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We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.

Background and objective The objective of this study was to enumerate total cells and the number of inflammatory cell differentials in large airways (LAs) versus small airways (SAs) of mild-moderate COPD, and against appropriate controls. Methods For LA, we used endobronchial biopsies and for SA resected lung tissues. Immunostaining was enumerated (cells per mm2) for macrophages, neutrophils, CD4 and CD8 T cells in the lamina propria (LP) up to 150 µM deep for LA and full wall thickness for SA. Results We confirmed hypocellularity in the LA and in the SA wall in smokers and COPD (P

Background and objective COPD is characterized by poorly reversible airflow obstruction usually due to cigarette smoking. Transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of COPD, and in particular a process called epithelial mesenchymal transition (EMT), which may well be an intermediatory between smoking and both airway fibrosis and lung cancer. The downstream classical or ‘canonical’ TGF-β1 pathway is via the phosphorylated (p) Smad transcription factor system. Methods We have investigated TGF-β1 expression and its ‘pSmad fingerprint’ in bronchoscopic airway biopsies from patients with COPD, and in smoking and non-smoking controls. A cross-sectional immunohistochemical study compared TGF-β1 and pSmad 2, 3 (excitatory) and 7 (inhibitory) expression in cells and blood vessels of three compartments of large airways: epithelium (especially the basal region), reticular basement membrane (Rbm) and underlying lamina propria (LP). Results TGF-β1 expression was generally higher in COPD subjects throughout the airway wall (P

Background and objective PAFr is a cell adhesion site for specific bacteria, notably non-typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. We previously published that PAFr expression is significantly upregulated in the large airways of smokers, especially in COPD. We have now investigated PAFr expression in the epithelium and Rbm of small airways and in the alveolar compartment in smokers and patients with both COPD and small airway disease. Methods We evaluated PAFr expression cross-sectionally in resected lung tissue from: eight smokers with normal lung function (NLFS); 10 with smoking-related small airway narrowing only; eight COPD smokers; 10 COPD ex-smokers, and compared these with nine control tissues. Anti-PAFr immunostaining was quantified using computer-aided image analysis. Results Significantly increased PAFr expression in small airway epithelium of all clinical groups was found compared with controls (P

Ultraviolet radiation (UVR) exposure increases malignant melanoma (MM) risk, but in the context of acute, not cumulative exposure. CT and CCTT changes make up the overwhelming majority of single base substitutions (SBS) in MM DNA, as both precursor melanocytes and melanocytic lesions have incurred incidental exposures to sunlight. To study the mutagenic mechanisms by which acute sunburn accelerates MM, we sequenced the exomes of spontaneous and neonatal UVB-induced Cdk4-R24C::Tyr-NRASQ61K mouse MMs. UVR-induced MMs carried more SBSs than spontaneous MMs, but the levels of genomic instability, reflected by translocations and copy number changes, were not different. CT/GA was the most common SBS in spontaneous and UVR-induced MMs, only modestly increased in the latter. However, they tended to occur at the motif A/GpCpG (reflecting CT transition due to spontaneous deamination of cytosine at CpG) in spontaneous MMs, and T/CpCpC/T (reflecting the effects of pyrimidine dimers on either side of the mutated C) in UVR-induced MMs. Unlike MMs associated with repetitive exposures, we observed no CCTT changes. In addition, we also found UVR 'footprints' at TA/ATs (at NpTpT) and TC/AG (at CpTpC). These footprints are also present in MMs from a chronic UVR mouse model, and in some human MMs, suggesting that they may be minor UVR signature changes. We found few significantly somatically mutated genes (~6 per spontaneous and 15 per UVR-induced melanoma) in addition to the Cdk4 and NRAS mutations already present. Trp53 was the most convincing recurrently mutated gene; however, in the UVR-induced MMs no Trp53 mutations were at CT/GA, suggesting that it was probably mutated during tumour progression, not directly induced by UVR photoproducts. The very low load of recurrent mutations convincingly induced by classical UVB-induced dimer photoproducts may support a role for cell extrinsic mechanisms, such as photoimmunosuppression and inflammation in driving MM after acute UVB exposure.

COPD is characterised by poorly reversible airflow obstruction usually due to cigarette smoking. The transcription factor clusters of β-catenin/Snail1/Twist has been implicated in the process of epithelial mesenchymal transition (EMT), an intermediate between smoking and airway fibrosis, and indeed lung cancer. We have investigated expression of these transcription factors and their “cellular localization” in bronchoscopic airway biopsies from patients with COPD, and in smoking and non-smoking controls. An immune-histochemical study compared cellular protein expression of β-catenin, Snail1 and Twist, in these subject groups in 3 large airways compartment: epithelium (basal region), reticular basement membrane (Rbm) and underlying lamina propria (LP). β-catenin and Snail1 expression was generally high in all subjects throughout the airway wall with marked cytoplasmic to nuclear shift in COPD (P

NSCLC is a leading cause of morbidity and mortality worldwide. It includes adeno- and squamous cell carcinoma. In the background, COPD and smoking play a vital role in development of NSCLC. Local progression and metastasis of NSCLC has been associated with various mechanisms, but in particular by a process called epithelial mesenchymal transition (EMT), which is implicated in COPD pathogenesis. In this study, we have investigated whether expression of EGFR (activation marker) and S100A4, vimentin and N-cadherin (as EMT) is different both in central and leading edge of NSCLC and to what extent related to EMT activity of both small and large airways, stage and differentiation of NSCLC. We have investigated EMT biomarkers (S100A4, vimentin, and N-cadherin), an epithelial activation marker (EGFR) and a vascularity marker (Type-IV collagen) in surgically resected tissue from patients with NSCLC (adeno- and squamous cell carcinoma), and compared them with expression in the corresponding non-tumorous airways. EGFR, S100A4, vimentin, N-cadherin expression was higher in tumor cells located at the peripheral leading edge of NSCLC when compared with centrally located tumor cells of same subjects (P < 0.01). Type-IV collagen-expressing blood vessels were also more at the leading edge in comparison with central parts of NSCLC. EGFR and S100A4 expression was related to differentiation status (P < 0.05) and TNM stage (P < 0.05) of NSCLC. Moreover, EMT markers in the leading edge were significantly related to airway EMT activity, while peripheral edge vascularity of squamous cell carcinoma only was significantly related to large airway Rbm vascularity (P < 0.05). EGFR- and EMT-related protein expression was markedly high in the peripheral leading edge of NSCLCs and related to tumor characteristics associated with poor prognosis. The relationships between EMT-related tumor biomarker expression and those in the airway epithelium and Rbm provide a background for utility of airway changes in clinical settings.

