Abnormal keratinocyte differentiation in the nasal planum of Labrador Retrievers with hereditary nasal parakeratosis (HNPK).

Hereditary nasal parakeratosis (HNPK) is an inherited disorder described in Labrador Retrievers and Greyhounds. It has been associated with breed-specific variants in the SUV39H2 gene encoding a histone 3 methyltransferase involved in epigenetic silencing. Formalin-fixed biopsies of the nasal planum of Labrador Retrievers were screened by immunofluorescence microscopy for the presence and distribution of epidermal proliferation and differentiation markers. Gene expression of these markers was further analysed using RNA sequencing (RNA-seq) and ultrastructural epidermal differences were investigated by electron microscopy. Differentiation of the nasal planum in the basal and suprabasal epidermal layers of HNPK-affected dogs (n = 6) was similar compared to control dogs (n = 6). In the upper epidermal layers, clear modifications were noticed. Loricrin protein was absent in HNPK-affected nasal planum sections in contrast to sections of the same location of control dogs. However, loricrin was present in the epidermis of paw pads and abdominal skin from HNPK dogs and healthy control dogs. The patterns of keratins K1, K10 and K14, were not markedly altered in the nasal planum of HNPK-affected dogs while the expression of the terminal differentiation marker involucrin appeared less regular. Based on RNA-seq, LOR and IVL expression levels were significantly decreased, while KRT1, KRT10 and KRT14 levels were up-regulated (log2fold-changes of 2.67, 3.19 and 1.71, respectively) in HNPK-affected nasal planum (n = 3) compared to control dogs (n = 3). Electron microscopical analysis revealed structural alterations in keratinocytes and stratum corneum, and disrupted keratinocyte adhesions and distended intercellular spaces in lesional samples (n = 3) compared to a sample of a healthy control dog (n = 1). Our findings demonstrate aberrant keratinocyte terminal differentiation of the nasal planum of HNPK-affected Labrador Retrievers and provide insights into biological consequences of this inactive SUV39H2 gene variant.

Enolase-1 expression in the stratum corneum is elevated with parakeratosis of atopic dermatitis and disrupts the cellular tight junction barrier in keratinocytes.

OBJECTIVE: Previous studies have shown that enolase-1 (ENO1) in the stratum corneum (SC) is more highly expressed in patients with atopic dermatitis (AD) than in healthy individuals, suggesting that it is a novel biomarker for evaluating skin condition in patients with AD. However, the mechanism underlying high ENO1 expression in the SC and its pathological relevance in AD are unclear. In this study, the relationship between ENO1 expression and keratinization of epidermis was investigated, and the role of high ENO1 expression in keratinocytes was characterized. METHODS: ENO1 expression and morphological characteristics were examined in SC from the cheeks of 24 patients with AD. Additionally, the localization of ENO1 in the excised human epidermis was observed. Moreover, to analyse the role of ENO1 in cellular barrier function, tight junction proteins (TJs) and transepithelial electrical resistance (TEER) in keratinocytes with ENO1 overexpression were evaluated. Furthermore, the localization of ENO1 and plasminogen in keratinocytes was evaluated by immunostaining, and the cellular barrier function in keratinocytes was examined after treatment with tranexamic acid (TXA). RESULTS: ENO1 expression was substantially correlated with the rate of nucleated corneocytes in AD. In addition, ENO1 localized in the basal to spinous layers, but was its expression dramatically decreased in healthy human SC. ENO1 overexpression in human epidermal keratinocytes reduced the expression of TJs (claudin-4, E-cadherin, tricellulin, and occludin) and TEER, and treatment with anti-ENO1 IgG reversed these effects. ENO1 colocalized with plasminogen in keratinocytes. Treatment with TXA rescued the ENO1-induced reductions in TJ and TEER expression. CONCLUSION: We found a substantial correlation between ENO1 expression and the rate of nucleated corneocytes in AD and decreased ENO1 expression with nuclear disappearance. These results suggest that high ENO1 expression in the SC of AD is caused by deficient keratinization, which is an AD characteristic. Moreover, ENO1 overexpression in keratinocytes promoted dysfunction of TJ dynamics, leading to reduced integrity of the cellular barrier, and these effects might be mediated by plasmin activity. We propose that ENO1 is a useful indicator of parakeratosis and might have a potential role in cellular TJ barrier function in the epidermis.

