onwards. It gradually decreases from gastrulation and disappears at the tailbud stage. The NvB24 17.1 clone was isolated from an ovary expression library of the newt Notophthalmus viridescens and then sequenced: the open reading frame is capable of encoding a polypeptide of 744 amino acids. Northern blot experiments have shown that the 17.1 clone recognizes a single transcript of about 3 Kb in the ovary. In situ hybridization experiments showed that B24 mRNA transcription starts from previtellogenic oocytes, and is followed by the appearance and gradual accumulation of B24 protein in germinal vesicles of medium and large size oocytes. Keeping in mind the sequence similarity shown by the B24 protein to the mouse P1 protein as well as to the budding yeast Mcm3 and fission yeast cdc21 proteins, B24 protein can be speculated to play a role in the events of DNA replication during early amphibian embryogenesis. As B24 antigen is located in the sphere organelles both inserted on the lampbrush chromosomes and free in the oocyte nucleoplasm, an additional possible role of B24 protein could be related to assembling and/or storing snRNPs during oogenesis.

Cellular proviral HIV-DNA decline and viral isolation in naïve subjects with <5000 copies/ml of HIV-RNA and >500 x 10(6)/l CD4 cells treated with highly active antiretroviral therapy.

OBJECTIVE: To evaluate the decay rate of cellular proviral HIV-DNA and viral replication in patients receiving highly active antiretroviral therapy (HAART) in the very early phase of infection. METHODS: Thirty-four patients treated with HAART and retrospectively selected for progressive decline of plasma viraemia up to undetectable levels (< 20 copies/ml), were stratified according to CD4+ cell count and plasma viraemia at base line: > 500 x 10(6) cells/l with < 5000 copies/ml (group 1) or with > 5000 copies/ml (group 2), > 5000 copies/ml with 300-500 x 10(6) cells/l (group 3) or with < 300 x 10(6) cells/l (group 4). Plasma HIV-RNA and proviral HIV-DNA were analysed at baseline and after 1, 2, 3, 6, 9 and 12 months of treatment. RESULTS: After 1 year of treatment, a significant decrease of proviral DNA titre was observed in all patients and a decrease > 1 log was achieved in 24 of 29 subjects of the first three groups. The more pronounced decay of HIV-DNA (half-life 28 weeks) up to < 50 HIV-DNA copies/10(6) CD4+ cells was detected in patients of group 1. At the year's endpoint, five patients (four in group 1 and one in group 2) had < 20 HIV-DNA copies. However, HIV strains sensitive to antiretroviral drugs were isolated from peripheral lymphocytes of 16 out of 34 patients. CONCLUSION: In patients with undetectable plasma viraemia after 1 year of HAART, the highest reduction of proviral DNA up to < 50 copies/10(6) CD4+ cells was obtained only in subjects in the early asymptomatic phase of infection. Nevertheless, a replication-competent virus can be detected in all phases of antiretroviral therapy.

Reduction of IFN-gamma and IL-2 production by peripheral lymphocytes of HIV-exposed seronegative subjects.

OBJECTIVES: In order to evaluate the role played by cytokine profile as a co-factor involved in the resistance to HIV infection in couples serodiscordant for HIV, we studied HIV-seronegative subjects with multiple unprotected sexual exposures. DESIGN AND METHODS: Twenty-one HIV-exposed seronegative subjects (HEPS), their 21 HIV-seropositive partners and 10 HIV-seronegative unexposed individuals were studied for T helper (Th) types 1-2 cell pattern and CCR5 receptor. RESULTS: Twelve out of 21 HIV-seropositive partners of HEPS showed a CD4 cell count below 200 lymphocytes/microl. HIV strains were isolated from peripheral blood mononuclear cells (PBMC) in 17 patients (81%): seven subjects with syncytium-inducing strains and 10 with non-syncytium-inducing isolates. Low Th1 cytokine production and high levels of IL-4 were detected in HIV-seropositive subjects. A significant reduction of IL-2 and IFN-gamma expression in the CD4 and CD8 cells of HEPS was found compared with HIV-seronegative unexposed individuals. Similar levels of low IL-4 were present in both HEPS and controls. The partial deletion of a single allele (wild type/delta32) of CCR5 was found in only one HEPS. CONCLUSION: The downregulated Th1 profile we observed in HEPS could be related to a cellular anergy state with a protective role in the transmission rate of HIV. Low levels of IL-2 and IFN-gamma could be involved in a low-grade activation state of CD4 lymphocytes. A decrease of IFN-gamma levels could render macrophage cells incapable of antigen presentation, thus resulting in a reduction of the cell-to-cell spread of infection.

Phenotypic and functional properties of gamma delta T cells from patients with Guillain Barré syndrome.

In this study we have examined the phenotypic and functional properties of circulating gamma delta T cells in patients with Guillain Barre syndrome (GBS), in normal healthy controls, and in patients with active multiple sclerosis (MS). Cells expressing the Vdelta2 T cell receptor showed elevated expression of the C-lectin receptor NKRP1A in both GBS and MS, suggestive of an activated state. However, in patients with GBS these cells failed to respond to pyrenil-pyrophosphate derivatives and Vdelta2 + T cell clones derived from these patients released lower levels of IFNgamma than Vdelta2 + clones derived from controls and MS patients. In contrast, in patients with GBS the Vdelta1 + subset was expanded, showed elevated expression of NKRPIA and Vdelta1 + clones derived from these patients secreted high levels of IL-4. Our findings of expanded NKRP-1A +, IL-4-producing Vdelta1 T cells in the GBS patients suggests the possibility that these cells are activated by the recognition of non-protein antigens in an MHC-unrestricted manner and contribute to the humoral response to glycolipids that is a hallmark of this disease.

