Mitochondrial complex I activity in microglia sustains neuroinflammation.

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.

Mitochondrial reverse electron transport in myeloid cells perpetuates neuroinflammation.

Sustained smouldering, or low grade, activation of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis (MS) 1 . Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells 2 . However, how these metabolic features act to perpetuate neuroinflammation is currently unknown. Using a multiomics approach, we identified a new molecular signature that perpetuates the activation of myeloid cells through mitochondrial complex II (CII) and I (CI) activity driving reverse electron transport (RET) and the production of reactive oxygen species (ROS). Blocking RET in pro-inflammatory myeloid cells protected the central nervous system (CNS) against neurotoxic damage and improved functional outcomes in animal disease models in vivo . Our data show that RET in myeloid cells is a potential new therapeutic target to foster neuroprotection in smouldering inflammatory CNS disorders 3 .

Invasive melanoma in vivo can be distinguished from basal cell carcinoma, benign naevi and healthy skin by canine olfaction: a proof-of-principle study of differential volatile organic compound emission.

BACKGROUND: Volatile organic compounds (VOCs) are continuously released by the body during normal metabolic processes, but their profiles change in the presence of cancer. Robust evidence that invasive melanoma in vivo emits a characteristic VOC signature is lacking. OBJECTIVES: To conduct a canine olfactory, proof-of-principle study to investigate whether VOCs from invasive melanoma are distinguishable from those of basal cell carcinoma (BCC), benign naevi and healthy skin in vivo. METHODS: After a 13-month training period, the dog's ability to discriminate melanoma was evaluated in 20 double-blind tests, each requiring selection of one melanoma sample from nine controls (three each of BCC, naevi and healthy skin; all samples new to the dog). RESULTS: The dog correctly selected the melanoma sample on nine (45%) occasions (95% confidence interval 0·23-0·68) vs. 10% expected by chance alone. A one-sided exact binomial test gave a P-value of < 0·01, supporting the hypothesis that samples were not chosen at random but that some degree of VOC signal from the melanoma samples significantly increased the probability of their detection. Use of a discrete-choice model confirmed melanoma as the most influential of the recorded medical/personal covariates in determining the dog's choice of sample. Accuracy rates based on familiar samples during training were not a reliable indicator of the dog's ability to distinguish melanoma, when confronted with new, unknown samples. CONCLUSIONS: Invasive melanoma in vivo releases odorous VOCs distinct from those of BCC, benign naevi and healthy skin, adding to the evidence that the volatile metabolome of melanoma contains diagnostically useful biomarkers.

Elimination of breast tumor-associated chondroitin sulfate promotes metastasis.

Breast cancer is one of the leading causes of cancer-related deaths amongst women in the USA. The tumor microenvironment has been suggested to be an attractive therapeutic target for treatment of cancers. The glycosaminoglycan chondroitin sulfate, as part of the cellular microenvironment, consists of long linear chains of repeating disaccharide units, which are covalently attached to core proteins to form chondroitin sulfate-proteoglycans. In vitro studies have implicated chondroitin sulfate in various aspects of carcinogenesis, whereas the in vivo roles of chondroitin sulfate are less clear. Drastically elevated levels of chondroitin sulfate have been observed within the stromal compartment of many solid tumors, including human breast carcinomas, the significance of which is unknown. We examined the role of tumor-associated chondroitin sulfate in breast cancer progression. Enzymatic elimination of endogenous chondroitin sulfate by intra-tumor injections of chondroitinase ABC leads to the development of secondary tumors and increased lung metastases, while primary orthotopic tumor growth was not affected. These results establish a metastasis-inhibiting effect of primary breast tumor-associated chondroitin sulfate, which may open novel carbohydrate-based therapeutic strategies to combat breast cancer.

Identification and characterization of TGFbeta-dependent and -independent cis-regulatory modules in the C4ST-1/CHST11 locus.