Degree of Skin Barrier Disruption Affects Lipid Organization in Regenerated Stratum Corneum.

Previously, a skin barrier repair model was developed to examine the effect of formulations on the lipid properties of compromised skin. In this model, the lipid organization mimics that of several skin diseases with impaired skin barrier and less dense lateral lipid organization. In addition, parakeratosis was occasionally observed. The present study investigated whether the extent of initial barrier disruption affects lipid organization and parakeratosis in regenerated stratum corneum. After barrier disruption and stratum corneum regeneration the fraction of lipids adopting a less dense lateral organization gradually increased with increasing degree of barrier disruption. Only when 75% of the stratum corneum was removed, were parakeratosis and a change in lamellar organization observed. This demonstrates the possibility of using the skin barrier repair model to study the effects of formulations on compromised skin in which the presence of parakeratosis and lipid organization can be modified by the extent of barrier disruption.

Response of the Endothelium to the Epicutaneous Application of Leukotriene B4.

BACKGROUND: Vascular changes, both endothelial and functional, are crucial events in inflammatory responses. OBJECTIVES: To investigate the dynamics of endothelial cell (EC) and functional changes during acute inflammation in an in vivo model of the skin using leukotriene B4. METHODS: EC proliferation, vascular network size, vessel diameter (VD), and hypoxia-inducible factor (HIF)-1α were studied by immunohistochemical CD31/Ki67 double staining and single staining of HIF-1α. Cutaneous perfusion (CP) was assessed using the Twente Optical Perfusion Camera. RESULTS: The initial phase illustrated an increase in VD, Ki67+ EC, and HIF-1α expression and late-phase vascular expansion. The HIF-1α and Ki67+ EC expression was limited. CP and VD were augmented after 24 h. CONCLUSION: The early phase of inflammation is characterized by EC proliferation and HIF-1α expression. Vascular expansion continues over time. CP and VD are seen in both phases of inflammation. Angiogenesis, vascular network formation, and perfusion are time-dependent processes which are mutually related during inflammation.

Human sebum extract induces barrier disruption and cytokine expression in murine epidermis.

BACKGROUND: Previous studies have shown that human sebum may play a role in barrier function but with much debate. OBJECTIVE: To elucidate the effects of human sebum on skin barrier function. METHODS: We used hairless mouse skin to study the functional and morphological alternation of epidermis after the application of human sebum. RESULTS: The results showed a significant increase in transepidermal water loss and erythema value, and a decrease in skin hydration, accompanied by epidermal hyperplasia with parakeratosis following sebum application. Nile red staining together with electron microscopic examination confirmed the underlying mechanisms for sebum-induced barrier disruption are related directly to the interaction of sebum with the intracellular lipid lamellae of the SC, thereby leading to the increase in the fluidity of SC intracellular lipids as demonstrated by ATR-FTIR measurement. An inflammatory reaction characterized by an enhanced cytokine cascade, including up-regulation of TNF-α, IL-1α and IL-6, was also observed. On the other hand, there were insignificant expression of thymic stromal lymphopoietin and unchanged serum levels of IgE, suggesting non-immunogenic stimulation by sebum treatment. CONCLUSION: It may be concluded that inflammation induced by excess amount of sebum is more likely an irritant contact dermatitis rather than an allergic one. Moreover, these findings implicated possible relationships between sebum, irritant contact dermatitis, and seborrheic dermatitis.

Retention of corneodesmosomes and increased expression of protease inhibitors in dandruff.