Susceptibility to most common cancers is likely to involve interaction between multiple low risk genetic variants. Although there has been great progress in identifying such variants, their effect on phenotype and the mechanisms by which they contribute to disease remain largely unknown. We have developed a mouse melanoma model harboring two mutant oncogenes implicated in human melanoma, CDK4(R24C) and NRAS(Q61K). In these mice, tumors arise from benign precursor lesions that are a recognized strong risk factor for this neoplasm in humans. To define molecular events involved in the pathway to melanoma, we have for the first time applied the Collaborative Cross (CC) to cancer research. The CC is a powerful resource designed to expedite discovery of genes for complex traits. We characterized melanoma genesis in more than 50 CC strains and observed tremendous variation in all traits, including nevus and melanoma age of onset and multiplicity, anatomical site predilection, time for conversion of nevi to melanoma and metastases. Intriguingly, neonatal ultraviolet radiation exposure exacerbated nevus and melanoma formation in most, but not all CC strain backgrounds, suggesting that genetic variation within the CC will help explain individual sensitivity to sun exposure, the major environmental skin carcinogen. As genetic variation brings about dramatic phenotypic diversity in a single mouse model, melanoma-related endophenotype comparisons provide us with information about mechanisms of carcinogenesis, such as whether melanoma incidence is dependent upon the density of pre-existing nevus cells. Mouse models have been used to examine the functional role of gene mutations in tumorigenesis. This work represents their next phase of development to study how biological variation greatly influences lesion onset and aggressiveness even in the setting of known somatic driver mutations.

Background ICAM-1 is a major receptor for ~60% of human rhinoviruses, and non-typeable Haemophilus influenzae, two major pathogens in COPD. Increased cell-surface expression of ICAM-1 in response to tobacco smoke exposure has been suggested. We have investigated epithelial ICAM-1 expression in both the large and small airways, and lung parenchyma in smoking-related chronic airflow limitation (CAL) patients. Methods We evaluated epithelial ICAM-1 expression in resected lung tissue: 8 smokers with normal spirometry (NLFS); 29 CAL patients (10 small-airway disease; 9 COPD-smokers; 10 COPD ex-smokers); Controls (NC): 15 normal airway/lung tissues. Immunostaining with anti-ICAM-1 monoclonal antibody was quantified with computerized image analysis. The percent and type of cells expressing ICAM-1 in large and small airway epithelium and parenchyma were enumerated, plus percentage of epithelial goblet and submucosal glands positive for ICAM- 1. Results A major increase in ICAM-1 expression in epithelial cells was found in both large (p

Ultraviolet B (UVB) irradiation of the skin has the benefit of causing the local production of previtamin D3 but also results in cutaneous DNA damage and suppression of the skin immune system (SIS). Strains of mice differ in their ability to be suppressed by UVB irradiation: BALB/c mice are considered "resistant" and C57BL/6 "sensitive". This study evaluated whether vitamin D-replete (D+) and deficient (D-) BALB/c and C57BL/6 mice differed in their cutaneous response to UVB irradiation. Immunosuppression was assessed by measuring the contact hypersensitivity (CHS) response, DNA damage and repair determined by counting thymine dimer positive keratinocyte nuclei, and cutaneous inflammation and epidermal hyperplasia evaluated by light microscopy. The suppression in the CHS response induced by the UVB irradiation was reduced in the D+ C57BL/6 mice compared with the D- C57BL/6 mice. Similarly there was a reduction in DNA damage and promotion of its repair in the D+ C57BL/6 mice compared with the D- C57BL/6 mice. A reduction in inflammation in female D+ C57BL/6 mice compared with D- C57BL/6 females also occurred. In contrast, the suppression in the CHS response, DNA damage and its repair, and inflammation induced by UVB irradiation were similar in the D+ and D- BALB/c mice. These results indicate that dietary vitamin D3 can reduce UVB-induced suppression of the CHS response depending on the genetic background of the mice, an effect that may relate to the reduction in DNA damage and an increase in its rate of repair.

Introduction: TGF-b 1 has been implicated in the pathogenesis of COPD, through epithelial mesenchymal transition (EMT), which may be an intermediatory process between smoking and both airway fibrosis and lung cancer.The downstream classical TGF-b1 pathway is via the Smad transcription factor system. Methods: An immunohistochemical study was done to compare TGF-β1 and Smad 2, 3 (excitatory) and 7 (inhibitory) expression in cells and blood vessels of 3 major compartments of large airway biopsies: the basal epithelium, reticular basement membrane (Rbm) and lamina propria (LP),from patients with COPD, and in smoking and non-smoking controls. Results: TGF-b1 expression was higher generally in COPD subjects throughout the airway, (P 1 expression, was related to airflow obstruction and EMT biomarker (S100 A4) expression. Conclusion: The TGF-b1-Smad 2/3 pathway is up-regulated in smokers and COPD patients, and is linked to EMT activity and loss of lung function. Reduce in expression of inhibitory Smad-7 is also seen in COPD, and may also contribute to initiation of EMT. This study facilitates the understanding of EMT-associated pathogenesis of COPD as a result of smoking-induced intracellular downstream Smad transcriptional pathways.

Deregulation of p16INK4A is a critical event in melanoma susceptibility and progression. It is generally assumed that the major effect of loss of p16 function is mediated through the CDK-cyclin pathway via its influence on the pocket protein (PP) pRb. However, there are also two other PPs, p107 and p130, which, when phosphorylated by CDK-cyclin complexes, play a role in permitting cell progression. Cohorts of mice carrying melanocyte-specific knockouts (KOs) of various combinations of the three PPs were generated. Mice null for pRb, p107, p130 or any combination of double mutants did not develop melanoma. Surprisingly, melanocyte-specific loss of all three PPs facilitated melanoma development (median age of onset 308 days, penetrance 40% at 1 year). Tumorigenesis was exacerbated by Trp53 co-deletion (median age of onset 275 days, penetrance 82% at 1 year), with cell culture studies indicating that this difference may result from the apoptotic role of Trp53. Melanomas in PP;Trp53-deficient mice lacked either Ras or Braf mutations, and hence developed in the absence of constitutive MAPK pathway activation. The lag period between induction of total PP or PP/Trp53 KO and melanoma development indicates that additional genetic or epigenetic alterations may account for neoplastic progression. However, exome sequencing of PP;Trp53 KO melanomas failed to reveal any additional recurrent driver mutations. Analysis of the putative mutation signature of the PP;Trp53 KO melanomas suggests that melanocytes are primed for transformation via a mutagenic mechanism involving an excess of T>G substitutions, but not involving a preponderance of C>T substitutions at CpG sites, which is the case for most spontaneous cancers not driven by a specific carcinogen. In sum, deregulation of all three PPs appears central to neoplastic progression for melanoma, and the customary reference to the p16INKA/CDK4/pRB pathway may no longer be accurate; all PPs are potentially critical targets of CDK-cyclins in melanoma.