Chondroitin-4-sulfotransferase-1(C4ST-1)/carbohydrate sulfotransferase 11 (CHST11) is a Golgi-bound enzyme involved in the biosynthesis of the glycosaminoglycan chondroitin sulfate. The sulfation pattern of chondroitin is tightly regulated during development, injury and disease, with the temporal and spatial expression of chondroitin sulfotransferase genes believed to be a crucial determinant of the fine balance of chondroitin sulfation. We have previously identified mouse C4st-1 as a target gene of ligands of the TGFbeta superfamily of growth factors, which could positively regulate C4st-1 expression in a number of cell types. We have also shown that a gene trap loss-of-function mutation in C4st-1 leads to severe skeletal abnormalities during mouse embryogenesis. In addition, we described a highly specific temporal and spatial expression pattern of C4st-1 during mouse embryogenesis. However, the transcriptional regulatory mechanisms that control C4st-1 gene expression remain unexplored. In order to gain knowledge on the transcriptional regulation of C4ST-1, we used a bioinformatical approach to identify conserved putative long-range cis-regulatory modules in a region of 120 kb spanning the 5' end of the C4ST-1 gene. Luciferase reporter assays in human HEK293T and mouse NmuMG cells identified a functional C4ST-1 promoter, as well as a number of cis-regulatory modules able to positively and negatively regulate C4ST-1 expression. Moreover, we identified TGFbeta- responsive regulatory modules that can function in a cell type-specific fashion. Taken together, our results identify TGFbeta-dependent and -independent cis-regulatory modules of the C4ST-1 gene.

External auditory canal pH in chronic otitis externa.

Several risk factors have been postulated to play a part in the progression of acute into chronic otitis externa, including changes towards alkalinity of the skin pH of the external auditory canal. These changes have been previously reported to occur in the acute stage, and their persistence may predispose to a chronic status of this condition. This prospective control study was designed to look at this possible relationship in more depth, by comparing the external auditory canal pH of individuals with chronic otitis externa, but with no current exacerbation, with an age-/sex-matched control group. Analysis of the data revealed a significantly higher external auditory canal pH in the chronic otitis externa group (P < 0.004) when compared with the controls, with no concomitant difference in body skin pH. Within this chronic otitis externa cohort, a statistically significant correlation was found between external auditory canal pH and the severity of the condition, as assessed using a multiparameter scoring system (r = 0.562; P < 0.02). Importantly, the pH was not related to the length of time since the last exacerbation. There was a significant age relationship present within our sample. Younger patients displayed more severe chronic otitis externa(r = -0.813; P < 0.001), with correspondingly higher external auditory canal pH values (r = -0.550; P < 0.02). The results suggest that alkaline ear canal skin is a local risk factor for the progression into chronic otitis externa, occurring, in this study, with greater severity in younger individuals.

Variability in responsiveness to irritants: thoughts on possible underlying mechanisms.

Patch testing with chemical irritants almost always produces a striking variability in the intensity of reaction between individuals, even amongst normal, healthy subjects. Whilst there have been many attempts to define factors which predispose to heightened or, conversely, to diminished reactivity, the underlying cellular mechanisms responsible for the variability remain poorly understood. In this review, a number of possible explanations are proposed, with a particular emphasis on those which relate to the influence of pre-existing disease or to the genetic regulation of certain immunological and inflammatory processes.

Sensitive skin: an epidemiological study.

BACKGROUND: There is a growing awareness that some individuals exhibit heightened skin sensitivity, particularly on the face, and have a high incidence of adverse reactions to cosmetics and toiletries. OBJECTIVES: To carry out an epidemiological study to assess the prevalence of sensitive skin and cosmetic-related adverse events in a U.K. population, and to examine possible factors that may be associated with sensitive skin. METHODS: Self-assessment questionnaires were sent out to 3300 women and 500 men, randomly selected, who were over the age of 18 years and lived within a 10-mile radius of High Wycombe (Bucks.). Fifty non-responder women were also questioned by telephone to ensure that the postal responders were representative of the population as a whole. RESULTS: The response rates were 62% for women and 52% for men, with the incidence of self-reported skin sensitivity being 51.4% and 38.2%, respectively. Ten per cent of women and 5.8% of men described themselves as having very sensitive skin. Fifty-seven per cent of women and 31.4% of men had experienced an adverse reaction to a personal product at some stage in their lives, with 23% of women and 13.8% of men having had a problem in the last 12 months. Among the women, symptoms of cosmetic-induced subjective sensory skin discomfort (burning, stinging, itching etc.) occurred more commonly in the sensitive skin cohort (53%) than in those who regarded themselves as non-sensitive (17%). An atopic diathesis in women did not appear to be a predictive factor for sensitive skin, the incidence of self-perceived sensitive skin being equivalent for atopics (49%) and non-atopics (51%). Furthermore, some 34% of atopic women described themselves as being non-sensitive. Nevertheless, the incidence of atopy was higher among the women in the sensitive skin group (49%) than among those in the non-sensitive group (27%). Dry skin and a predilection for blushing/flushing were associated factors for sensitive skin. CONCLUSIONS: Our survey indicates that sensitive facial skin is a common problem for women and men in the U.K. and points to the need for the development of personal products designed for this skin phenotype.

