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Epidermolysis bullosa (EB) is an inherited, heterogeneous group of rare genetic dermatoses characterized by mucocutaneous fragility and blister formation, inducible by often minimal trauma. A broad phenotypic spectrum has been described, with potentially severe extracutaneous manifestations, morbidity and mortality. Over 30 subtypes are recognized, grouped into four major categories, based predominantly on the plane of cleavage within the skin and reflecting the underlying molecular abnormality: EB simplex, junctional EB, dystrophic EB and Kindler EB. The study of EB has led to seminal advances in our understanding of cutaneous biology. To date, pathogenetic mutations in 16 distinct genes have been implicated in EB, encoding proteins influencing cellular integrity and adhesion. Precise diagnosis is reliant on correlating clinical, electron microscopic and immunohistological features with mutational analyses. In the absence of curative treatment, multidisciplinary care is targeted towards minimizing the risk of blister formation, wound care, symptom relief and specific complications, the most feared of which - and also the leading cause of mortality - is squamous cell carcinoma. Preclinical advances in cell-based, protein replacement and gene therapies are paving the way for clinical successes with gene correction, raising hopes amongst patients and clinicians worldwide.

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

Expression of the integrin αvβ6 is upregulated in a variety of carcinomas where it appears to be involved in malignant progression, although the biology of this integrin is not fully explored. We have generated oral carcinoma cells that express αvβ6 composed of wild-type αv and a mutant β6 that lacks the unique C-terminal 11 amino acids (aa). We found that these residues, although not required for αvβ6-dependent adhesion or migration, are essential for αvβ6-dependent invasive activity. We have used a proteomic approach to identify novel binding partners for the β6 subunit cytoplasmic tail and report that psoriasin (Psor) (S100A7) bound preferentially to the recombinant β6 cytoplasmic domain, though not in the absence of the unique C-terminal 11aa. Endogenous cellular Psor co-precipitated with endogenous β6 and colocalised with αvβ6 at the cell membrane and intracellular vesicles. Knockdown of Psor, with small interfering RNA, had no effect on αvβ6-dependent adhesion or migration but abrogated αvβ6-mediated oral carcinoma cell invasion both in Transwell and, the more physiologically relevant, organotypic invasion assays, recapitulating the behaviour of the β6-mutant cell line. Membrane-permeant Tat-peptides encoding the unique C-terminal residues of β6, bound directly to recombinant Psor and inhibited cellular Psor binding to β6; this blocked αvβ6-dependent, but not αvβ6-independent, invasion. These data identify a novel interaction between Psor and β6 and demonstrate that it is required for αvβ6-dependent invasion by carcinoma cells. Inhibition of this interaction may represent a novel therapeutic strategy to target carcinoma invasion.

Exposure to drug-paired cues can trigger addicts to relapse into drug seeking. Although the molecular mechanisms underlying cue-elicited cocaine seeking are incompletely understood, the protein kinase extracellular signal-regulated kinase (ERK) is known to have an important role. Psychostimulants and their associated cues can activate ERK in medium spiny neurons of the nucleus accumbens core (AcbC). These medium spiny neurons can be classified according to their projections (to ventral pallidum and/or substantia nigra) and by their mRNA expression. The present experiments were designed to determine which distinct set of AcbC projection neurons expresses phosphorylated ERK (pERK) in response to cocaine-paired contextual cues. Combined use of the retrograde label Flurogold with immunohistochemical staining of pERK was used to show that the AcbC pERK accompanying preference for cocaine-paired contexts occurs in both the accumbens (Acb)-nigral and Acb-pallidal projections. The gene expression characteristics of the neurons expressing pERK in response to cocaine-paired cues was further investigated using combined in situ hybridization and immunocytochemistry to show that AcbC pERK+ cells correspond to D1, but not preproenkephalin, mRNA+ cells. Furthermore, intra-AcbC infusion of the D1-antagonist SCH23390 attenuated cue-induced AcbC pERK expression. In aggregate, these results indicate that (i) the D1-expressing AcbC neurons evidence long-term plasticity related to drug-cue memories and (ii) local dopamine D1 receptors are necessary for the expression of cocaine-paired cue-induced pERK in these AcbC neurons.

A growing body of evidence indicates that protracted use of methamphetamine (mAMPH) causes long-term impairments in cognitive function in humans. Aside from the widely reported problems with attention, mAMPH users exhibit learning and memory deficits, particularly on tasks requiring response control. Although binge mAMPH administration to animals results in cognitive deficits, few studies have attempted to test behavioral flexibility in animals after mAMPH exposure. The aim of this study was to evaluate whether mAMPH would produce impairments in two tasks assessing flexible responding in rats: a touchscreen-based discrimination-reversal learning task and an attentional set shift task (ASST) based on a hallmark test of executive function in humans, the Wisconsin Card Sort. We treated male Long-Evans rats with a regimen of four injections of 2 mg/kg mAMPH (or vehicle) within a single day, a dosing regimen shown earlier to produce object recognition impairments. We then tested them on (1) reversal learning after pretreatment discrimination learning or (2) the ASST. Early reversal learning accuracy was impaired in mAMPH-treated rats. MAMPH pretreatment also selectively impaired reversal performance during ASST testing, leaving set-shifting performance intact. Postmortem analysis of [(125)I]RTI-55 binding revealed small (10-20%) but significant reductions in striatal dopamine transporters produced by this mAMPH regimen. Together, these results lend new information to the growing field documenting impaired cognition after mAMPH exposure, and constitute a rat model of the widely reported decision-making deficits resulting from mAMPH abuse seen in humans.