Soltani A, Muller H K, Sohal S S, Reid D W, Weston S, Wood-Baker R & Walters E H (2012) Histopathology 60, 964–970 Distinctive characteristics of bronchial reticular basement membrane and vessel remodelling in chronic obstructive pulmonary disease (COPD) and in asthma: they are not the same disease Aims: This study compared reticular basement membrane (Rbm) and vascular remodelling within the bronchial mucosa of subjects with chronic obstructive pulmonary disease (COPD) with those from patients with asthma, to test the ‘Dutch hypothesis’ of whether these are essentially the same or different pathological conditions. Methods and results: Bronchoscopic biopsies were stained with anti-collagen IV antibody; 18 current smoking COPD, 10 symptomatic asthmatics and 13 healthy non-smoking controls were studied. The Rbm in COPD was fragmented, non-homogeneous, variable in thickness and hypervascular, whereas in asthma the Rbm was compact and homogeneous with no evidence of increased vascularity compared to controls. Length of Rbm splitting presented as percentage of Rbm length was used to measure fragmentation; it was greater in COPD than in controls and asthmatics [median (range) 20.7% (0.4–68.5) versus 5.3% (0.0–21.7) versus 1.5% (0.0–15.1), P

Malik Quasir Mahmood,1,* Sukhwinder Singh Sohal,1,2,* Shakti Dhar Shukla,1 Chris Ward,3 Ashutosh Hardikar,4 Wan Danial Noor,1 Hans Konrad Muller,1 Darryl AKnight,5 Eugene Haydn Walters1 1NHMRC Centre of Research Excellence for Chronic Respiratory Disease and Lung Ageing, School of Medicine, University of Tasmania, Hobart, TAS, Australia; 2School of Health Sciences, Faculty of Health, University of Tasmania, Launceston, TAS, Australia; 3Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK; 4Royal Hobart Hospital, Hobart, TAS, Australia; 5School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia *These authors contributed equally to this work Background: Small airway fibrosis is the main contributor in airflow obstruction in chronic obstructive pulmonary disease. Epithelial mesenchymal transition (EMT) has been implicated in this process, and in large airways, is associated with angiogenesis, ie, Type-3, which is classically promalignant. Objective: In this study we have investigated whether EMT biomarkers are expressed in small airways compared to large airways in subjects with chronic airflow limitation (CAL) and what type of EMT is present on the basis of vascularity. Methods: We evaluated epithelial activation, reticular basement membrane fragmentation (core structural EMT marker) and EMT-related mesenchymal biomarkers in small and large airways from resected lung tissue from 18 lung cancer patients with CAL and 9 normal controls. Tissues were immunostained for epidermal growth factor receptor (EGFR; epithelial activation marker), vimentin (mesenchymal marker), and S100A4 (fibroblast epitope). Type-IV collagen was stained to demonstrate vessels. Results: There was increased expression of EMT-related markers in CAL small airways compared to controls: EGFR (P

SUMMARYCutaneous tolerance to antigens may be induced in mice through application of antigen during the first few days following birth. The mechanism governing this neonatally induced tolerance remains uncertain. We employed a contact hypersensitivity model to analyse dendritic cell (DC) function and the expression of classical and non-classical lymphocyte populations within the neonate. Examination of draining lymph node DC after antigenic challenge of the skin revealed these DC to be significantly deficient in their ability to stimulate antigen-specific T cell proliferation. Co-stimulatory molecule (CD40, CD80 and CD86) expression of these cells was deficient in comparison to adult DC, and functional tests revealed these cells to possess a critical absence of CD40 signalling. A numerical analysis of classical and non-classical lymphocyte expression demonstrated that while the neonatal spleen is devoid of T cells, the lymph nodes have a normal repertoire of T, B, γδ and CD4+CD25+ lymphocytes but an increased expression of natural killer (NK) cells. This study indicates that functionally deficient DC are likely contributors to neonatally induced cutaneous tolerance.

Shakti Dhar Shukla,1,* Sukhwinder Singh Sohal,1,* Malik Quasir Mahmood,1 David Reid,2 Hans Konrad Muller,1 Eugene Haydn Walters1 1NHMRC Centre for Research Excellence for Chronic Respiratory Disease and Lung Ageing, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia; 2Queensland Institute of Medical Research, Iron Metabolism Laboratory, Brisbane, Queensland, Australia *Shakti Dhar Shukla and Sukhwinder Singh Sohal are joint first authors Background: We recently published that platelet-activating factor receptor (PAFr) is upregulated on the epithelium of the proximal airways of current smokers and also in bronchial epithelial cells exposed to cigarette smoke extract. These treated cells also showed upregulation of Streptococcus pneumoniae adhesion. Bacterial wall phosphorylcholine specifically binds to PAFr expressed on airway epithelium, thus facilitating adherence and tissue invasion, which may be relevant to chronic obstructive pulmonary disease (COPD). Moreover, the use of inhaled corticosteroids (ICS) in COPD patients is associated with an increased risk of invasive respiratory pneumococcal infections. Objective: In this study, we have investigated whether PAFr expression is especially upregulated in airway epithelium in COPD patients and whether this expression may be modulated by ICS therapy. Methods: We cross-sectionally evaluated PAFr expression in bronchial biopsies from 15 COPD patients who were current smokers (COPD-smokers) and 12 COPD-ex-smokers, and we compared these to biopsies from 16 smokers with normal lung function. We assessed immunostaining with anti-PAFr monoclonal antibody. We also used material from a previous double-blinded randomized placebo-controlled 6-month ICS intervention study in COPD patients to explore the effect of ICS on PAFr expression. We employed computer-aided image analysis to quantify the percentage of epithelium stained for PAFr. Results: Markedly enhanced expression of PAFr was found in both COPD-smokers (P

Exposure of the skin to environmental stimuli, such as chemical or physical carcinogens, modifies the local skin environment, including depletion of epidermal Langerhans' cells (LC). Any subsequent exposure of the LC-depleted skin to antigen results in the generation of antigen-specific tolerance. In this study we evaluated the antigen-bearing cells in the draining lymph nodes by capitalizing on the fluorescent nature of the contact sensitizer, fluorescein isothiocyanate (FITC). When FITC was applied to the skin of normal mice, two distinct populations of antigen-bearing cells were identified in the draining lymph nodes. They were classified as either FITC(hi) or FITC(lo) on the basis of their fluorescence intensity and thus the amount of antigen they internalized. Only FITC(lo) cells were detected in the lymph nodes draining FITC-treated murine skin that had been depleted of epidermal LC by prior treatment with the complete carcinogen 9,10-dimethyl 1,2-benzanthracene (DMBA). Functional analysis of these cells revealed that the FITC(hi) cells, but not the FITC(lo) cells, induced antigen-specific T- cell proliferation. Further analysis of the FITC(lo) cells from the DMBA- treated mice demonstrated that these cells had reduced levels of CD80 expression, had substantially reduced levels of CD86 expression and performed poorly as costimulator cells in an anti-CD3-mediated proliferative assay. Nonetheless these cells still induced early signs of T-cell activation and interleukin-12 production. Consequently the FITC(lo) cells migrating from the LC-depleted skin, through a combination of reduced antigen presentation and reduced co-stimulatory activity, induced a state of unresponsiveness or anergy in the responder T cells in a similar manner to that observed when antigen presentation occurs in the absence of costimulation. We propose that these unresponsive, or anergic cells, account for the antigen-specific tolerance observed in these experiments.