Reduced levels of glutathione S-transferases in patch test reactions to dithranol and sodium lauryl sulphate as demonstrated by quantitative immunocytochemistry: evidence for oxidative stress in acute irritant contact dermatitis.

There is increasing evidence that oxidative stress plays a role in the pathogenesis of acute irritant contact dermatitis. As part of on-going studies into the effect of irritant chemicals on the anti-oxidant enzyme systems in the skin, we have examined the changing levels of two classes of glutathione S-transferase in patch test reactions to dithranol and sodium lauryl sulphate, using quantitative immunocytochemistry. Although no changes were evident after 6 hrs, significant reductions in the density of staining for glutathione S-transferase alpha were seen with both irritants after 48 hrs and 96 hrs. Glutathione S-transferase pi levels were reduced to a lesser degree, reaching significance for dithranol at the 96 hrs time point only, and for sodium lauryl sulphate at 48 hrs only. The results support the hypothesis that oxidative stress plays a role in chemically-induced inflammation, not only in the case of irritants such as dithranol which are known to directly generate reactive oxygen species, but also with chemicals not generally associated with free radical generation.

The influence of patch test size and design on the distribution of erythema induced by sodium lauryl sulfate.

Patch testing is an invaluable tool for the experimental induction of acute irritant contact dermatitis (ICD), with a variety of chamber systems available for use. Ideally, the inflammatory reactions produced should be of uniform intensity across the test area, thereby facilitating grading of the response and tissue sampling for histopathological studies. Unfortunately, annular, follicular and/or blotchy erythema frequently occur. In this study, we set out to compare the performance of 5 patch test systems (8 mm, 12 mm and 18 mm Finn Chambers; 19 mm and 25 mm Hilltop chambers) when testing normal healthy volunteers with sodium lauryl sulfate at concentrations selected to produce mild, moderate and moderately severe reactions. Visual assessment of the patch test sites revealed good dose responses with all 5 chamber types. Uniformity of erythema across the test site was more closely linked to the actual intensity of response than the delivery system itself, mild reactions being far less likely to display homogeneous erythema than moderately severe reactions. Extra large chambers did not perform significantly better than smaller chambers. Balancing the need for a uniform reaction pattern and adequate tissue sampling area, against the exposure risk, we conclude that 12 mm Finn Chambers represent the optimum patch test system for acute SLS-induced irritation where histopathological investigations are the ultimate aim.

Keratin 17 is expressed during the course of SLS-induced irritant contact dermatitis, but unlike keratin 16, the degree of expression is unrelated to the density of dividing keratinocytes.

The aims of this study were to utilize quantitative immunocytochemical techniques to determine the densities of keratin 16 (K16) and keratin 17 (K17) expressed by keratinocytes during the course of acute patch test reactions to sodium lauryl sulfate (SLS), and to relate these to the proliferative state of the epidermis, as assessed by Ki-67 immunolabelling. Significantly increased numbers of dividing keratinocytes were present in 48h and 96h reactions, concurrent with high levels of expression of K16 and more moderate expression of K17. Statistical analysis indicated a good correlation between K16 expression and the density of Ki-67+ keratinocytes present in the epidermis (r=0.843). This was not the case for K17 (r=0.396). The results demonstrate that both K16 and K17 expression are features of acute irritant contact dermatitis reactions, but suggest that the factors which influence and control their expression differ.

