Infrared densitometry: a fast and non-destructive method for exact stratum corneum depth calculation for in vitro tape-stripping.

The investigation of drug penetration into the stratum corneum (SC) by tape-stripping requires an accurate measure of the amount of SC on each tape-strip in order to determine the depth inside the SC. This study applies infrared densitometry (IR-D) to in vitro tape-stripping using the novel Squame Scan(R) 850A. The device had recently been shown to provide accurate measurements of the SC depth for tape-stripping in vivo. Furthermore, the suitability of IR-D for determining the endpoint of tape-stripping, i.e. complete SC removal, was tested. The SC depth was computed from the IR-D data of sequential tape-strips and compared to the results of a protein assay as gold standard. IR-D provided accurate depth results both for freshly excised skin and for skin stored frozen for up to 3 months. In addition, the lower limit of quantification of IR-D indicates the complete removal of the SC (less than 5% of the total SC remaining) and can be used for adjusting the number of tapes applied in situ. Therefore, IR-D is an accurate, fast and non-destructive method for SC depth determination.

Absence of epidermal growth factor receptor expression in squamous cell carcinoma of the uterine cervix is an indicator of limited tumor disease.

The expression of growth factors is considered as an important diagnostic and prognostic feature in tumor pathology. We investigated the value of the immunohistochemical EGF-receptor expression (EGF-R) in 30 squamous cell carcinomas of the uterine cervix, treated by radical hysterectomy and lymphadenectomy according to the Wertheim-Meigs-Okabayashi technique. Immunohistochemical reactions were performed on 4 microm sections from paraffin-embedded tissue, using an indirect peroxidase method. The staining results were evaluated semiquantitatively as negative (n=9; 30%) or as slightly, moderately or severely positive (n=21; 70%). The EGF-R-negative tumors were found in less advanced tumor stages. None had invaded into the parametrium (100%), eight were staged as T1 (89%), seven as N0 (78%), and seven showed no evidence for lymphangiosis carcinomatosa (78%). The respective values for the EGF-R-positive tumors ranged from 52% to 67%. However, only the difference in parametral invasion (EGF-R-negative: 0%, EGF-R-positive: 38%) was statistically significant (p=0.0306), probably due to the small number of cases. The EGF-R-expression was not correlated to histomorphological tumor grading. The results of this study indicate an inverse correlation between EGF-R expression and tumor spread. Assuming that this trend could be confirmed by a larger group of patients, immunostaining for EGF-R in a tumor biopsy could be useful to adapt surgical strategies and adjuvant therapy in the individual patient. Moreover, the EGF-R is an interesting target for immunotherapeutic approaches in squamous cell cervical carcinoma.

Immunohistochemical differentiation between inflammatory linear verrucous epidermal nevus (ILVEN) and psoriasis.

Inflammatory linear verrucous epidermal nevus (ILVEN) is a rare skin disorder with a clinical and histological resemblance to psoriasis. In the past clinical and histological criteria have been defined. However, there remains a discussion as to whether ILVEN is a disease entity distinct from linear psoriasis. Our objective was to compare by quantitative immunohistochemistry the subsets of T-lymphocytes and markers for epidermal growth and keratinisation in biopsies taken from skin lesions of 4 patients with psoriasis and 3 patients with ILVEN: 1. patients with psoriasis (case 1-4) 2. patient with ILVEN cum psoriasis (case 5) 3. patients with ILVEN sine psoriasis (case 6 and 7). Our aim was to delineate ILVEN from psoriasis. Four patients with active psoriasis and three patients with signs and symptoms of ILVEN are described in this case report. Two patients of the ILVEN group had only linear verrucous lesions (ILVEN sine psoriasis), and one patient had linear lesions combined with widespread psoriasis outside the linear verrucous lesion (ILVEN cum psoriasis). The following markers were investigated in skin biopsies taken from the aforementioned patients by quantitative immunohistochemistry: CD2, CD4, CD8, CD25, CD161, CD94, CD45RO, CD45RA, HLA-DR, Keratin-10, Ki-67. In patients with ILVEN (cum and sine psoriasis) the number of Ki-67 positive nuclei, tended to be lower, the number of keratin-10 positive cells and HLA-DR expression higher as compared to psoriasis. In ILVEN sine psoriasis all T-cell subsets and cells expressing NK receptors were reduced as compared to psoriasis, except for CD45RA+ cells, whereas in the patient with ILVEN cum psoriasis the number of these T cell subsets had an intermediary position. In particular the density of CD8+, CD45RO+ and CD2+, CD94 and CD161 showed a marked difference between ILVEN sine psoriasis and psoriasis. In addition to the increased keratin 10 expression in ILVEN sine psoriasis, T cells relevant in the pathogenesis of psoriasis are markedly reduced in ILVEN sine psoriasis as compared to psoriasis. T-cell subsets in ILVEN cum psoriasis had an intermediary position.

