Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

Asymmetry in genitalia does not increase the rate of their evolution.

Left-right asymmetry is a frequently encountered phenomenon in the copulation organs of insects. While various causes have been proposed for genital asymmetry, we raise the question of whether asymmetry might facilitate, or even accelerate, morphological divergence of genitalia between species. We tested this hypothesis in the scarab chafer genus Schizonycha, which comprises species with symmetric as well as asymmetric male genitalia. Morphometric analyses were conducted in the context of their phylogeny, inferred from mitochondrial and nuclear ribosomal DNA sequence data (cox1, rrnL, and 28S) for a sample of 99 South African specimens, including 34 species and 5 outgroup taxa. Trees were reconstructed with maximum likelihood and Bayesian analysis. The extent of asymmetry and the variation of male copulation organs were analyzed with Generalized Procrustes analysis (GPA), by quantifying shape divergence of the parameres. We found a continuous transition in the degree of asymmetry among the investigated species. Ancestral state reconstruction revealed multiple origins and a high degree of evolutionary plasticity of paramere asymmetry in Schizonycha. However, no significant correlation between evolutionary rates of paramere shape divergence and the degree of paramere asymmetry was found, and so we conclude that asymmetric genitalia in Schizonycha do not increase the rate of genital shape divergence.

Age-related changes in auditory nerve-inner hair cell connections, hair cell numbers, auditory brain stem response and gap detection in UM-HET4 mice.

This study compared the timing of appearance of three components of age-related hearing loss that determine the pattern and severity of presbycusis: the functional and structural pathologies of sensory cells and neurons and changes in gap detection (GD), the latter as an indicator of auditory temporal processing. Using UM-HET4 mice, genetically heterogeneous mice derived from four inbred strains, we studied the integrity of inner and outer hair cells by position along the cochlear spiral, inner hair cell-auditory nerve connections, spiral ganglion neurons (SGN), and determined auditory thresholds, as well as pre-pulse and gap inhibition of the acoustic startle reflex (ASR). Comparisons were made between mice of 5-7, 22-24 and 27-29 months of age. There was individual variability among mice in the onset and extent of age-related auditory pathology. At 22-24 months of age a moderate to large loss of outer hair cells was restricted to the apical third of the cochlea and threshold shifts in the auditory brain stem response were minimal. There was also a large and significant loss of inner hair cell-auditory nerve connections and a significant reduction in GD. The expression of Ntf3 in the cochlea was significantly reduced. At 27-29 months of age there was no further change in the mean number of synaptic connections per inner hair cell or in GD, but a moderate to large loss of outer hair cells was found across all cochlear turns as well as significantly increased ABR threshold shifts at 4, 12, 24 and 48 kHz. A statistical analysis of correlations on an individual animal basis revealed that neither the hair cell loss nor the ABR threshold shifts correlated with loss of GD or with the loss of connections, consistent with independent pathological mechanisms.

Interleukin-33 promotes the proliferation of mouse mast cells through ST2/MyD88 and p38 MAPK-dependent and Kit-independent pathways.

Interleukin-33 (IL-33), a member of the IL-1 cytokine family, is emerging as a new modulator of immune and inflammatory responses. Although IL-33 and its associated receptor ST2 are reportedly expressed in mast cells (MCs), the precise role of IL-33 in modulating MC function has not been determined. In the present studies, we explored IL-33 effects on MCs in vivo and in vitro. IL-33 increased the number of peritoneal and skin MCs in vivo. IL-33 also resulted in increased proliferation of MCs in vitro, as explored by WST assay. Cell cycle analysis further confirmed this result by showing increased G2 cell populations in MCs stimulated with IL-33. We found that IL-33-mediated MC proliferation requires ST2 and MyD88, is independent of Kit, and is mediated through a p38 MAPK-dependent pathway. IL-33 did not induce degranulation and was not cytotoxic for MCs. This novel mechanism for increasing MC proliferation and numbers further defines the role of IL-33 in MC-dependent diseases including allergies and may help to develop novel approaches for the treatment of these disorders.