To assess the role of the p53 tumor suppressor gene in skin carcinogenesis by UV radiation, mice constitutively lacking one or both copies of the functional p53 gene were compared to wild-type mice for their susceptibility to UV carcinogenesis. Heterozygous mice showed greatly increased susceptibility to skin cancer induction, and homozygous p53 knockout mice were even more susceptible. Accelerated tumor development in the heterozygotes was not associated with loss of the remaining wild-type allele of p53, as reported for tumors induced by other carcinogens, but in many cases was associated with UV-induced mutations in p53. Tumors arose on the ears and dorsal skin of mice of all three genotypes, and homozygous knockout mice also developed ocular tumors, mainly melanomas. Skin tumors in the p53 knockout mice were predominately squamous cell carcinomas and were associated with premalignant lesions resembling actinic keratoses, whereas those in the heterozygous and wild-type mice were mainly sarcomas. These results demonstrate the importance of p53 in protecting against UV-induced cancers, particularly in the eye and epidermis.

The expression of HDAC2 is reported as reduced in chronic obstructive pulmonary disease (COPD). We assessed HDAC2 expression within the airways of smokers and subjects with COPD and effects of inhaled corticosteroids (ICS), using immuno-histology to contrast with previous molecular methodology. Endobronchial biopsies (ebb) from current smokers with COPD (COPD-CS; n = 15), ex-smokers with COPD (COPD-ES; n = 17), smokers with normal lung function (NS; n = 16) and normal controls (NC; n = 9) were immunostained for HDAC2. A double-blinded, randomized, placebo-controlled 6 months intervention study assessed effects of ICS on HDAC2 in 34 COPD subjects. There was no difference in epithelial HDAC2 staining in all groups. There was a significant reduction in total cell numbers in the lamina propria (LP) in COPD-CS and NS (p

The ability of the chemical carcinogen dimethylbenz(a)anthracene (DMBA) to deplete Langerhans' cells (LC) from murine skin is crucial to the development of antigen-specific suppression. This depletion is a consequence of the LC recognizing the DMBA as antigenic and migrating to the draining lymph nodes to attempt to elicit T-cell activation. This depletion also occurred following exposure to high doses of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 2,4,6-trinitrochlorobenzene (TNCB) and fluorescein isothiocyanate (FITC). However, LC depletion was not significant at lower doses, even though these doses were sufficient to induce strong contact sensitivity responses. Application of the contact sensitizer, DNFB, through skin depleted of LC (by pretreatment with either the carcinogen DMBA or the antigen TNCB) failed to induce contact sensitivity. This immune non-responsiveness was antigen specific, and could be transferred by spleen cells to naive mice, which were unable to respond to DNFB. Mouse skin treated with doses of TNCB, that did not cause LC depletion but still induced a normal contact hypersensitivity, retained its ability to initiate a normal immune response to DNFB. Together these findings demonstrate that carcinogens share some properties with antigens as they both cause LC depletion and interact with the immune system. Furthermore, it is this LC depletion, rather than carcinogen treatment, that is a critical factor which leaves the skin immunologically compromised and favours the induction of antigen-specific suppression.

Cell-mediated immune responses were studied in 12 Antarctic and sub-Antarctic wintering groups at quarterly intervals over the period 1984-1992, using the cutaneous CMI Multitest. These populations are among the most isolated on earth. While the sub-Antarctic population at Macquarie Island had levels of responsiveness and hypoergy (9%) comparable to healthy populations in temperate zones, the Antarctic Continental group showed a level of hypoergy of 36%. There was no seasonal variation in the pattern of responses. It is concluded that the extreme and isolated environment and stress factors are responsible for the decreased immunological responsiveness but the mechanisms are presently unclear. On review, one factor appears to be perceived anxiety. The high rate of hypoergy in Antarctica, where medical care is limited, may have health implications. These groups provide an excellent analogue for immunological investigations in longer term space flight.

Chemical carcinogens reduce cutaneous immunity, an event accompanied by alterations to the number and morphology of the resident epidermal Langerhans cell (LC) population. This study aimed to examine the functional capacity of LC and other dendritic cells (DC) that are migrating from carcinogen-treated skin via afferent lymphatic vessels. Generation and subsequent cannulation of prefemoral pseudoafferent lymphatic vessels in sheep allowed continuous collection of DC migrating from a defined area of carcinogen-treated skin. The ability of metrizamide-enriched afferent lymph DC to present antigen to autologous primed peripheral blood lymphocytes was used as an indicator of DC function. Topical application of the complete carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene abrogated the stimulatory capacity of migrating DC for periods of 8 weeks and 5 weeks, respectively, whereas the tumor promoter 12-O-tetradecanoylphorbol-13-acetate reduced DC function for less than 1 week. These findings favor tumor development in carcinogen-treated skin being enhanced due to impairment of DC immunological surveillance.

The immune status of 29 members of the Australian National Antarctic Research Expeditions (ANARE) was investigated before, during, and after a 56 day summer voyage to Antarctica and correlated with psychological and physiological parameters. All subjects were healthy. Expedition personnel demonstrated decreased cell mediated immune responses (CMI) assessed by the CMI Multi-test; 21% were hypoergic. The major associated observation was a significant negative correlation with anxiety in Antarctica. However, perceived anxiety was greater before and after the voyage. No significant changes were found in T and B lymphocyte subsets, immunoglobulin and complement components and cutaneous blood flow, nor was there any clinical evidence of illness. Of the hormones examined only cortisol was low predeparture which may reflect increased perceived anxiety at that time. Changes in immune control mechanism were apparent as shown by reduced CMI responses and lowered tetanus antibody levels. Stress factors are postulated to induce depression of the immune response in Antarctica. The association with anxiety suggests that brain peptides or associated cytokines may have a role in mediating these immune events. Such alterations in immune status have implications for health management in isolated and extreme conditions.