BACKGROUND: Dandruff is a common, relapsing and uncomfortable scalp condition affecting a large proportion of the global population. The appearance of flakes on the scalp and in the hair line, and associated itch are thought to be consequences of a damaged skin barrier, altered corneocyte cohesion and abnormal desquamation in dandruff. The balance between skin proteases and protease inhibitors is essential for driving the key events, including corneodesmosome degradation, in the desquamation process and to maintain stratum corneum (SC) barrier integrity. OBJECTIVES: To investigate the distribution of corneodesmosomes, the key component of the SC cohesivity and barrier function, and the protease inhibitors lympho-epithelial Kazal-type-related inhibitor (LEKTI-1) and squamous cell carcinoma antigen (SCCA1) in the scalp of dandruff-affected participants. METHODS: The methods utilized were immunohistochemistry, scanning immunoelectron microscopy, phase-contrast microscopy, Western blotting and serine protease activity assay on tape-stripped SC or scalp skin biopsies. RESULTS: In SC samples from healthy subjects, corneodesmosomes were peripherally located in the corneocytes. In samples of dandruff lesions, corneodesmosomes were located both peripherally and on the entire surface area of the corneocytes. LEKTI-1 and SCCA1 protein levels and parakeratosis were found to be highly elevated in the lesional samples. CONCLUSIONS: The persistence of nonperipheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in dandruff. The increased expression levels of LEKTI-1 and SCCA1 are consistent with the view that the dandruff condition is characterized by an imbalance in protease-protease inhibitor interaction in the SC.

Circumscribed palmar or plantar hypokeratosis: first report on a nonacral site with unique histologic features.

Circumscribed palmar or plantar hypokeratosis was first described by Pérez et al in 2002 as a unique entity of the skin in which they reported 10 patients who presented with well-circumscribed areas of erythematous depressed or eroded skin mostly over the thenar or hypothenar eminences of the palms and less commonly on the soles. Histologically, the lesions demonstrated an abrupt drop-off in the cornified layer resulting in a broad area of hypokeratosis. Pérez et al hypothesized that these lesions were a distinctive epidermal malformation. There have been several reports since, some of which implicate trauma as an etiologic agent; however, the exact etiology remains unclear. The authors present the first case of circumscribed palmar or plantar hypokeratosis on a nonacral site (chest of a 63-year-old man) with novel histological features, including granular parakeratosis and evidence of trauma (subepidermal fibrin and ulcerations).

Parakeratosis in skin is associated with loss of inhibitor of differentiation 4 via promoter methylation.

Parakeratosis refers to incomplete maturation of epidermal keratinocytes, resulting in abnormal retention of nuclei in the stratum corneum. It occurs in many diseases of the skin, particularly in psoriasis. Down-regulation of inhibitor of differentiation 4 messenger RNA has been demonstrated in psoriatic skin, but the specificity and mechanism for this finding are unknown. In this study, we addressed specificity by immunohistochemical staining for inhibitor of differentiation 4 protein in skin disorders showing parakeratosis, including: psoriasis (n = 9), chronic eczema (n = 6), and squamous cell carcinoma (n = 7). In these conditions, parakeratotic keratinocytes in the upper layers of the skin lacked inhibitor of differentiation 4 protein expression, whereas keratinocytes in the lower layers were densely stained, in contrast to diffuse expression in normal skin. Because promoter hypermethylation of inhibitor of differentiation 4 has been described in several cancers, we determined the methylation pattern of the inhibitor of differentiation 4 promoter in psoriasis and compared this with squamous cell carcinoma. We found a novel methylation pattern of the inhibitor of differentiation 4 promoter in both conditions. Inhibitor of differentiation 4 promoter methylation was significantly increased in psoriasis (34.8%) and squamous cell carcinoma (21.8%), compared with normal skin (0%). Moreover, cells in the upper and lower parts of psoriatic epidermis were, respectively, hypermethylated and nonmethylated, at the inhibitor of differentiation 4 promoter. Comparable studies in several cell lines confirmed that hypermethylation of the promoter was associated with loss of inhibitor of differentiation 4 messenger RNA and protein expression. Our study demonstrates a previously unreported link between gene-specific promoter hypermethylation and abnormal cellular differentiation in several skin diseases. This mechanism might provide clues for novel therapies for skin disorders characterized by parakeratosis.

Efficacy study of vesicular gel containing methotrexate and menthol combination on parakeratotic rat skin model.

