Intralesional photodynamic therapy induces apoptosis in basal cell carcinoma and Bowen's disease through caspase 3 and granzyme B.

BACKGROUND: Photodynamic therapy (PDT) is used to treat cutaneous cancers. It may induce cell death through direct and indirect means, including apoptosis, inflammation and certain immune mechanisms, with the depth of penetration as a potential modifying factor. OBJECTIVES: To examine the pathways of apoptosis in the intralesional PDT of basal cell carcinoma (BCC) and intraepidermal squamous cell carcinoma (Bowen's disease). METHODS: Sixteen patients with superficial or nodular BCC and Bowen's disease were treated with intralesional aminolevulinic acid-PDT. Biopsies were taken at baseline and 24 h post-PDT, and sections were examined by immunohistochemistry for the expression of markers of apoptosis, such as caspase 3, involved in the intrinsic apoptotic pathway, granzyme B, a caspase-independent apoptotic mediator, and the proapoptotic markers BAX and BAK. RESULTS: Apoptotic cells stained with TUNEL showed statistically significant staining at 24 h post PDT (p < 0.01 in both BCC and Bowen's lesions). Caspase 3 (p < 0.01 in BCC and p < 0.05 in Bowen's) and granzyme B (p < 0.01 in BCC and p < 0.01 in Bowen's) were significantly increased at 24 h post-PDT. BAX expression was apparently increased compared to baseline in Bowen's lesions at 24 h post-PDT, whereas Bak was upregulated both in BCC and Bowen's disease at baseline and at 24 h post-PDT. CONCLUSION: Intralesional PDT induces apoptosis in BCC and Bowen's disease via common and alternative apoptotic pathways involving granzyme B. Proapoptotic factors Bak in both BCC and Bowen and Bax in Bowen's disease appear to increase by intralesional PDT at 24 h.

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.

Conventional vs. daylight photodynamic therapy for patients with actinic keratosis on face and scalp: 12-month follow-up results of a randomized, intra-individual comparative analysis.

INTRODUCTION: Daylight PDT (DLPDT) is a new PDT procedure. Several trials demonstrate that DLPDT achieves similar response rates with conventional PDT (CPDT) in the treatment of non-hyperkeratotic actinic keratoses (AKs) in a nearly painless way. It seems that DLPDT represents a more convenient and equally effective treatment modality. Data on long-term efficacy of DLPDT are limited. OBJECTIVE: To compare short- and long-term efficacy, safety and tolerability of DLPDT with that of CPDT in face and scalp AKs. METHODS: The study, an intra-individual right-left comparison study, was conducted in three centres in North, Center and South Greece. Eligible patients received either DLPDT or CPDT randomly allocated to alternate sides of face or scalp. Patients were evaluated at baseline, 3 and 12 months after treatment. Assessments included lesion response at 3 and 12 months, PDT-associated pain during PDT session, local skin reactions 3 days after treatment as well as patients' preference 3 months after treatment. RESULTS: A total of 46 patients completed the study. Three months after treatment, the overall lesion complete response rate was 78% for DLPDT and 80.6% for CPDT. At the 12-month follow-up, response rate decreased to 71.8% and 73.7% for DLPDT and CPDT accordingly. Regarding response based on lesion grade, response rates obtained in grade-I lesions were higher with DLPDT, while treatment with CPDT resulted to higher rates of cured grade-II lesions at both follow-up visits. Results were not supported by statistical significance. DLPDT was associated with significantly lower pain and reduced severity of local skin reactions. Patients' preference favoured DLPDT. CONCLUSIONS: Our study demonstrated that DLPDT is similar to CPDT in terms of long-term efficacy and recurrence rates in the treatment of face and scalp AKs. DLPDT demonstrated a better tolerability profile as it was associated with lower pain and less severe adverse events.

Hydrodynamic gene delivery in human skin using a hollow microneedle device.

Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVß and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50µL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVß reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery.

Recycled sand in lime-based mortars.

The increasing awareness of the society about safe guarding heritage buildings and at the same time protecting the environment promotes strategies of combining principles of restoration with environmentally friendly materials and techniques. Along these lines, an experimental program was carried out in order to investigate the possibility of producing repair, lime-based mortars used in historic buildings incorporating secondary materials. The alternative material tested was recycled fine aggregates originating from mixed construction and demolition waste. Extensive tests on the raw materials have been performed and mortar mixtures were produced using different binding systems with natural, standard and recycled sand in order to compare their mechanical, physical and microstructure properties. The study reveals the improved behavior of lime mortars, even at early ages, due to the reaction of lime with the Al and Si constituents of the fine recycled sand. The role of the recycled sand was more beneficial in lime mortars rather than the lime-pozzolan or lime-pozzolan-cement mortars as a decrease in their performance was recorded in the latter cases due to the mortars' structure.

Biomarkers of opiate use.