ROS-dependent phosphorylation of Bax by wortmannin sensitizes melanoma cells for TRAIL-induced apoptosis.

The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive. Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways. Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression. The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns. Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced. Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here. Characteristically, production of ROS was seen early in response to wortmannin and MK-2206. Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation. This correlated with reduced Bax phosphorylation at threonine-167. The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation. The pathway may be considered for proapoptotic, anticancer strategies.

Influence of hepatitis C virus infection and high virus serum load on biliary complications in liver transplantation.

BACKGROUND: Biliary complications (BCs) and recurrent hepatitis C virus (HCV) infection are among the major causes of morbidity and graft loss following liver transplantation. The influence of HCV on BCs has not been definitely clarified. PATIENTS AND METHODS: We performed a retrospective cohort study to analyze risk factors and outcome of post orthotopic liver transplantation (OLT) BCs in 352 liver transplant recipients over 12 years in Munich, Germany (n = 84 with HCV; living donor and re-OLT were excluded). BCs diagnosed with imaging techniques and abnormal liver enzyme pattern, requiring an intervention, were considered. RESULTS: In a multivariate analysis, HCV serostatus and a high pre-and post-surgery HCV RNA serum load were independent risk factors for anastomotic strictures. HCV positivity and BCs alone did not alter graft loss. HCV-positive patients with BCs, however, had a significantly worse graft outcome (P = 0.02). Non-anastomotic strictures, bile leaks, and the number of interventions needed to treat bile leaks led to worse graft outcome in all patients. CONCLUSION: HCV positivity and a high HCV RNA serum load were risk factors for anastomotic strictures. BCs and HCV had an additive effect on graft loss.

Sensitization of melanoma cells for TRAIL-induced apoptosis by BMS-345541 correlates with altered phosphorylation and activation of Bax.

Resistance to TRAIL (TNF-related apoptosis-inducing ligand)- induced apoptosis limits its therapeutic use. Different strategies of TRAIL sensitization and a dependency on Bax have been reported, but common principles of TRAIL resistance and the way of Bax activation remained poorly understood. Applying a melanoma model of TRAIL-sensitive and -resistant cell lines, efficient sensitization for TRAIL-induced apoptosis is demonstrated by the kinase inhibitor BMS-345541 (N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-1,2-ethanediamine hydrochloride), which targets IκB (inhibitor of κB proteins) kinase ß (IKKß). This effect was completely abrogated by Bax knockout as well as by Bcl-2 overexpression, in accordance with a Bax dependency. Early loss of the mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspases) clearly indicated the activation of mitochondrial apoptosis pathways. Of note, BMS-345541 alone resulted in an early Bax activation, seen by conformational changes and by Bax translocation. The synergistic effects can be explained by Bid activation through TRAIL, which inhibits Bcl-2, and the activation of Bax through BMS-345541. The critical roles of XIAP (X-chromosome-linked inhibitor of apoptosis protein), Smac and Bid were clearly proven by overexpression and siRNA knockdown, respectively. The way of Bax activation by BMS-345541 was unraveled by establishing new assays for Bax activation. These showed reduction of the inactivating Bax phosphorylation at serine-184, while the activating Bax phosphorylation at threonine-167 was enhanced. Thus, modulation of Bax phosphorylation appeared as tightly related to TRAIL sensitivity/resistance in melanoma cells, and therapeutic strategies may be considered.

Is there a benefit to a routine preoperative screening of infectivity for HIV, hepatitis B and C virus before elective orthopaedic operations?