Exposure of murine skin to suberythernal doses of ultraviolet-B (UVB) radiation before contact sensitization alters the activity of antigen-presenting cells (APC) in the draining lymph nodes (DLN), decreases the contact hypersensitivity (CHS) response, and induces hapten-specific Ts cells. We determined whether in vivo UVB irradiation alters the ability of hapten-bearing APC from the DLN to form dusters with hapten-specific T lymphocytes. When APC from UV-irradiated, fluorescein isothiocyanate (FITC)-sensitized mice were mixed with a FITC-specific T-cell line, significantly fewer clusters formed compared with FITC+ APC from unirradiated mice. A higher percentage of clusters formed with APC from UV-irradiated mice were Mac-1+ and bound both CD4+ and CD8+ T cells, whereas FITC+ APC from nonirradiated mice bound CD4+ cells. These results suggest that UVB irradiation interferes with the induction of CHS by altering the functional interaction between APC and T cells, perhaps by altering the population of APC in the skin.

OBJECTIVE To explore immune function in patients with chronic post-traumatic stress disorder (PTSD). DESIGN A case-control study using the standardised Cell-mediated Immunity (CMI) Multitest. PARTICIPANTS Cases were 25 Vietnam combat veterans undergoing treatment for clinically diagnosed PTSD. Controls had no diagnosed psychiatric disorders: 28 were civilians and 20 were servicemen who had had South-east Asian postings but no combat experience. MAIN OUTCOME MEASURES The sum score, the number of reactions and the compound scores from the CMI Multitest were assessed as a measure of immune function. RESULTS Psychometric tests distinguished between cases and controls at a highly significant level (P < 0.001). Veterans with PTSD had enhanced cell-mediated immunity compared with civilians (P = 0.008) and servicemen (P = 0.02). CONCLUSION PTSD in combat veterans is associated with enhanced cell-mediated immune responsiveness. This could have wide implications for psychiatry and general medicine.

RATIONALE: Airway remodeling complicates the course of chronic inflammatory airway diseases and angiogenesis is one of its main features. Published data on angiogenesis in bronchial biopsies (BB) from subjects with chronic obstructive pulmonary disease (COPD) is limited, and especially so for effects of medication. METHODS: BB from current-smoker COPD (S-COPD), ex-smoker COPD (ES-COPD) and smokers with normal lung function (S-N) were stained with collagen IV and vascular endothelial growth factor (VEGF) antibodies and compared with healthy non-smokers (H-N). Then COPD subjects entered a double blind controlled trial comparing fluticasone propionate (FP, 0.5 mg/twice daily) with placebo (2:1 randomization) for 6 months and their BB were compared before and after treatment. RESULTS: (Table 1). 66 subjects were recruited in the cross-sectional study and 22 mild to moderate COPD patients had enough pair tissues for the clinical trial (FP, n=15 and placebo, n=7). The intervention groups were well matched at baseline. The Rbm was significantly fragmented in all 3 clinical groups compared with H-N; fragmentation was measured as length of splits (Figure 1 A). The Rbm was also hypervascular (Figure 1 A) but the lamina propria (LP) in contrast was hypovascular in both current smoker groups but not in ES-COPD. Staining for VEGF in the Rbm was significantly greater in the three clinical groups compared with H-N. Rbm splitting regressed significantly with FP (p

SUMMARY Prostaglandins have been implicated in the immune suppression associated with the development of some tumours. Application of the prostaglandin synthetase inhibitor indomethacin, to murinc skin prior to treatment with the chemical carcinogens benzo(a)pyrene (BP) or7, 12 dimethylbenz(a)anthracene (DMBA), restored contact sensitivity responses to2,4-dinitrofluorobenzene in BP- but not DM BA-treated mice. However, indomethacin failed to restore antibody responses in either group of mice. Prolonged treatment with BP or DMBA led to cutaneous tumour formation. Indomethacin was found to delay the onset and reduce the size of tumours in BP- but not DM BA-treated mice. It is proposed that prostaglandin-induced suppression of cellular cutaneous immunity may play a role in BP- but not DMBA-induced cutaneous carcinogenesis.

To ascertain the influence of vitamin D3 and its metabolites on the function of the skin immune system and the induction of the contact hypersensitivity (CHS) response, a population of vitamin D3-deficient BALB/c mice was established, through dietary vitamin D3 restriction and limitation of exposure to UVB irradiation. Vitamin D3 normal female mice had higher CHS responses than their male counterparts, and dietary vitamin D3 deficiency significantly increased the CHS responses in male, but not in female, mice. This change in the vitamin D3-deficient male mice was not due to an alteration in skin dendritic cell function including antigen carriage, migration or costimulatory molecule expression. In addition, 18 h after sensitisation, the lymph node populations in the vitamin D3-deficient and normal male mice showed similar proliferation and IFN-gamma production. However, during the sensitisation phase of CHS, there was lower lymphocyte recruitment to the skin draining lymph nodes of the vitamin D3-deficient and normal male mice compared with their female counterparts which could account for the difference between the sexes in the extent of the CHS response. These results indicate the vitamin D system can influence cutaneous immune responses in male mice, but this did not occur through the modulation of the dendritic cell functions analysed.

Background Skin develops through a process of epidermal proliferation, maturation, and remodelling of the epidermis and dermis. This period also involves the maturation of the skin immune system, such that antigen applied though the skin of a neonatal mouse always results in immunosuppression, whereas in adults, immunity will occur. Objectives Using proteomics, to identify proteins uniquely involved in the development of the skin and skin immune system. Methods Proteins were extracted from whole skin of mice aged 4 and 21 days, and separated using two-dimensional electrophoresis. Results Of the 25 proteins that were sequenced by peptide mass fingerprinting with matrix-assisted laser desorption/ionization–time of flight–mass spectrometry, three were known markers of keratinocyte differentiation and proliferation. These were cyclophilin A, epidermal fatty acid binding protein 5 and stefin A. Of interest were the two isoforms of stefin A, an intracellular protease inhibitor, found in neonatal skin. The strong expression of stefin A in neonates was confirmed by immunohistochemical analysis, suggesting an important role in the development of the epidermis. Additionally, Western blotting identified two larger isoforms in adult skin, revealing a change in the stefin A during development. Conclusions We propose that stefin A is involved in development of the skin, that development of the skin and of immune function is linked, and that stefin A has an important function in neonatal skin and potentially the neonatal immune response.

To identify possible genetic interactions between the mechanisms of tumor suppression of menin and pRb, we intercrossed mice with targeted deletions of Men1 and Rb1, and compared tumor development in cohorts of animals carrying single or dual mutations of these tumor-suppressor genes. In mice lacking one copy of Men1, pancreatic islet and anterior pituitary adenomas are common. In animals lacking one copy of Rb1, intermediate pituitary and thyroid tumors occur at high frequency, with less frequent development of pancreatic islet hyperplasia and parathyroid lesions. In mice heterozygous for both Men1 and Rb1, pancreatic hyperplasia and tumors of the intermediate pituitary and thyroid occurred at high frequency. Serum measurements of calcium and glucose did not vary significantly between genotypic groups. Loss of heterozygosity at the Rb1 locus was common in pituitary and thyroid tumors, whereas loss of menin was observed in pancreatic and parathyroid lesions. The tumor spectrum in the double heterozygotes was a combination of pathologies seen in each of the individual heterozygotes, without decrease in age of onset, indicating independent, non-additive effects of the two mutations. Together with the lack of increased tumor spectrum, this suggests that menin and pRb function in a common pathway of tumor suppression.