Expression of FGF2 in the limb blastema of two Salamandridae correlates with their regenerative capability.

Limb regenerative potential in urodeles seems to vary among different species. We observed that Triturus vulgaris meridionalis regenerate their limbs significantly faster than T. carnifex, where a long gap between the time of amputation and blastema formation occurs, and tried to identify cellular and molecular events that may underlie these differences in regenerative capability. Whereas wound healing is comparable in the two species, formation of an apical epidermal cap (AEC), which is required for blastema outgrowth, is delayed for approximately three weeks in T. carnifex. Furthermore, fewer nerve fibres are present distally early after amputation, consistent with the late onset of blastemal cell proliferation observed in T. carnifex. We investigated whether different expression of putative blastema mitogens, such as FGF1 and FGF2, in these species may underlie differences in the progression of regeneration. We found that whereas FGF1 is detected in the epidermis throughout the regenerative process, FGF2 onset of expression in the wound epidermis of both species coincides with AEC formation and initiation of blastemal cell proliferation, which is delayed in T. carnifex, and declines thereafter. In vitro studies showed that FGF2 activates MCM3, a factor essential for DNA replication licensing activity, and can be produced by blastemal cells themselves, indicating an autocrine action. These results suggest that FGF2 plays a key role in the initiation of blastema growth.

Phenotypic changes of monocytes induced by HIV-1 gp120 molecule and its fragments.

Several phenotypic and functional changes of monocytes (M phi) have been described in HIV-1+ subjects and AIDS patients. Some of these changes that are pertinent for immunopathogenesis of the disease may be induced by HIV-1 envelope glycoprotein 120 (gp120). In the present study the effect of recombinant full length gp120 (FLgp120) and its two fragments: rp120cd (aa 410-511) and rp120 (aa 446-511) on the expression of the surface molecules of M phi cultured in vitro was determined. The FLgp120 and rp120cd caused upregulation of CD14 and CD44. The rp120cd peptide significantly increased the expression of CD16 (Fc gamma receptor type III) and TNF receptor type II. In contrast, the rp120 downregulated HLA-DR, CD64 (Fc gamma RI), interferon gamma receptor and induced IL-10 production by M phi. This study indicates that gp120 molecule and its fragments may induce several phenotypic changes of M phi in particular the increased proportion of CD14+CD16+ cells that is observed in the blood of AIDS patients. These results provide further evidence for variable response of M phi to gp120 which may explain the variability of phenotypic changes and heterogeneity of M phi subsets seen in HIV-1 disease.

Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists.

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.

Mycobacterium tuberculosis enhances iNOS mRNA expression and HIV replication in human astrocytoma cells.

The aim of this study was to investigate whether Mycobacterium tuberculosis (MTB) infection affects human immunodeficiency virus (HIV) replication in T67 human astrocytoma cells and whether HIV and MTB infections induce iNOS mRNA expression in T67 cells. T67 cells were susceptible to both HIVLai and MTB infections, and MTB was able to increase HIV infection in T67 cells, as demonstrated by a marked increase in p24 release. Furthermore, both HIV and MTB infections strongly induced inducible nitric oxide synthase (iNOS) mRNA expression, as verified by RT-PCR. These findings suggest that HIV and MTB-induced iNOS expression of astroglial cells may be involved in the neuronal damage associated with HIV infection, particularly in the presence of opportunistic infections such as tuberculosis.

Management of primary sarcomas of the retroperitoneum.

We here report on our 10-year experience of surgery in 25 patients with primary retroperitoneal sarcoma and make an appraisal of the more effective available clinical approach. Ten patients had leiomyosarcoma, eight liposarcoma, and seven had other tumor types. Histological grading was G1 in seven patients, G2 in six patients and G3 in 12. Ten patients (40%) underwent wide tumor excision, five (20%) marginal excision, four (16%) partial excision and six (24%) wedge biopsy at laparotomy. Adjacent organ resection was required in 10 of the 15 patients who underwent wide or marginal resection, seven of whom had adjuvant chemoradiotherapy. After wide resection, local recurrences were observed in 2/10 patients; the 10-year survival rate was 33%. Three out of five patients who underwent marginal resection had local recurrences; one is alive 10 years after surgery and chemo-radiotherapy. Histological grading was the most important prognostic factor. Radical resection is the only effective available treatment for retroperitoneal sarcomas, and patients with low-grade tumors benefit most from it with respect to survival and local recurrence rate. The efficacy of adjuvant treatment has yet to be clarified.

P2X7 receptors and apoptosis in tuberculosis infection.

Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. Evidence accumulated has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. Triggering of apoptosis, in response to intracellular infection, has been identified for a wide range of pathogens and host organisms, and there is now emerging interest about mechanism mediating host cell death and its role in pulmonary tuberculosis. The physiological meaning of P2X receptor-dependent cell death is not completely understood, but and involvement in immune-mediated reactions is postulated.

Tests of toxicity and teratogenicity in biphasic vertebrates treated with heavy metals (Cr3+, Al3+, Cd2+).

Developmental toxicity of chromium(III), aluminium(III) and cadmium(II) were evaluated by examining abnormalities and mortality in embryos belonging to different species of amphibians. Cr(III) and Al(III) are lethal at 1.5 mM concentration, and seriously affect the differentiation of central nervous system, skeleton and eye, and cause cephalic and trunk oedemas at lower concentrations, being aluminium significantly more harmful than chromium. Cd(II), tested only in P. waltl, is highly toxic: embryos exposed to concentrations ranging from 0.18 to 50 microM display malformations, delay and arrest of development in a dose dependent manner.