PURPOSE: Before elective operations, particularly orthopaedic surgery, national guidelines in Germany recommend testing for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) to reduce the risk of transmission of the virus through a needlestick or cutting injury. Such testing is expensive. The number of new and unknown diagnoses of viral infections that can be detected by routine screening has not yet been evaluated. METHODS: The aim of our department of orthopaedic surgery is to screen every adult patient listed for an operation for HBV, HCV and HIV. We retrospectively analysed the number of operations in this single centre from 2001 to 2010, correlated this number with the total number of screens and calculated the number of newly diagnosed infections. An additional cost:benefit ratio was calculated. RESULTS: A total of 20,869 operations were performed by the department between 2001 and 2010. After exclusion of all interventions in children and all patients who had multiple operations, 15,482 patients remained. Test results were found for 10,011 of these patients during this period (screening rate 65 %). Of those screened, in only four cases (0.4 ‰) was a previously unknown infection detected. CONCLUSIONS: Two-thirds of the patients included in our study actually underwent screening; this rate was lower than expected. The incidence of newly detected infections was low, putting the benefit of a routine preoperative screening for HBV, HCV and HIV into question. From an economic point of view the low detection rate is a strong argument in favour of omitting routine preoperative screening. Screening only those patients with risk factors may be as safe as screening every patient and would help reduce costs.

Disruption of the VDAC2-Bak interaction by Bcl-x(S) mediates efficient induction of apoptosis in melanoma cells.

The proapoptotic B-cell lymphoma (Bcl)-2 protein Bcl-x(S) encloses the Bcl-2 homology (BH) domains BH3 and BH4 and triggers apoptosis via the multidomain protein Bak, however, the mechanism remained elusive. For investigating Bcl-x(S) efficacy and pathways, an adenoviral vector was constructed with its cDNA under tetracycline-off control. Bcl-x(S) overexpression resulted in efficient apoptosis induction and caspase activation in melanoma cells. Indicative of mitochondrial apoptosis pathways, Bcl-x(S) translocated to the mitochondria, disrupted the mitochondrial membrane potential and induced release of cytochrome c, apoptosis-inducing factor and second mitochondria-derived activator of caspases. In melanoma cells, Bcl-x(S) resulted in significant Bak activation, and Bak knockdown as well as Bcl-x(L) overexpression abrogated Bcl-x(S)-induced apoptosis, whereas Mcl-1 (myeloid cell leukemia-1) knockdown resulted in a sensitization. With regard to the particular role of voltage-dependent anion channel 2 (VDAC2) for inhibition of Bak, we identified here a notable interaction between Bcl-x(S) and VDAC2 in melanoma cells, which was proven in reciprocal coimmunoprecipitation analyses. On the other hand, Bcl-x(S) showed no direct interaction with Bak, and its binding to VDAC2 appeared as also independent of Bak expression. Suggesting a new proapoptotic mechanism, Bcl-x(S) overexpression resulted in disruption of the VDAC2-Bak interaction leading to release of Bak. Further supporting this pathway, overexpression of VDAC2 strongly decreased apoptosis by Bcl-x(S). New proapoptotic pathways are of principle interest for overcoming apoptosis deficiency of melanoma cells.

Gastrointestinal chemosensation: chemosensory cells in the alimentary tract.

Sensing potentially beneficial or harmful constituents in the luminal content by specialized cells in the gastrointestinal mucosa is an essential prerequisite for governing digestive processes, initiating protective responses and regulating food intake. Until recently, it was poorly understood how the gastrointestinal tract senses and responds to nutrients and non-nutrients in the diet; however, the enormous progress in unraveling the molecular machinery underlying the responsiveness of gustatory cells in the lingual taste buds to these compounds has been an important starting point for studying intestinal chemosensation. Currently, the field of nutrient sensing in the gastrointestinal tract is evolving rapidly and is benefiting from the deorphanization of previously unliganded G-protein-coupled receptors which respond to important nutrients, such as protein degradation products and free fatty acids as well as from the FACS-assisted isolation of distinct cell populations. This review focuses on mechanisms and principles underlying the chemosensory responsiveness of the alimentary tract. It describes the cell types which might potentially contribute to chemosensation within the gut: cells that can operate as specialized sensors and transducers for luminal factors and which communicate information from the gut lumen by releasing paracrine or endocrine acting messenger molecules. Furthermore, it addresses the current knowledge regarding the expression and localization of molecular elements that may be part of the chemosensory machinery which render some of the mucosal cells responsive to constituents of the luminal content, concentrating on candidate receptors and transporters for sensing nutrients.

Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome.

Despite recent therapeutic improvements, the prognosis for patients suffering from Sézary syndrome (SS), a disseminated form of cutaneous T-cell lymphomas, is still poor. We identified bi- and monoallelic deletions of the tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) in a high proportion of SS patients as well as biallelic A20 deletion in the SS-derived cell line SeAx. Furthermore, we demonstrate that inhibition of A20 activates the NF-κB pathway thereby increasing the proliferation of normal T lymphocytes. On the other hand, the reconstitution of A20 expression slowed down the cell cycle in SeAx cells. Recently A20 inactivation has been reported in various B-cell lymphomas. In this study, we show that A20 is also a putative tumor suppressor in the T-cell malignancy-SS.

[cFLIP diminishes CD95-induced apoptosis of CD30-stimulated cutaneous anaplastic large T cell lymphoma (cALCL) cells]. / Kutanes anaplastisches großzelliges Lymphom. Apoptoseresistenz durch CD30-induzierte cFLIP-Expression.

Stimulation of the TNF receptors CD30 and CD95 exerts opposite effects. Crosstalk of both receptors is unknown. We aimed to reveal regulatory mechanisms of CD30-induced effects on CD95 signaling of cALCL cell lines. "CD30/CD95 crosstalk analysis" was performed in cALCL cell lines by comparison of CD30 or CD95 stimulation and CD30/CD95 costimulation. Receptor expression and induction of apoptosis was investigated by flow cytometry. mRNA expression of CD30-inducible genes (cFLIP, TRAF1, cIAP2, and A20) was compared by semiquantitative reverse transcription (RT-RQ-) PCR in stimulated and unstimulated cells. Protein expression of IκBα, p100/p52, caspase-8, caspase-3, and cFLIP was analyzed by immunoblotting. A lentiviral-based shRNA-mediated approach was used to inhibit cFLIP expression. CD30/CD95 crosstalk experiments revealed that CD30 ligation leads to NFκB-mediated cFLIP upregulation in cALCL cells, which in turn enhanced resistance to CD95-mediated apoptosis. This effect is based on the CD30-induced upregulation of cFLIP. Knockdown of cFLIP restores sensitivity to CD95-mediated apoptosis. We conclude that the anti-apoptotic function of CD30 antibodies should be kept in mind if CD30 antibody-based therapeutic concepts for ALCL lymphoma are considered.

Influence of antiretroviral therapy on immunogenicity of simultaneous vaccinations against influenza, pneumococcal disease and hepatitis A and B in human immunodeficiency virus positive individuals.

OBJECTIVES: Immune response to many vaccinations is impaired in human immunodeficiency virus (HIV) positive patients. METHODS: A total of n = 131 HIV positive patients were vaccinated against influenza, pneumococcal disease, hepatitis A and B, with n = 82 patients (62.6%) receiving 2 or more simultaneous vaccinations. Safety and immunogenicity of simultaneous vaccinations were assessed. Current antiretroviral therapy (ART) regimens were evaluated as potential predictors for antibody response. RESULTS: Immune response rates were 45% (influenza), 68% (pneumococcus), 63.6% (hepatitis A) and 62.5% (hepatitis B). Adverse reactions after vaccination were documented in 2 of 131 patients (1.5%). No statistically significant difference between pre- and post-vaccination CD4+ T-cell counts (CD4) and HIV plasma load was observed. 85% of patients received ART containing nucleotide reverse transcriptase inhibitors, non-nucleotide reverse transcriptase inhibitors and/or protease inhibitors (PI). Higher ratio of CD4 to CD8 and intake of PI were statistically significant, independent predictors for antibody response after influenza vaccination (OR 1.9 and 2.8, p = 0.01 and 0.04, respectively). CONCLUSIONS: Simultaneous vaccinations in HIV positive patients were safe and well tolerated. The positive effect of PI on antibody response after influenza vaccination should be confirmed in larger studies.