This article reviews many of the complex events that occur after cutaneous ultraviolet (UV) exposure. The inflammatory changes of acute exposure of the skin include erythema (sunburn), the production of inflammatory mediators, alteration of vascular responses and an inflammatory cell infiltrate. Damage to proteins and DNA accumulates within skin cells and characteristic morphological changes occur in keratinocytes and other skin cells. When a cell becomes damaged irreparably by UV exposure, cell death follows via apoptotic mechanisms. Alterations in cutaneous and systemic immunity occur as a result of the UV-induced inflammation and damage, including changes in the production of cytokines by keratinocytes and other skin-associated cells, alteration of adhesion molecule expression and the loss of APC function within the skin. These changes lead to the generation of suppressor T cells, the induction of antigen-specific immunosuppression and a lowering of cell-mediated immunity. These events impair the immune system's capacity to reject highly antigenic skin cancers. This review gives an overview of the acute inflammatory and immunological events associated with cutaneous UV exposure, which are important to consider before dealing with the complex interactions that occur with chronic UV exposure, leading to photocarcinogenesis.

Sukhwinder Singh Sohal,1,* Amir Soltani,1,* David Reid,1,2 Chris Ward,1,3 Karen E Wills,1,4 H Konrad Muller,1 Eugene Haydn Walters1 1National Health and Medical Research Council Centre of Research Excellence for Chronic Respiratory Disease, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia; 2Iron Metabolism Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia; 3Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK; 4Department of Biostatistics, Menzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia *These authors contributed equally to this workBackground: We recently reported that epithelial–mesenchymal transition (EMT) is active in the airways in chronic obstructive pulmonary disease (COPD), suggesting presence of an active profibrotic and promalignant stroma. With no data available on potential treatment effects, we undertook a blinded analysis of inhaled corticosteroids (ICS) effects versus placebo on EMT markers in previously obtained endobronchial biopsies in COPD patients, as a “proof of concept” study.Methods: Assessment of the effects of inhaled fluticasone propionate (FP; 500 µg twice daily for 6 months) versus placebo in 34 COPD patients (23 on fluticasone propionate and eleven on placebo). The end points were epidermal growth factor receptor (EGFR; marker of epithelial activation) and the biomarkers of EMT: reticular basement membrane (Rbm) fragmentation (“hallmark” structural marker), matrix metalloproteinase-9 (MMP-9) cell expression, and S100A4 expression in basal epithelial and Rbm cells (mesenchymal transition markers).Results: Epithelial activation, “clefts/fragmentation” in the Rbm, and changes in the other biomarkers all regressed on ICS, at or close to conventional levels of statistical significance. From these data, we have been able to nominate primary and secondary end points and develop power calculations that would be applicable to a definitive prospective study.Conclusion: Although only a pilot “proof of concept” study, this trial provided strong suggestive support for an anti-EMT effect of ICS in COPD airways. A larger and fully powered prospective study is now indicated as this issue is likely to be extremely important. Such studies may clarify the links between ICS use and better clinical outcomes and protection against lung cancer in COPD.Keywords: pilot trial, reticular basement membrane, S100A4, EGFR, MMP-9, lung cancer

Depletion of dendritic cells from UV-B-irradiated sheep skin was investigated by monitoring migration of these cells towards regional lymph nodes. By creating and cannulating pseudoafferent lymphatic vessels draining a defined region of skin, migrating cells were collected and enumerated throughout the response to UV-B irradiation. In the present study, the effects of exposing sheep flank skin to UV-B radiation clearly demonstrated a dose-dependent increase in the migration of Langerhans cells (LC) from the UV-B-exposed area to the draining lymph node. The range of UV-B doses assessed in this study included 2.7 kJ/m2, a suberythemal dose; 8 kJ/m2, 1 minimal erythemal dose (MED); 20.1 kJ/m2; 40.2 kJ/m2; and 80.4 kJ/m2, 10 MED. The LC were the cells most sensitive to UV-B treatment, with exposure to 8 kJ/m2 or greater reproducibly causing a significant increase in migration. Migration of gammadelta+ dendritic cells (gammadelta+ DC) from irradiated skin was also triggered by exposure to UV-B radiation, but dose dependency was not evident within the range of UV-B doses examined. This, in conjunction with the lack of any consistent correlation between either the timing or magnitude of migration peaks of these two cell types, suggests that different mechanisms govern the egress of LC and gammadelta+ DC from the skin. It is concluded that the depression of normal immune function in the skin after exposure to erythemal doses of UV-B radiation is associated with changes in the migration patterns of epidermal dendritic cells to local lymph nodes.

The damaging effects of UVB light have been described previously and include a number of changes to multiple cell types. At previous meetings of this society, we have shown that Langerhans' cells are the most susceptible to UVB induced damage which can be shown as ultrastructural changes in dendrites, nucleus and cytoplasm by transmission electron microscopy. We have also shown that their patterns of migration from skin to regional lymph node and their ability to present antigens to autologous T cells have been profoundly altered by UVB irradiation. The aim of this work was to establish if it was possible to reverse any of the damage done to Langerhans' cells by UVB exposure by topical application of a DNA repair enzyme such as T4N5 endonuclease. These experiments were undertaken in a sheep model that allowed collection of cells as they migrate from the skin. This allowed for a direct examination of the migration characteristics and ultrastructural features of all Langerhans' cells before, during, and for 2 weeks after exposure to a single dose of UVB. Results obtained from this project indicate that treatment by topical application of DNA repair enzyme immediately after UVB irradiation may restore a number of normal immune parameters associated with the structure and function of migrating Langerhans' cells. It appears that there is a dose related correction of the increased tempo of cell migration and some improvements in the number of ultrastructurally damaged Langerhans' cells have also been associated with application of higher doses of DNA repair enzyme. These preliminary findings indicate that some potential therapeutic benefits are associated with the use of such agents in reversing the immunological damage caused by exposure to erythemal doses of UVB light.