A di-leucine motif and an upstream serine in the interleukin-6 (IL-6) signal transducer gp130 mediate ligand-induced endocytosis and down-regulation of the IL-6 receptor.

The interleukin-6 (IL-6) receptor complex is composed of two different subunits, the IL-6 binding protein (IL-6R, gp80) and the signal transducing component gp130. Our previous studies revealed that the 10-amino acid sequence TQPLLDSEER within the intracellular domain of gp130 is crucial for the efficient internalization of IL-6. Since this sequence contains a putative di-leucine internalization motif, we further analyzed this region by constructing two additional deletions and a series of point mutants. Analyses of these mutants showed that the di-leucine pair (Leu-145 and Leu-146) is essential for ligand internalization, with leucine 145 being less resilient to exchanges. Furthermore, when a chimeric protein (Tac-STQPLL) composed of the Tac antigen fused to the hexapeptide STQPLL of gp130 was studied, we found that this sequence is sufficient to mediate endocytosis and lysosomal targeting of the chimera. Mutational analysis of three serine residues upstream of the di-leucine motif revealed that mutation of serine 139 to an alanine reduces the initial internalization rate by 50%. This finding suggests that a serine phosphorylation may be important for rapid endocytosis.

Expression of heat shock protein 70 (HSP70) at the interface of polymer-implants in vivo.

Synthetic polymer meshes are widely applied in the modern surgical approach for repairing abdominal wall defects. The implanted material is often observed leading to post-operative complications such as deficient abdominal wall mobility and adhesion formation with the abdominal cavity and/or abdominal organs. However, the functioning of the implant is primarily affected by the wound healing process guided by inflammatory events occurring at the tissue-material interface. This could presumably be influenced by the physicochemical properties of the polymer. With regard to it, the cellular and molecular processes involved in the successful restoration of the abdominal wall function are poorly understood. The present in vivo study, therefore, exemplary investigated in a rat model, the commercially available polymer-meshes Prolene (polypropylene, PP), Mersilene (polyester, PE) and Vicryl (polyglactin 910), as well as new mesh variants consisting either of PP (EB) or a combination of PP and polyglactin 910 (A plus or Vypro). The implanted material was evaluated by light and electron microscopy, immunohistochemistry as well as morphometry over an implantation period of 90 days. The data show that polymers induce heat shock protein (HSP)70, and its expression at the interface correlates inversely with the activity of the inflammatory reaction in vivo. Further, an ascent in HSP70 expression parallels the increasing implantation period and evolving foreign-body granulomas. Accordingly, a major role for HSP70 in modulating the local acceptance of polymers and as an additional marker for in vivo testing of polymers is suggestive.

Internalization of the interleukin 6 signal transducer gp130 does not require activation of the Jak/STAT pathway.

Signalling receptors often undergo receptor-mediated endocytosis. In many cases this internalization is stimulated by ligand binding and activation of intrinsic receptor tyrosine kinases, resulting in a receptor down-regulation. We have analysed whether internalization of the interleukin 6 signal transducer gp130 is dependent on the activation of receptor-associated Jak kinases. By using a chimaeric receptor system we found that receptor mutants that lack box1 and therefore are not capable of activating Jak and signal transducer and activator of transcription (STAT) proteins are still endocytosed efficiently. A chimaeric receptor with the recently identified dileucine internalization motif being replaced by two alanine residues was not efficiently internalized but still capable of recruiting STATs. Furthermore an antagonistic antibody that inhibits the signalling of all interleukin-6-type cytokines via gp130 was internalized as efficiently as an agonistic one that activates the Jak/STAT pathway. Our findings suggest that the endocytosis of gp130 is signal-independent.

Expression of the extracellular matrix proteins collagen I, collagen III and fibronectin and matrix metalloproteinase-1 and -13 in the skin of patients with inguinal hernia.