Prevention of non-melanoma skin cancer in organ transplant patients by regular use of a sunscreen: a 24 months, prospective, case-control study.

BACKGROUND: Skin cancers represent a major challenge within the ever growing group of long time surviving organ transplant recipients (OTR) world wide. Especially UV-induced non-melanoma skin cancers (NMSC) like invasive squamous cell carcinomas (SCC) and actinic keratoses (AK), and basal cell carcinoma (BCC), outnumber every other form of cancer in organ transplant recipients. Despite encouraging reports of protective effects of broad-spectrum sunscreens in immunocompetent patients, evidence for the prevention of NMSC in immunocompromised patients is still missing. OBJECTIVES: To assess preventive effects of regular sun-screen use on AK, SCC and BCC in chronically immunocompromised organ transplant recipients. METHODS: Hundred and twenty matched (age, sex, skin type, graft, transplant duration, previous post-transplant skin malignancies) organ transplant recipients (40 heart, 40 kidney, 40 liver grafted) were recruited for this prospective, single-center study. Both groups received equally written and oral information on sun protection measures. Sixty patients were provided with a free broad spectrum study-sunscreen (SPF>50, high-UVA absorption) for daily application of 2 mg cm(-2) to the head, neck, forearms, and hands. RESULTS: All 120 patients completed the 24 months study. Within this 24 month study interval 42 of the 120 patients developed 82 new AK (-102 sunscreen group vs. +82 control; P<0.01), 8 new invasive SCC (0 vs. 8; P<0.01) and 11 BCC (2 vs. 9; ns). In spite of equal numbers of AK at baseline, a marked difference in favor of the intent-to-treat sunscreen group was recorded after 24 months (89 vs. 273; P<0.01, mean difference 3.07 [1.76-4.36]) and the lesion count was significantly lower as compared to the initial visit (89 vs. 191; P<0.01, mean difference 1.7 [0.68-2.72]). With an average of 5.6 applications per week throughout the 24 months the study sunscreen was generally well tolerated. Serum 25-hydroxy vitamin D levels as marker for vitamin D status were decreased in all patients without adequate substitution and 25(OH)D was found to be lower in the sunscreen-group as compared to the control group (mean value 53 ng mL(-1) vs. 60 ng mL(-1)). INTERPRETATION: Regular use of sunscreens, as part of a consequent UV-protection strategy, may prevent the development of further AK and invasive SCC and, to a lesser degree, BCC in immune-compromised organ transplant recipients.

UV-induced squamous cell carcinoma--a role for antiapoptotic signalling pathways.

The incidence of nonmelanoma skin cancer including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) has dramatically increased in the last decades, and chronic sun exposure was identified as a main etiologic agent. UV radiation may produce DNA damage either directly or through reactive oxygen species (ROS). As mutations caused by UV may lead to skin cancer due to oncogene activation and tumor suppressor gene inactivation, efficient safeguard mechanisms have been developed during evolution. These enclose induction of apoptosis and formation sunburn cells aiming at the removal of premalignant cells. The keratinocyte apoptotic machinery in response to UV consists of both intrinsic/mitochondrial and extrinsic/death receptor-mediated cell-death pathways, which are particularly regulated by mitogen-activated protein kinases (MAPKs, JNK and p38) and the tumor-suppressor protein p53. For development of skin cancer, it appears that critical steps in apoptosis control are dysregulated leading to resistance both to death ligand-mediated and intrinsic proapoptotic pathways. These particularly include inactivation of p53, as well as activation of EGFR, COX-2 and MAPKs, which result in specific regulation of Bcl-2 proteins, death ligands and death receptors. The final unravelling of apoptosis regulation in epithelial skin cancer may allow the development of new targeted therapeutic strategies.

Actinic keratosis is an early in situ squamous cell carcinoma: a proposal for reclassification.