Depletion of epidermal Langerhans cells (LC) and the concomitant depression of the skin immune system after excessive exposure to ultraviolet B light (UVB) has been established in the international literature for some time. Our investigations were intended to determine whether or not these phenomena occurred as a direct result of increased LC migration being triggered by the UVB exposure. To test this hypothesis, a sheep model was established in which the lymphatic vessels draining a defined region of skin were cannulated and the cells migrating towards the regional lymph node continuously collected. Cell populations in these collections were identified and enumerated by indirect immunofluorescence and flow cytometry. These experiments showed there was a significant, dose-dependent increase in the rate of LC migration from sheep skin after exposure to doses of UVB light exceeding 1 minimal erythemal dose (MED). In a series of parallel experiments, the functional characteristics of dendritic cells (DC) migrating from normal or UVB irradiated sheep were studied. To assay them, enriched preparations of DC were collected via cannulated afferent lymphatic vessels and pulsed with antigen prior to incubation with autologous peripheral blood lymphocytes. The relative efficiency of antigen presentation was determined by the ability of DC to induce T cell proliferation. Our data clearly demonstrate that there is a profound loss of normal antigen-presenting cell function after exposure to UVB light. Various experiments were undertaken to determine the mechanism(s) associated with these changes in migration kinetics and cellular function. Electron microscopic examinations of LC migrating from normal or UVB irradiated skin have demonstrated a profound loss of dendritic processes after UVB exposure. This provides a possible explanation for the changes in skin immunity after UVB exposure.

BALB/c mice thymectomized 3 days after birth (3dnTx) are prone to the development of autoimmune gastritis. As this outcome may be a consequence of altered immunoregulatory mechanisms, we set out to determine the immunological status of these mice and their capacity to acquire antigen-specific peripheral tolerance. The latter was assessed by the capacity of these mice to suppress a contact sensitivity response to 2,4,6-trinitrochlorobenzene (TNCB) following treatment of the skin by the carcinogen, DMBA. The 3dnTx mice had a reduced number of CD4 + and CD8 + T cells and a reduced lymphocyte proliferative response to PHA, but a normal contact sensitivity response to TNCB. After treatment of the skin with DMBA these mice failed to develop contact sensitivity to TNCB. Adoptive transfer of splenocytes from these mice to naive mice transfered antigen-specific suppression, irrespective of whether the 3dnTx mice had developed autoimmune gastritis. We conclude that despite thymectomy at day 3 and the attendant immunosuppression, the capacity of BALB/c mice to generate antigen-specific peripheral tolerance to TNCB was retained. These results suggest that precursor T cells which mediate suppression to antigens such as TNCB are present in 3dnTx mice and that these cells are likely to have developed in the thymus and exported to the periphery before 3 days after birth.

This study examined the effects of chronic and current sun exposure on the number of Langerhans cells in epidermal sheets of UV-exposed and unexposed skin of the arms and assessed the effect of sunscreens. Participants were enrolled in a skin cancer prevention trial and had been using sunscreen daily for the previous 3 years. There were significantly fewer Langerhans cells on the exposed (463 cells/mm2) than on the unexposed forearm (528 cells/mm2) (P = 0.0001). High sun exposure in the previous 2 weeks and a history of predominantly outdoor occupations were both associated with a reduced number of Langerhans cells, although age and other biological indicators of chronic exposure were not associated. Sunscreen use was protective against the effects of current but not chronic sun exposure, with a suggestion of a greater effect at higher levels of exposure. Unexpectedly, people with a past history of nonmelanoma skin cancer had more Langerhans cells in both the exposed and the unexposed skin. These results emphasize the need for continued public health education to protect the immune system from the damaging effects of UV radiation.

The ability to produce antigen-specific down-regulation of an established immune response was investigated in 2,4,6-trinitrochlorobenzene (TNCB)-immune mice by delivery of antigen through chemical carcinogen- or ultraviolet B (UVB)-treated skin. When TNCB-immune mice were treated on the dorsal trunk skin with 7,12-dimethylbenz(a)anthracene (DMBA) followed by TNCB there was an antigen-specific reduction in both contact sensitivity and antibody production. Further, immune mice that received spleen cells from naive syngeneic donors treated with DMBA followed by TNCB also exhibited a reduction in both contact sensitivity and antibody production. In contrast, mice treated with UVB irradiation followed by TNCB had a reduction in contact sensitivity but not antibody production. These results provide evidence that an ongoing immune response can be manipulated by immunization through a modified skin immune system. This may provide a beneficial approach for the treatment of autoimmune disease.

Epidermal Langerhans cells (LC) are an integral component of the skin immune system as they initiate immune responses to a variety of antigens, including tumor antigens. When skin is exposed to carcinogenic doses of ultraviolet-B irradiation, chemical carcinogens or tumor promoters there is a significant reduction of LC density. This causes the skin to be immunocompromised and provides an opportunity for aberrant cells to escape immune detection and develop into tumors. Consequently LC depletion is a key event associated with the pathogenesis of skin cancer. We propose that LC depletion contributes to tumor promotion and therefore any agents that reduce LC number, e.g. the contact sensitizing antigen 2,4,6-trinitrochlorobenzene (TNCB), may also contribute to tumor promotion. This proposal was evaluated in cutaneous carcinogenesis by treating mouse skin with a tumor initiating dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter. The initiating dose of DMBA did not cause LC depletion or tumor development. However, if the DMBA-treated skin was then exposed to a concentration of TNCB that caused LC depletion, skin tumors developed. This is analogous to the classical initiator/ promoter system with an LC-depleting dose of TNCB contributing to tumor promotion. Further, this promotion effect was independent of the commencement time of the promoter application, as 2% TNCB applied either 1 or 12 weeks after DMBA initiation induced tumor development. Analysis of the association of LC depletion with immunosuppression and tumor promotion, showed that these events were linked, irrespective of the agent that caused the depletion. It is therefore concluded that LC depletion and local immunosuppression are important aspects of tumor promotion in cutaneous carcinogenesis and non-carcinogenic agents may have tumor promoter activities.

Stressful environmental conditions are a major determinant of immune reactivity. This effect is pronounced in Australian National Antarctic Research Expedition populations exposed to prolonged periods of isolation in the Antarctic. Alterations of T cell function, including depression of cutaneous delayed-type hypersensitivity responses and a peak 48.9% reduction of T cell proliferation to the mitogen phytohaemagglutinin, were documented during a 9-month period of isolation. T cell dysfunction was mediated by changes within the peripheral blood mononuclear cell compartment, including a paradoxical atypical monocytosis associated with altered production of inflammatory cytokines. There was a striking reduction in the production by peripheral blood mononuclear cells of the predominant pro-inflammatory monokine TNF-alpha and changes were also detected in the production of IL-1, IL-2, IL-6, IL-1ra and IL-10. Prolonged Antarctic isolation is also associated with altered latent herpesvirus homeostasis, including increased herpesvirus shedding and expansion of the polyclonal latent Epstein-Barr virus-infected B cell population. These findings have important long-term health implications.