Immunocytochemical demonstration of reduced Cu,Zn-superoxide dismutase levels following topical application of dithranol and sodium lauryl sulphate: an indication of the role of oxidative stress in acute irritant contact dermatitis.

Oxidative stress is known to be implicated in the inflammation induced by the anti-psoriatic irritant, dithranol. In this study, we wished to investigate whether this is reflected in the levels of the antioxidant enzyme, Cu,Zn-superoxide dismutase, as detected by quantitative immunocytochemistry, and whether similar changes also occur following exposure to an irritant not normally associated with reactive oxygen species generation, namely sodium lauryl sulphate. Analysis of biopsies from patch test sites revealed that significant reductions in the epidermal levels of Cu,Zn-superoxide dismutase were induced by both dithranol and sodium lauryl sulphate, although the time course of diminished activity was different for each irritant. Our findings suggest that oxidative stress plays a general role in the pathophysiology of acute irritant contact dermatitis.

Differential patterns of epidermal leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants.

In previous studies, we showed that a number of aspects of the histopathology of irritant contact dermatitis are profoundly influenced by the chemical nature of the irritant applied. We report here that this phenomenon also extends to the infiltration of leukocytes into the epidermis. Healthy volunteers were patch tested with the following irritants and their appropriate controls: benzalkonium chloride, sodium lauryl sulphate, croton oil, dithranol, nonanoic acid, and propylene glycol. After visually grading the intensity of the resulting inflammation, biopsies were removed and the major phenotypic classes of leukocytes identified immunocytochemically. Dermal and epidermal cell densities were determined, and the expression of several activation/proliferation antigens studied. We found a similar pattern of cellular infiltration in the dermis of all irritant groups; the densities of most of the cell types rising in line with the intensity of inflammation. Within the epidermis, however, there were marked differences in the patterns of cellular infiltration between the irritant groups, leading to poorer correlations between leukocyte density and visual grading. The greatest disparity occurred between croton oil and nonanoic acid biopsies, the former being characterized by the influx of large numbers of leukocytes, the latter showing remarkably little exocytosis. Infiltration of neutrophils occurred to varying degrees with all irritants, but a disproportionately large number were present in sodium lauryl sulphate biopsies. All control groups showed a rise in CD4+ cells, with distilled water also producing increases in CD11c+ cells and neutrophils. A selective influx of CD25+ cells occurred in the epidermis of both irritant and control groups. Our observations further highlight the heterogeneous nature of irritant contact dermatitis, and confirm previous findings that visually negative control patch tests show marked cellular reactivity.

Differential effects of structurally unrelated chemical irritants on the density of proliferating keratinocytes in 48 h patch test reactions.

Alterations in the proliferative capacity of human epidermis following topical exposure to structurally unrelated chemical irritants were investigated, with the aim of improving our understanding of the cellular changes that take place during the development of irritant contact dermatitis (ICD). Healthy volunteers were patch tested for 48 h with the following six irritants and their appropriate vehicle and occlusion controls: 5% sodium lauryl sulphate (SLS), 0.5% benzalkonium chloride, 80% nonanoic acid (NAA), 0.02% dithranol, 0.8% croton oil, and 100% propylene glycol (PG). After the degree of inflammation induced was visually graded, biopsy samples were removed and the dividing keratinocytes were identified immunocytochemically by using the monoclonal antibody Ki-67, with quantification being performed on the basis of the number of positive cells/100 basal keratinocytes. Statistically significant increases in the density of proliferating cells occurred in the reactions to SLS, NAA, and PG, whereas, in contrast, dithranol caused a marked decrease in the number of dividing keratinocytes. Overall, the density of proliferating keratinocytes did not show a linear relationship with the visually assessed intensity of inflammation, indicating that the changes observed were related to the chemical nature of the individual irritants and their specific biochemical interactions with the keratinocytes, rather than being the consequence of a generalized inflammatory response. Differential release of epidermal cytokines and mediators by the six irritants may account for these varying states of keratinocyte proliferation. Application of the Spearman rank coefficient of correlation revealed that the changes in mitotic activity of keratinocytes were unrelated either to the total density of leukocytes infiltrating the epidermis and dermis, or to the individual densities of the major phenotypic classes of inflammatory cells present. This makes it unlikely that the localized release of cytokines by infiltrating leukocytes is, by itself, the primary factor in the alteration in epidermal cell kinetics seen in ICD. Our results provide a further demonstration of the diverse actions of different chemical irritants on human skin and emphasize the need to regard ICD as a heterogeneous disorder.

Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis.

The expression of three immunoregulatory surface antigens by epidermal keratinocytes was studied in irritant contact dermatitis (ICD), in order to assess whether keratinocytes have a modulatory role in the pathogenesis of this disorder. Biopsies were taken from 48-h patch test reactions to six structurally unrelated irritants, and frozen sections immunolabeled with monoclonal antibodies to the major histocompatibility complex class II antigen, HLA-DR, intercellular adhesion molecule-1 (ICAM-1), and the 88-Kd glycoprotein CD36 (OKM5), as well as to the CD3 (T cells) and CD11a (lymphocyte function associated antigen-1, LFA-1) antigens. We found that there was very limited expression of HLA-DR by keratinocytes, with no correlation between the extent of HLA-DR positivity and the degree of T cell infiltration into the epidermis and dermis, suggesting that interferon gamma may not be a significant mediator of ICD at 48 h. In contrast, keratinocytes showed extensive upregulation of ICAM-1, with an excellent spatial association between ICAM-1 expression and LFA-1 positive leucocytes in the epidermis. This indicates that keratinocyte ICAM-1 induction is not restricted to diseases in which antigen presentation is pivotal, but that it has a generalized role in cutaneous inflammatory reactions, promoting the infiltration of leucocytes into the epidermis. Immunolabeling with OKM5 revealed that CD36 is present to a variable degree on keratinocytes in normal skin. Differential changes in the pattern of keratinocyte expression occurred between irritants, in a manner that suggested that the CD36 antigen does not act as an adhesion molecule in ICD, but rather that its expression is related to the proliferative state of the epidermis. The results of this study demonstrate that immune-associated antigens are selectively expressed on the surface of keratinocytes in 48-h ICD biopsies, implying that these cells play an important regulatory role in the development of the inflammatory response to irritant chemicals.

Differential effects of structurally unrelated chemical irritants on the density and morphology of epidermal CD1+ cells.

In order to gain a greater insight into the complex mechanisms of action of different irritant chemicals on the skin, we have studied the behavior of epidermal CD1+ cells in experimentally induced irritant contact dermatitis. Healthy, human volunteers were patch tested for 48 h with the following six chemically unrelated irritants and their appropriate vehicle controls; benzalkonium chloride, sodium lauryl sulphate, dithranol, nonanoic acid, croton oil, and propylene glycol. After visually assessing and grading the resulting inflammatory reactions, punch biopsies were taken and the morphology and density of CD1+ cells in the epidermis studied using immunocytochemical techniques in combination with image analysis and electron microscopy. Statistically significant decreases in the epidermal density of CD1+ cells occurred in the responses to dithranol (p less than 0.05) and nonanoic acid (p less than 0.01). Importantly, these changes in density were not simply due to variations in the intensity of inflammatory response (r = 0.1157). Alterations in the length of the dendritic processes of CD1+ cells were also induced, and semi-quantitative analysis revealed significant decreases in dendrite length in the reactions to sodium lauryl sulphate (p less than 0.05), nonanoic acid (p less than 0.001), croton oil (p less than 0.05), and dithranol (p less than 0.005). Unlike epidermal density, however, this effect on cell morphology was directly related to the severity of inflammation (r = -0.74, p less than 0.01). Morphologic evidence of cellular injury to Langerhans cells was seen by electron microscopy in the majority of biopsies, although relatively few cells were affected in sodium lauryl sulphate and propylene glycol reactions. Benzalkonium chloride, unlike the other irritants, also induced a state of metabolic activation in a high proportion of epidermal Langerhans cells. Lymphocyte/Langerhans cell apposition was observed in most samples, but was particularly prevalent in the reactions to dithranol. The results of this study demonstrate that significant changes in the morphology and density of Langerhans cells occur in irritant contact dermatitis, some of which are directly influenced by the chemical nature of the irritant.