Methamphetamine (mAMPH) is an addictive drug that produces memory and recall impairments in humans. Animals subjected to a binge mAMPH dosing regimen that damages brain dopamine and serotonin terminals show impairments in an object recognition (OR) task. Earlier research demonstrated that preceding a single-day mAMPH binge regimen with several days of increasing mAMPH doses greatly attenuates its neurotoxicity in rats. The escalating dose (ED) paradigm appears to mimic the human pattern of escalating drug intake. The current aim was to test whether an ED plus binge mAMPH regimen produces OR impairments. In addition to its translational value, this experiment helps address whether monoaminergic neurotoxicity accounts for OR impairments seen after mAMPH administration. To further address this issue, a separate experiment investigated both OR impairments and monoamine transporter integrity in groups of rats treated with a range of mAMPH doses during a single day. An ED mAMPH regimen attenuated the acute hyperthermic response to the subsequent mAMPH binge and prevented the OR impairments and reductions in [125 I]RTI-55 binding to monoamine transporters in striatum, hippocampus (HC), and perirhinal cortex (pRh) that otherwise occur 1 week after the mAMPH binge. Single-day mAMPH regimens (4 x 1mg/kg to 4 x 4 mg/kg, s.c.) dose-dependently produced acute hyperthermia and, 1 week post-mAMPH, produced dose-dependent impairments in OR and reductions in monoamine transporter binding. The OR impairments of single-day mAMPH-treated rats correlated with monoaminergic transporter loss in ventral caudate-putamen, HC, and pRh. In aggregate, these findings suggest a correspondence between mAMPH-induced monoaminergic injury and the resulting OR deficits.

Repeated moderate doses of methamphetamine (mAMPH) damage forebrain monoaminergic terminals and nonmonoaminergic cells in somatosensory cortex, and impair performance in a novelty preference task of object recognition (OR). This study aimed to determine whether the memory deficit seen after a neurotoxic mAMPH regimen results from damage to dopamine (DA) and/or serotonin (5-HT) terminals. Animals were given a neurotoxic regimen of mAMPH, p-chloroamphetamine (PCA, preferentially damages 5-HT terminals), d-amphetamine (d-AMPH, preferentially damages DA terminals), or saline. After 1 week, animals were trained and tested for OR memory. Rats treated with mAMPH showed no recognition memory during the short-term memory (STM) test, whereas both PCA- and d-AMPH-treated rats showed OR STM scores comparable to controls. After behavioral testing, the specificity of monoaminergic lesions was determined by postmortem [125I]RTI-55 binding to dopamine (DAT) and serotonin (SERT) transporter proteins. Tissue from a separate group of animals killed 3 days after drug treatment was processed for Fluoro-Jade (F-J) fluorescence histochemistry to detect damaged cortical neurons. mAMPH-treated rats showed reductions in striatal DAT and hippocampal (HC) and perirhinal (pRh) SERT, as well as degeneration of neurons in primary somatosensory cortex. In PCA-treated rats, HC and pRh SERT were substantially depleted, but striatal DAT and cortical neuron survival were unaffected. By contrast, d-AMPH-treated animals showed marked depletions in striatal DAT and cortical neurodegeneration, but HC and pRh SERT were unaffected. This pattern of results indicates that no single feature of mAMPH-induced neurotoxicity is sufficient to produce the OR impairments seen after mAMPH treatment.

Mice deficient in TNF-alpha (TNF-alpha(-/-) mice) are resistant to skin carcinogenesis and expression of MMP-9 is inhibited in TNF-alpha(-/-) mice during skin tumour development. In the early stages of tumour promotion, MMP-9 protein initially localized to the follicular epidermis but subsequently began to accumulate in the interfollicular epidermis of wild-type but not TNF-alpha(-/-) mice. Inhibition of TNF-alpha or MMP-9 function reduced keratinocyte migration in vitro. In addition, a deficiency of TNF-alpha delayed re-epithelialization in vivo and this correlated with reduced MMP-9 expression. Collectively, these data suggest that MMP-9 regulates keratinocyte migration in a TNF-alpha-dependent manner. Expression profiling of genes that control cell adhesion and migration revealed markedly lower levels of the integrin subunits alphav and beta6 in TNF-alpha(-/-) compared with wild-type keratinocytes in vitro. alphavbeta6 expression was upregulated by keratinocytes in vitro and during tumour promotion in vivo in a TNF-alpha-dependent manner. Furthermore, alphavbeta6 blockade significantly inhibited keratinocyte migration and TNF-alpha-stimulated MMP-9 expression in vitro. These data illustrate a novel TNF-alpha-dependent mechanism for the control of alphavbeta6 expression and suggest one pathway for TNF-alpha regulation of MMP-9. Increased MMP-9 and alphavbeta6 expression may stimulate epithelial cell migration during tumour formation and may be one mechanism whereby TNF-alpha acts as an endogenous tumour promoter.

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100-200 ng, from this microdissected tissue required use of the Atlas SMART trade mark Probe Amplification Kit to synthesize and amplify cDNA and make (33)P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.

Twelve patients with schizophrenia received positron emission tomography scans with 18F-deoxyglucose before and after 4 to 6 weeks of treatment with clozapine or thiothixene. Both stereotaxic and magnetic resonance image template methods were used to position regions of interest for metabolic rate analysis. Clozapine increased and thiothixene decreased metabolic rates in the basal ganglia; these effects were most marked on the right side. Within the basal ganglia, a superior to inferior gradient in drug effect was found for thiothixene but not clozapine. This gradient resembled in some respects observations on regional differences in D2 receptors in human autoradiography. Baseline metabolic rates also predicted clinical medication response, with right inferior caudate metabolic rates differentiating clozapine and thiothixene responders. Larger sample studies are needed to replicate and extend these initial findings.