Methotrexate (MTX) is indicated in the symptomatic control of severe, recalcitrant, and disabling psoriasis. The oral or parenteral route of administration causes systemic toxicity. The topical route of delivery, though, reduces systemic toxicity and has limited applicability due to restricted permeability. Liposomal and niosomal MTX topical formulations have also been investigated with limited success to achieve drug localization in the skin. Menthol has been suggested in conditions of psoriasis, in addition to its skin-penetration-enhancing effect on drugs. The present work aimed at investigating the potential benefits of combining menthol with MTX in a vesicular gel base for not only improving the penetration and dermal availability of MTX, but also to render such a formulation more effective with greater patient acceptability. MTX liposomes were prepared by thin-film hydration, and the vesicles were characterized for drug-entrapment efficiency, size, and morphology. These liposomal vesicles were incorporated in a gel base, and this vesicular gel was evaluated for transdermal drug permeation and extent of drug accumulation in the skin, using a rat skin ex vivo model. Skin histology studies were carried out to investigate any structural changes caused by the permeation enhancers. Antipsoriatic efficacy of the formulations was tested in vivo, using the rat tail model. The results indicated that the vesicular gel containing menthol could cause maximum drug retention in the skin. The skin treated with menthol had a disrupted epidermis and microcavities. The in vivo studies also ascertained the effectiveness of the formulation in inducing a normal pattern of differentiation in the rat tail skin that initially showed parakeratosis, which is also characteristic of psoriatic epidermis. These results show the potential of vesicular gel containing MTX and menthol to improve penetration into the skin and cause drug retention in skin appendages.

Up-regulation of serpin SCCA1 is associated with epidermal barrier disruption.

BACKGROUND: Parakeratosis, the persistent presence of nuclei in the stratum corneum (SC) is associated with serious disruption of skin barrier function. Squamous cell carcinoma antigen 1 (SCCA1) is strongly up-regulated in inflamed and parakeratotic skin. OBJECTIVE: To find a biochemical marker for the SC barrier disruption, especially the disruption associated with parakeratosis. METHODS: An ELISA assay system was established to quantify SCCA1 in the extract of tape-stripped cornified cells. Transepidermal water loss (TEWL) and other skin parameters were measured and compared with the amount of SCCA1. Localization of SCCA1 was investigated immunohistochemically in various skin diseases with parakeratosis. Nuclei and SCCA1 on the skin surface were detected by staining of corniocytes collected on an adhesive-coated slide glass. RESULTS: SCCA1 showed strong up-regulation in lesional skin with psoriasis (466-fold), hayfever skin caused by Japanese ceder pollen (232-fold) and sun-exposed skin of healthy individuals (90-fold) compared to their normal sun-protected skin. The increased levels of SCCA1 were well correlated with increased values of TEWL and the number of parakeratotic cells in the SC. Furthermore, subjects with high levels of SCCA1 in the epidermis were more susceptible to barrier disruption by external stimuli, and this was accompanied with a further increase of SCCA1. We confirmed that localization of SCCA1 was limited to parakeratotic areas by using the skin surface staining technique. Immunohistochemical study also demonstrated that SCCA1 was always present at high levels in parakeratotic epidermis. CONCLUSION: All of our findings indicate that SCCA1 plays an important role in the induction of epidermal barrier disruption. SCCA1 may be a critical determinant of barrier function in the epidermis.

Irritated seborrheic keratosis with coarse keratohyalin granules.

AIMS: The viral etiology of certain types of seborrheic keratosis (SK) has been a controversial subject in literature, with different molecular results. On the contrary, to the molecular approach, some have suggested that certain types of SK are indeed warts, due to their morphologic features. We decided to investigate the presence of coarse keratohyalin granules in cases of irritated SK. MATERIAL, METHODS AND RESULTS: We examined the last 60 cases with such a diagnosis in our Service of Anatomic Pathology and found these granules in eight cases (7.5%). The granules were evidenced in squamous eddies in four cases, while they were seen in foci of hypergranulosis from the top part of the epidermis in five cases. These granules were evidenced in a few foci in three cases while they were seen in multiple foci in five cases. In these eight cases, we also looked for other morphologic signs suggesting a viral origin, such as papilated, exo-endophytic configuration, parakeratosis at the tips of digitations, dilated vessels in the papillae and koilocytes. While six cases presented at least any of these other features, in two of the eight cases (25%), the only clue suggesting a viral origin was the evidence of the thick granules of keratohyalin. CONCLUSIONS: We discuss the meaning of such a finding as described in literature, and conclude that it should be a specific feature to look out for, in cases of irritated SK, in order to exclude a diagnosis of verruca vulgaris.