Epidermal damage induced by irritants in man: a light and electron microscopic study.

Irritant contact dermatitis may be induced by many chemicals and has a far greater incidence than allergic contact dermatitis. Despite this, it receives relatively little attention and its pathogenesis remains poorly understood. To gain a greater understanding of the interaction of irritants with the skin, we investigated the histopathological changes resulting from the topical application of a series of structurally unrelated irritants. Human volunteers were patch-tested with appropriate concentrations of nonanoic acid, sodium lauryl sulphate, dithranol, benzalkonium chloride, croton oil, and propylene glycol, which produced generally mild to moderate responses. Biopsy specimens were taken after 48 h and examined by light and electron microscopy. Spongiosis and the infiltration of predominantly mononuclear cells were observed in the epidermis of the majority of biopsy specimens, and were particularly pronounced and extensive in croton oil reactions. In addition, several irritants induced distinct and characteristic patterns of keratinocyte damage. Nonanoic acid and sodium lauryl sulphate caused morphologic changes indicative of disturbances in keratinocyte metabolism and differentiation, giving rise to dyskeratosis and parakeratosis respectively, while dithranol induced marked swelling of keratinocytes in the upper epidermis. The results suggest that there is a diversity and specificity in the histopathology of irritant contact dermatitis, reflecting the different ways in which chemicals may interact with components of the skin.

Assessment of erythema in irritant contact dermatitis. Comparison between visual scoring and laser Doppler flowmetry.

Assessment of erythema in experimentally-induced irritant contact dermatitis has been performed visually and using the laser Doppler flowmeter (LDF). A close correlation was shown between the 2 methods (r = 0.9079, p less than 0.001), with the LDF producing mean blood flow values which were able to discriminate between the different visual scores. Of the 100 patch tests evaluated, 3 gave poor correlations between their visual and LDF readings, including 2 dithranol reactions and 1 sodium hydroxide response. Patch tests with no visible erythema had blood flow values similar to those of normal untreated skin. Although the LDF was an easy instrument to operate, it was not considered suitable for use in the routine patch test clinic, due mainly to the unacceptable length of time required to measure each patch test.

Experimentally-induced irritant contact dermatitis. Determination of optimum irritant concentrations.

Patch testing with 7 irritants has been performed on a panel of 42 healthy volunteers, with the aim of determining concentrations which would induce mild to moderate reactions in at least 75% of individuals tested. The irritants studied and their optimum concentrations were as follows: benzalkonium chloride, 0.5%; sodium lauryl sulphate, 5%; croton oil, 0.8%; dithranol, 0.02%; nonanoic acid, 80%; propylene glycol, 100%; sodium hydroxide, 2%. Responder rates lower than 75% had to be accepted for benzalkonium chloride and sodium hydroxide in order to prevent overly severe reactions, whilst propylene glycol proved to have only marginal irritant properties.

Immunopathological and ultrastructural findings in human allergic and irritant contact dermatitis.

The histopathological features of allergic contact dermatitis were compared with those of irritant contact dermatitis in a group of 17 subjects. Each patient received simultaneous patch tests of a known allergen and a standardized irritant (benzalkonium chloride). The cellular changes occurring between 3 h and 7 days after patch test application were studied by light and electron microscopy and immunocytochemistry. No differences were observed between the induced allergic contact dermatitis (ACD) and the irritant contact dermatitis (ICD), either in the responding cell types or the sequence of cellular events. Both reactions showed a predominantly T lymphocyte infiltrate with no polymorphonuclear leukocyte involvement. Apposition of Langerhans cells to lymphocytes in the epidermis was seen in both types of response. Considerable variability in the intensity of reaction to irritant and allergen occurred within individuals. There was no statistically significant difference between the intensity of the reactions to the irritant and the allergen.