The activation of T cells through presentation of antigen by dendritic cells (DC) relies on many factors, including the correct balance of cytokines in the immediate microenvironment. Antigen presentation by DC migrating from carcinogen-treated skin is impaired as evidenced by the failure of antigen-pulsed DC to initiate specific T cell proliferation. To elucidate mechanism(s) of DC dysfunction, DC migrating from carcinogen-treated skin were collected, pulsed with OVA, and cultured with antigen-specific autologous lymphocytes. Supernatants were assayed for the costimulatory cytokine IL-1 beta which influences the outcome of DC:T cell interactions. The dendritic cells migrating from carcinogen-treated skin that failed to induce T cell proliferation were unable to produce IL-1 beta. This may account for the abrogation of DC function following exposure to chemical carcinogens and provides an explanation for the inability of DC to induce a protective immune response to carcinogen-induced tumours.

Introduction and Objectives We recently reported 1, 2 that EMT is active in the airways of COPD patients; in this process epithelial cells change shape and become motile, then digest through the reticular basement membrane (Rbm) which becomes fragmented and transition to a mesenchymal fibroblast-like cell. We also demonstrated that the Rbm is hyper-vascular, a combined picture specifically suggesting active EMT-type-III, which is a dangerous, pre-malignant condition. This may well be the link between COPD and lung cancer. In this study, we have assessed the effects of ICS on markers of EMT in endobronchial biopsies (ebb) in COPD. Methods A double-blinded, randomised, placebo-controlled study assessed the effects of inhaled fluticasone propionate (FP: 500µg twice daily) on EMT in 34 COPD patients. Ebb were assessed for EMT related Rbm fragmentation (core structural marker) and immunostained for the EMT signatures S100A4 (a fibroblast epitope), matrix-metalloproteinase-9 (MMP-9) and the epithelial activation marker, epidermal growth factor receptor (EGFR). Results Table 1. Conclusions This is the first study to report the positive effects of ICS on EMT markers in COPD. This may be the mechanistic link between ICS and its reported preventive action against smoking-related lung cancer in COPD. References Sohal SS, et al. Respirology 2010, 15 (6):930–938. Sohal SS, et al. Respir Res 2011, 12 (1):130.

Langerhans cells (LCs) are antigen-presenting cells of the skin that trap small contact-sensitizing molecules and induce cutaneous hypersensitivity. LCs can present larger molecules but the mechanisms of processing have required investigation. A system combining in vitro culture of antigen with epidermal cells in the presence of inhibitors, followed by fixation and transfer of these antigen/drug-treated epidermal cells to naive mice, was developed to investigate the steps of antigen processing. Langerhans cells undertake similar, but not identical, pathways for the processing of simple and complex molecules. Complex molecules such as trinitrophenyl conjugated to ovalbumin (TNP-OVA) were internalized and modification required a chloroquine-sensitive proteolysis step and a cycloheximide-sensitive protein synthesis step. This modified product was actively recycled to the cell membrane as presentation was inhibited by blocking receptor translocation with either monensin or cytochalasin B. Small contact sensitizers such as trinitrophenyl did not undergo modification but required internalization and presentation was also inhibited by blocking receptor translocation. J. Leukoc. Biol. 57: 891–896; 1995.

Chemical carcinogens have a recognised ability to reduce the status of cutaneous immunity. Antigen presentation through carcinogen-treated skin results in immune suppression rather than activation of effector mechanisms (reviewed by 1). Topical application of the complete chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) leads not only to the development of skin tumours but also induces a rapid depletion of Langeihans cells (LC) from the epidermis - up to 50% of the LC are depleted from the epidermis in the 3–4 days following DMBA application2.

Epidermal Langerhans' cell (LC) migration to the regional lymph node and beyond into central lymph was examined in sheep following topical application of the complete chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or the contact sensitizing antigen 2,4,6-trinitrochlorobenzene (TNCB). This was facilitated by cannulating previously constructed pseudoafferent lymphatic vessels draining the skin treated with these agents or alternatively, the efferent lymphatic vessel of the regional lymph node. Application of DMBA resulted in a biphasic increase in LC migration. There was an initial increase in LC migration at 25 h with the maximum response (3.6 x 10(7) LC/h) occurring approximately 5 days after DMBA treatment. In contrast, the contact sensitizing antigen TNCB caused enhanced LC migration within minutes of the application of antigen (3.3 x 10(6) LC/h) and peak migration at 8-12 h. Examination of efferent lymph cells from the regional lymph node after DMBA treatment showed uncharacteristically large numbers of LC traversing the lymph node. These LC migration patterns suggest different mechanisms may trigger the migration of LC from skin after the application of DMBA to those associated with the normal processes of antigen presentation.

Langerhans cells (LC) are skin dendritic cells that trap foreign antigens1 and migrate to the local lymph nodes where they present antigen to initiate an immune response. Maintenance of LC density is an essential component in the generation of an immune response as immunization through skin deficient in LC (either naturally depleted such as mouse tail skin or physically depleted by chemicals, UV light or “tape stripping”) results in immunological unresponsiveness2. We have documented LC depletion following carcinogen/tumour promoter treatment and reported that sensitization with 2,4-dinitrofluorobenzene (DNFB) through such depleted sites induced immunological tolerance by activating antigen specific T suppressor cells3. The immunosuppressive ability of carcinogens/tumour promoters suggests that LC depletion is a phenomenon unique to these compounds but depletion of LC following antigen (2,4,6 trinitrochlorobenzene; TNCB) application has also been demonstrated4. Experiments by our group have clearly shown a rapid and substantial migration of LC from the epidermis following either antigen (TNCB) or carcinogen (7,12 dimethylbenz(a)anthracene; DMBA) treatment. These observations raise the possibility that depletion of LC following carcinogen/tumour promoter application is due to a normal immunological response. LC recognise the carcinogens as antigens and respond accordingly by migrating to the draining lymph node.

Mental and physical health are both at risk under conditions of prolonged remoteness from home and removal from normal social support networks. This article examines relevant data from small groups isolated in (a) Antarctic summer stations, (b) a Transantarctic traverse expedition, and (c) a spacecraft simulator capsule.

Summary Treatment of murine skin with the polyaromatic hydrocarbon carcinogens benzo(a)pyrene (BP) or dimethylbenz(a)anthracene (DMBA) for 3 weeks resulted in an increase and a decrease in epidermal Langerhans cell (LC) numbers, respectively, compared with solvent-treated skin. Implantation of subcutaneous indomethacin pellets prior to carcinogen treatment prevented the changes in LC numbers and morphology in BP, but not DMBA-treated skin. Indomethacin treatment was also found to reduce elevated prostaglandin E2 (PGE)2 levels in the skin of BP-treated mice, whereas PGE2 levels were not significantly raised in DMBA-treated mice. There thus appears to be a link between altered prostaglandin levels and LC numbers in murine skin treated with BP, but not DMBA. In the latter, LC numbers were reduced by mechanisms not reversed by indomethacin. It is concluded that increased prostaglandin levels may contribute to the impairment of cutaneous immunity previously observed in BP-treated mice by altering LC density and morphology within the epidermis.