Although abnormal collagen metabolism has been ascribed an important role in the high recurrence rates after surgical hernia repair, knowledge on tissue sampled in the region affected by inguinal hernias is poor. In the present study, we determined collagen type I and type III in the skin of adult patients with indirect and direct inguinal hernias by both immunohistochemistry and Western blot analysis. In addition, we quantified the immunohistochemical expression of fibronectin and matrix metalloproteinase (MMP)-1 and -13. The results indicated that the ratio of collagen type I/III was significantly decreased in the skin of patients with either indirect (n = 9) or direct hernia (n = 7), with a concomitant increase in collagen type III (p < 0.001 vs. controls, n = 7, without affection of the inguinal region). There was no significant difference between patients with indirect and direct hernia (p > 0.05). MMP-13 was not expressed in any of the skin samples investigated, whereas MMP-1 was found in the epidermis. Fibronectin was predominantly detected at the epidermal-dermal junction. MMP-1, MMP-13 and fibronectin levels were significantly different between patients and controls (p > 0. 05). We conclude that in contrast to the unchanged expression of fibronectin and MMP-1 and MMP-13, the decreased ratios of collagen tpye I/III with the basically increased amount of collagen type III could be of significant importance for the pathophysiology of hernias. The specific ratio collagen I/III probably reflects the altered structural integrity and mechanical stability of the connective tissue in both indirect and direct hernias. Moreover, our findings stress that hernias should be regarded as the manifestation of a systemic disease in the inguinal region with a genetic background, explaining the high recurrence rates after repeated suture repair, as well as the usefulness of surgical meshes in this clinical setting.

Memory effector (CD45RO+) and cytotoxic (CD8+) T cells appear early in the margin zone of spreading psoriatic lesions in contrast to cells expressing natural killer receptors, which appear late.

BACKGROUND: An influx of immunocytes, increased epidermal proliferation and abnormal keratinization are hallmarks of the psoriatic lesion. T-lymphocyte subsets in particular activated effector memory T cells and natural killer (NK) T cells have been suggested to play an important role in the pathogenesis of psoriasis. OBJECTIVES: In the present study we investigated the number of T-cell subsets (CD4, CD8, CD45RO, CD45RA, CD2, CD25), cells expressing NK receptors (CD94 and CD161), the proliferation marker Ki67 and the keratinization marker keratin (K10) across the margin of the spreading psoriatic plaque: distant uninvolved skin, the outer margin (immediately outside the clinical edge), the inner margin (immediately inside the clinical edge) and the central area. PATIENTS AND METHODS: Eight patients with active psoriasis vulgaris participated in this study. Biopsies were taken from the spreading psoriatic lesion from the distant uninvolved skin, the outer margin, the inner margin and the central area. Biopsies were processed for immunohistochemical staining. RESULTS: In the outer margin CD8+ (cytotoxic T cells) and CD45RO+ (memory effector T cells) T lymphocytes invade the epidermis and in this early stage the activation markers CD2 and CD25 also show a substantial increase. The next phase, from the outer to the inner margin, shows a statistically significant increase of these markers, and especially, the cells expressing NK receptors (CD94 and CD161) show a massive increase together with a significant increase of epidermal proliferation (Ki67) and a decrease of the K10+ epidermal surface. CONCLUSIONS: CD8+, CD45RO+, CD2+ and CD25+ T cells have a role in the early phase of the psoriatic process, whereas CD94- and CD161-expressing cells together with epidermal proliferation and keratinization are involved in a later phase.

The effect of the combination of calcipotriol and betamethasone dipropionate versus both monotherapies on epidermal proliferation, keratinization and T-cell subsets in chronic plaque psoriasis.

Several reports have indicated that the combination of calcipotriol ointment and potent or ultrapotent corticosteroids are more effective and better tolerated, as compared to the monotherapies. The aim of the present study was to find out the effect of combination of calcipotriol ointment once daily and betamethasone dipropionate ointment once daily vs. the effect of twice-daily applications of each of the two treatments as monotherapy during a four-week treatment period. Seven patients with chronic plaque psoriasis were included for treatment with the three treatment schedules. Biopsies were taken before treatment and after four weeks of treatment, and markers for epidermal proliferation (Ki-67) and epidermal differentiation (keratin-10) were studied using a quantitative image analysis, and T-cell subsets in epidermis and dermis (CD4, CD8, CD25, CD45RO, CD45RA, CD94, CD161, and CD2) were studied using immunohistochemical scoring. The most impressive clinical result was reached with the combination. Calcipotriol proved to have a major effect on the proliferation marker Ki-67 and differentiation marker keratin-10, whereas the effect on T-cell subsets was more selective with major reductions of CD45RO(+) and CD8(+) T cells. In contrast, the effect of betamethasone dipropionate on the epidermis was restricted to a normalization of differentiation with a highly significant increase of keratin-10 positive epidermal surface without a significant effect on Ki-67 positive nuclei, and the effect on T-cell subsets was restricted to a reduction of natural killer T-cell receptors designated by CD94 and CD161 in the epidermis. The combination of the two treatments did not affect the proliferation marker Ki-67 and keratinization marker keratin-10, beyond the effect of calcipotriol monotherapy. However, the combination had a profound effect on, virtually, all T-cell subsets, beyond the effect of the monotherapies. It is concluded that the action spectra of calcipotriol and betamethasone on the psoriatic plaque are different and that the combination has effects on T-cell subsets, beyond the addition of the effects of monotherapies.