The interpretation of toxicological findings is critical for the thorough investigation of the use and abuse of psychoactive substances. A positive analytical result for a sample taken could usually result in criminal proceedings and a punitive outcome for the defendant whose sample was analysed. The detection of markers of illicit opiate misuse is important both in the management of substance misuse and in the postmortem identification of illicit opiate use. The aim of this study was to emphasise the role of opiate biomarkers available at the laboratory and in the clinical environment. Urine remains the biological tool of choice for qualitative detection of illicit drug use in a clinical setting, while quantitative accuracy remains strictly the domain of blood. Accurate interpretation of the screening tests within a clinical setting alongside other relevant information remains the key to the usefulness of any test. Moreover, the finding of a morphine/codeine concentration ratio in blood exceeding unity is a strong evidence that the person had used heroin, as opposed to having taken a prescription analgesic drug containing codeine.

Fundamental issues in forensic semen detection / Problemas fundamentales en la detección forense de semen

Sperm detection can be an important factor in confirming sexual assault in cases of rape. A large number of cases received in a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The objective of this paper is to provide an overview of the most important methods and tests used in the identification of spermatozoa or constituents ofseminal fluid during the investigation ofalleged sexual assault cases in forensic medical practice. Furthermore, this paper focusses on the basic knowledge that is necessary to the graduate students who wish to specialize in forensic sciences.

La detección de esperma puede ser un factor importante a la hora de confirmar un ataque sexual en los casos de violación. Un gran número de los casos recibidos en el laboratorio forense tienen relación con ofensas sexuales, lo cual hace necesario examinar muestras de presencia de manchas seminales. El objetivo de este trabajo es proporcionar una apreciación global de los métodos y pruebas más importantes usados en la identificación de espermatozoos o constituyentes del fluido seminal durante la investigación de supuestos casos de ataque sexual, en la práctica médica forense. Además, este trabajo presta atención al conocimiento básico necesario para los estudiantes graduados que desean especializarse en las ciencias forenses.

Fundamental issues in forensic semen detection.

Sperm detection can be an important factor in confirming sexual assault in cases of rape. A large number of cases received in a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The objective of this paper is to provide an overview of the most important methods and tests used in the identification of spermatozoa or constituents of seminal fluid during the investigation of alleged sexual assault cases in forensic medical practice. Furthermore, this paper focusses on the basic knowledge that is necessary to the graduate students who wish to specialize in forensic sciences.

Human exposure to endocrine disruptors and breast milk.

Endocrine system is one of the most sensitive communication networks of the human body which influences all aspects of human health and well-being, including reproductive potential, cognitive functions, thyroid and metabolism, digestion and hormonal balance. In recent years basic laboratory research has been focused on the potential relationship between environmental contaminants and cellular endocrine function. Environmental contaminants are ubiquitous in the environment, alter endocrine physiology and produce endocrine disruption without acting as classic toxicants. These endocrine disruptors (EDCs) are lipophilic and stored for long periods of time in the adipose tissue. Maternal exposure to EDCs during pregnancy and lactation has as a result the exposure of the fetus and neonate through placenta and breast milk. It has been recognized that human milk is the best natural food for neonates providing immunologic, developmental and practical advantages throughout childhood. However, contamination of human milk by the presence of environmental toxicants is widespread through the past decades due to inadequately controlled pollution. Persistent pesticides, chemical solvents and others tend to invade slowly the environment, to bioaccumulate in the food chain and to have long half-lives in animals and humans. During the past fifteen years, the scientific interest has been focused on xenoestrogens, i.e.,environmental chemicals with estrogen disrupting activity. Certain adverse health and reproductive outcomes are attributed to these chemicals in wildlife, in laboratory animals, as well as in humans. Although most toxic agents are hazardous in high doses, the human health risks associated with EDCs concern exposure to low doses. The human health risks that may be associated with these low-level but constant exposures are still largely unknown and highly controversial. In this paper, we review available data on environmental chemicals present in breast milk that may affect child health through breastfeeding. Specifically, we focused on the breast-feeding pharmacokinetic aspects related to infant exposure of chemical pollutants that have estrogen and antiandrogen activities, such as environmental estrogen disruptors or xenoestrogens.

Butyrylcholinesterase: biomarker for exposure to organophosphorus insecticides.

Butyrylcholinesterase (BuChE) is routinely measured to assess exposure to or effects of organophosphorus insecticides (OP). As a biomarker, it can be used to clarify the relation between exposure to OP and health impairment. The interpretation of BuChE inhibition data, particularly of small changes in enzymatic activity, sometimes presents significant complexities. These complexities are presented in this short communication and the factors that influence the degree of BuChE inhibition are discussed. Despite the complexities of their interpretation, BuChE measurements remain a mainstay for the fast initial screening of exposure to OP; thus, they are a useful tool in the protection of humans, domestic animals and wildlife from overexposure to these toxic agents.

DNA content of Tetrahymena pyriformis as a biomarker for different toxic agents.