The term actinic keratosis (AK) describes a sun-induced, clinical erythematous lesion covered with scale, but does not provide an understanding of the biology or histopathology of the lesion. Consequently, several classification systems for AK have been suggested, but as yet no consensus has been reached. These systems strive to correlate the pathological and clinical features to better provide physicians with the most accurate information to enable correct decisions to be made regarding treatments, Prognosis and metastatic potential. AK is a clinical description that has a histological diagnosis consistent with squamous cell carcinoma (SCC) in situ. We recommend an AK classification system that describes these lesions as squamous cell carcinomas (SCCs), using the terminology 'early in situ SCC Type AK I', 'early in situ SCC type AK II' and 'in situ SCC Type AK III', there by giving clinicians better guidance for diagnosis and specific treatment recommendations.

Apoptosis pathways as promising targets for skin cancer therapy.

Apoptosis pathways provide efficient safeguard mechanisms against cancer that are mediated via cell-intrinsic responses and immune-mediated extrinsic signals. Intrinsic pro-apoptotic pathways are largely controlled by p53 and Bcl-2 proteins, whereas the extrinsic induction of apoptosis is initiated by death ligands, such as tumour necrosis factor-alpha (TNF-alpha), CD95L/FasL and TNF-related apoptosis-inducing ligand (TRAIL), or by granzyme B. Initiation of these pathways results in the induction of a caspase cascade leading to cell death. The inactivation of pro-apoptotic pathways is elementary for tumourigenesis and may be responsible for therapy resistance. Thus, apoptosis-based strategies represent important tools for the development of effective tumour therapies. The aim of these therapies is to restore p53 activity, downregulate anti-apoptotic Bcl-2 proteins or NF-kappaB activity, and to upregulate extrinsic, death receptor-mediated pathways. The initial results of apoptosis-based strategies are proving promising. Also, topical treatments for actinic keratosis (AK), such as cyclo-oxygenase-2 inhibitors (e.g. diclofenac 3% gel), have been shown to trigger pro-apoptotic pathways. There is hope that pro-apoptotic strategies will lead to pronounced therapeutic success against skin cancer. Importantly, the involvement of the different pro-apoptotic pathways in specific tumour types needs to be unravelled and understood in order to evaluate drug effectiveness, as well as to modify and optimise therapeutic approaches.

The role of apoptosis in therapy and prophylaxis of epithelial tumours by nonsteroidal anti-inflammatory drugs (NSAIDs).

In addition to having anti-inflammatory activities, nonsteroidal anti-inflammatory drugs (NSAIDs) also inhibit neoplastic cell proliferation by inducing apoptosis. Diclofenac is the anti-neoplastic compound in diclofenac 3% gel (Solaraze) used for topical treatment of actinic keratosis (AK). Main target of NSAIDs seems to be the inhibition of cyclo-oxygenase-2 (COX-2), which is overexpressed in several epithelial tumours and catalyses the synthesis of prostaglandins. The precise mechanism of action of diclofenac in cutaneous cells is still unclear, but induction of apoptosis is a key effect of anti-neoplastic drugs, including NSAIDs. In this paper we give an overview of the anti-tumoural activities of NSAIDs with emphasis on induction of apoptosis. Cyclo-oxygenase-2-mediated synthesis of prostaglandin E(2) (PGE(2)) leads to activation of mitogen-activated protein kinase (MAPK), as well as phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Induction of the anti-apoptotic Bcl-2 and Mcl-1, as well as activation of the caspase-8 inhibitor cFLIP have been reported. In addition, altered lipid concentrations in the cytoplasmic membrane may modulate death receptor activities. Downregulation of both the intrinsic mitochondrial and the extrinsic pathways have been reported. Our data demonstrate induced apoptosis and activation of the caspase cascade in three of four cutaneous squamous cell carcinoma (SCC) cell lines, after treatment with diclofenac plus hyaluronic acid and diclofenac alone; one cell line remained nonresponsive. The effects were less pronounced in normal keratinocytes and cytotoxic effects were not seen. Detailed analysis of apoptosis pathways employed by diclofenac in these cells may help to improve therapeutic strategies and to overcome possible mechanisms that are involved in nonresponsiveness.