The cutaneous manifestations of atypical complete DiGeorge syndrome: a histopathologic and immunohistochemical study.

DiGeorge syndrome is a congenital anomaly with a constellation of findings that includes thymic hypoplasia. Only a small subset of patients with DiGeorge syndrome has complete athymia, classified as complete DiGeorge anomaly; one third of these patients show an eczematous dermatitis, oligoclonal T-cells and lymphadenopathy, known as atypical complete DiGeorge anomaly. Six biopsies from six patients with the distinctive clinical phenotype of atypical complete DiGeorge anomaly were studied. Every biopsy showed exocytosis (100%), parakeratosis, often confluent and spongiosis (100%). Neutrophilic abscesses (50%), dyskeratosis (67%) and satellite cell necrosis (50%) were seen. Perieccrine and perivascular inflammation were seen in half of the cases. Eosinophils were identified (83%); most commonly in both the epidermis and dermis. All of lymphocytes were CD3 positive. Most (83%) of cases contained T-cell intracellular antigen 1 (TIA-1) positive cells. Special testing of the selected patients using spectratyping identified oligoclonal T-cell populations. The presence of dyskeratotic keratinocytes, satellite cell necrosis and parakeratotic scale with neutrophils characterizes the cutaneous rash seen in this subset of complete DiGeorge syndrome patients. Such skin lesions from patients with DiGeorge anomaly should alert the pathologist to the potential diagnosis of atypical complete DiGeorge anomaly. The pathophysiologic role of the oligoclonal T-cells in this entity requires additional study.

Expression of epidermal N-methyl-D-aspartate receptors (NMDAR1) depends on formation of the granular layer--analysis in diseases with parakeratotic cornification.

Ionotrope glutamate receptors of the N-methyl-D-aspartate (NMDA) receptor type are expressed on keratinocytes and influence the intracellular calcium concentration. The importance of NMDA receptors in pathophysiological processes in the skin is, however, still unclear. Epidermal distribution patterns of NMDA receptors were investigated in dermatoses with parakeratotic cornification (psoriasis vulgaris and verrucae vulgares) and compared to the expression of filaggrin. The expression of NMDA receptors (R1 component) in paraffin-embedded normal epidermis (n = 22), psoriasis vulgaris (n = 21) and verrucae vulgares (n = 23) was examined and evaluated by means of digital image analysis. For quantitative characterization of the distribution patterns, a quotient was formed of the expression in the stratum granulosum and stratum basale ("NMDA ratio"). The distribution of NMDAR1 was compared to the immunohistochemical expression of filaggrin. Additionally the expression of filaggrin was investigated in HaCaT cells after treatment with the NMDA receptor antagonist MK-801. NMDA receptors were demonstrated in the epidermis of all preparations. In healthy skin, the highest receptor density was found in the stratum granulosum. This distribution pattern was basically also present in the dermatoses examined. Thus, the occurrence of parakeratosis in psoriasis vulgaris, but not in verrucae vulgares, was characterized by a significant reduction in the NMDA ratio (reduced expression of NMDAR1 in the upper epidermis). The immunohistochemical distribution of filaggrin was similar to that of NMDAR1. In HaCaT cells MK-801 suppressed the expression of filaggrin. NMDA receptors are expressed in human epidermis under physiological conditions especially in the stratum granulosum. Their reduced expression within parakeratotic epidermis in psoriasis vulgaris may be evidence of impaired intracellular calcium influx in this disease.

Histochemical localization of hyaluronan in psoriasis, allergic contact dermatitis and normal skin.