The toxicity of different substances was studied on the protozoan Tetrahymena pyriformis, using as an endpoint the DNA content of the macronucleus. Substances from various chemical classes were administered to the Tetrahymena cultures and then the DNA content of the protozoan macronuclei was measured by means of Image Analysis System. The increase in the DNA content of the nuclei is indicative of the stimulation of the mitotic process. Since mitogenic stimuli can substantially alter susceptibility to chemical carcinogenesis, the results of such experiments, which are cheap and easy to run, may contribute to the investigation of the toxic action of several substances on cellular level.

Health impacts of fire smoke inhalation.

Most fatalities from fires are not due to burns, but are a result of inhalation of toxic gases produced during combustion. Fire produces a complex toxic environment involving flame, heat, oxygen depletion, smoke and toxic gases. As a wide variety of synthetic materials is used in buildings (insulation, furniture, carpeting, and decorative items) the potential for severe health impacts from inhalation of products of combustion during building fires is continuously increasing. In forest fires the burning of biomass leads to smoke emissions, the composition of which, as well as the relative health impacts from their inhalation, depends on the ecosystem's chemical and physical features and the local environmental parameters. In the present review, health problems that appear to people exposed to fire smoke and especially to firefighters are described and suggestions for fire management and prevention are made.

Programmed cell death of keratinocytes in infliximab-treated plaque-type psoriasis.

BACKGROUND: Tumour necrosis factor (TNF)-alpha blockade using infliximab, a chimeric anti-TNF-alpha antibody, is an effective treatment for plaque-type psoriasis, inducing remission in about 80% of patients. OBJECTIVES: To examine infliximab-induced programmed cell death (PCD) of keratinocytes in psoriatic plaques on serial skin biopsy samples. METHODS: Five patients with moderate to severe plaque-type psoriasis received infliximab infusions intravenously (5 mg kg(-1)) at weeks 0, 2 and 6. Biopsies of nonlesional and lesional skin (days 0, 5, 14 and 21) were obtained. Conventional microscopy was used to examine the morphology of the psoriatic keratinocytes. In situ detection of apoptosis was performed by electron microscopy and by immunohistochemical staining with anti-p53 and anti-caspase-3 antibodies. Results Infusion of infliximab induced a clinical response in all five patients with psoriasis, with a mean Psoriasis Area and Severity Index improvement of 24.8% already at day 5. This was accompanied by significant histopathological changes in the skin biopsy samples after infliximab treatment. Light and electron microscopic evaluation revealed apoptosis-like morphological changes in lesional keratinocytes, i.e. nuclear condensation, chromatin fragmentation and cytoplasmic vesiculation, visible already after the first infusion. These damaged keratinocytes stained positively for p53, but not for active caspase-3. CONCLUSIONS: The effects of infliximab in psoriasis extend beyond merely anti-inflammatory actions, and may include caspase-independent PCD of lesional keratinocytes. The PCD of keratinocytes may be an important mechanism that could explain at least in part the rapid and sustained therapeutic effect of infliximab in psoriasis.

Zinc: a multipurpose trace element.

Zinc (Zn) is one of the most important trace elements in the body and it is essential as a catalytic, structural and regulatory ion. It is involved in homeostasis, in immune responses, in oxidative stress, in apoptosis and in ageing. Zinc-binding proteins (metallothioneins, MTs), are protective in situations of stress and in situations of exposure to toxic metals, infections and low Zn nutrition. Metallothioneins play a key role in Zn-related cell homeostasis due to their high affinity for Zn, which is in turn relevant against oxidative stress and immune responses, including natural killer (NK) cell activity and ageing, since NK activity and Zn ion bioavailability decrease in ageing. Physiological supplementation of Zn in ageing and in age-related degenerative diseases corrects immune defects, reduces infection relapse and prevents ageing. Zinc is not stored in the body and excess intakes result in reduced absorption and increased excretion. Nevertheless, there are cases of acute and chronic Zn poisoning.

Adverse effects of anabolic steroids in athletes. A constant threat.

Anabolic-androgenic steroids (AAS) are used as ergogenic aids by athletes and non-athletes to enhance performance by augmenting muscular development and strength. AAS administration is often associated with various adverse effects that are generally dose related. High and multi-doses of AAS used for athletic enhancement can lead to serious and irreversible organ damage. Among the most common adverse effects of AAS are some degree of reduced fertility and gynecomastia in males and masculinization in women and children. Other adverse effects include hypertension and atherosclerosis, blood clotting, jaundice, hepatic neoplasms and carcinoma, tendon damage, psychiatric and behavioral disorders. More specifically, this article reviews the reproductive, hepatic, cardiovascular, hematological, cerebrovascular, musculoskeletal, endocrine, renal, immunologic and psychologic effects. Drug-prevention counseling to athletes is highlighted and the use of anabolic steroids is must be avoided, emphasizing that sports goals may be met within the framework of honest competition, free of doping substances.