In suction blister fluid from active psoriatic lesions we have previously found elevated concentrations of hyaluronan. The aim of this investigation was to study the localization of hyaluronan with a histochemical method, in biopsy specimens from lesions of 13 patients with progressive psoriasis. Ten normal subjects and seven patients with allergic contact dermatitis were also studied. In normal epidermis the highest intensity of hyaluronan staining was found in the intercellular spaces in the middle and upper spinous layer, whereas the staining was much weaker in the basal layer. No hyaluronan was detected in the granular layer or in the orthokeratotic stratum corneum. In the dermis there was pronounced staining of the papillary dermis and around the sebaceous glands, sweat glands, hair follicles and blood vessels. In six of the 16 specimens from psoriatic lesions the normal epidermal meshwork of hyaluronan was partly absent and replaced by diffuse staining of both the spinous and the basal layer. In the remaining ten of these 16 specimens the same type of meshwork was found in stratum spinosum as in normal skin. The parakeratotic stratum corneum contained hyaluronan, in contrast to the normal stratum corneum, where no hyaluronan was present. The pattern of hyaluronan staining in the dermis of the psoriatic lesions did not differ from that in normal dermis. In the majority of the allergic patch test reactions the junction was less distinct than in normal skin between dermis and epidermis and the normal hyaluronan pattern of the basal layer was abolished and replaced by a diffuse staining throughout the layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Punctate porokeratotic keratoderma: some pathogenetic analyses of hyperproliferation and parakeratosis.

An 82-year-old Japanese woman had numerous palmoplantar keratotic plugs and pits, resembling 'music box spines'. Histological examination revealed compact columns of parakeratosis in the horny layer. Ultrastructually, the affected stratum corneum contained numberous variable-sized pyknotic nuclei, and cells in the stratum granulosum contained fewer keratohyalin granules. Autoradiographic analysis by [3H]thymidine [3H]TdR incorporation into epidermal cells of affected skin slices in organ culture revealed that only basal cells below the keratotic plug were stimulated to proliferate. Two-dimensional gel electrophoresis revealed that palmar keratotic plugs contained the keratin filaments that are specifically present in the plantar viable epidermal layer, or other hyperproliferative epithelial cells.

Monoclonal anti-interleukin 2 (15-2) antibody binding to granular layer keratinocytes of human skin.

Among several monoclonal antibodies (moABs) directed against human interleukin 2 (IL-2), the 15-2 moAB raised in our laboratory against unglycosylated recombinant IL-2 (produced in Escherichia coli) cross-reacted with a human skin epitope. This moAB gave a strong staining on the cell-surface membranes of keratinocytes from the granular layer of the epidermis. In addition, the 15-2 moAB stained 15% of epidermal cell suspensions obtained from suction blisters and reacted with cells from the spinous layer in parakeratosis and psoriasis, as well as with spinous epithelioma cells. Preincubation of the 15-2 moAB with pure human recombinant IL-2 abrogated skin binding, whereas a polyclonal antikeratin antiserum did not block 15-2 skin binding. Two other anti-IL-2 moABs, one directed against unglycosylated recombinant IL-2 (17-2 moAB) and one against glycosylated natural IL-2 (9B11 IE5 moAB), were unreactive on skin. Taken together, the data suggest that the 15-2 moAB binds to an epitope cross-reacting with, but different from, IL-2 which is located in the cell-surface membranes of granular layer cells. This cross-reactive epitope may provide a useful probe for the study of human epidermal cell differentiation.

Comparative investigation of keratin-filaments in normal tissues and tumours of skin, oral mucosa, salivary glands and thymus.

Antibodies against different fractions of keratins can be helpful in various fields of special pathology. Antibodies against "small" and "large" keratins permit to evaluate epithelial maturation in skin and oral mucosa. In addition, disturbances of keratinization during inflammatory processes and malignant transformation can be analyzed. The main application of antibodies against the entire fractions of keratins is the detection of the epithelial nature of a neoplasm. By this tool, particular problems in surgical pathology concerning differential diagnosis can be handled in an easier way. Among the different tissues and their neoplasms, examples of the analysis of thymus tumours and salivary gland tumours are presented. Immunoreactivity with keratin antibodies depends crucially on tissue processing. In the normal diagnostic procedure, good results are regularly obtained if cryostat or Bouin-fixed paraffin-embedded sections are used.