Enzymatic distinction between two subgroups of autosomal recessive lamellar ichthyosis

It has been proposed that the autosomal recessive lamellar ichthyoses may be divided into two subgroups, the erythrodermic (EARLI) and non-erythrodermic (NEARLI) forms. We report measurements of the enzymes beta-glucosidase, a recently described phosholipase, a short-chain carboxylesterase ("butyrase"), and a long-chain carboxylesterase ("palmitase") in aqueous extracts of scales from patients diagnosed according to clinical and micromorphologic criteria, and show that beta-glucosidase and phospholipase tend to be lower in the EARLI group, whereas butyrase is relatively low in the NEARLI group. The internal ratio of either butyrase/glucosidase or butyrase/phospholipase yields a clear separation of the two subgroups, supporting the concept of heterogeneity in this group of diseases.     

Colocalization of cystatin M/E and cathepsin V in lamellar granules and corneodesmosomes suggests a functional role in epidermal differentiation

Cystatin M/E is a cysteine protease inhibitor with two distinct binding sites for papain-like cysteine proteases (family C1) and the asparaginyl endopeptidase (AEP) legumain of family C13. We have previously demonstrated that deficiency of cystatin M/E in mice causes ichthyosiform skin changes and barrier disruption, which could be caused by unrestrained AEP activity. Recently, we provided biochemical evidence that human cathepsin V (CTSV) and cathepsin L (CTSL) are additional biological targets for human cystatin M/E. To address the possible role of these three proteases and their inhibitor in epidermal differentiation, we investigated the localization of these proteins in normal human skin. Whereas CTSL and AEP were broadly expressed in epithelial cells of the skin, we found a specific colocalization of cystatin M/E and CTSV in the stratum granulosum and in the root sheets of the hair follicle, using immunofluorescence microscopy. Immunoelectron microscopy revealed that cystatin M/E and CTSV are separately transported within the lamellar granules. Cystatin M/E was also found in the extracellular space in the stratum corneum associated with corneodesmosomes, where it was closely associated with CTSV. Based on the striking stratum-specific colocalization of cystatin M/E and CTSV, we propose that these molecules could have an important role in epidermal differentiation and desquamation.     

Tenascin-C degradation in chronic wounds is dependent on serine proteinase activity

Abstract Degradation of extracellular matrix (ECM) components by proteinases is part of the physiological remodelling process during normal wound healing. Excessive degradation of the ECM, however, is likely to create an environment that can no longer support keratinocyte migration and is thought to play a role in the impaired healing of chronic ulcers. Tenascin-C is an ECM component that is markedly upregulated in acute and chronic wounds. Here we report on our investigations into the degradation of tenascin-C in chronic venous leg ulcers. We found proteolytic fragments of tenascin-C in leg ulcer exudate. We also detected fragments of fibronectin in the wound fluid and in addition observed breakdown of fibronectin by wound fluid in vitro, as has previously been reported by others. Wound fluid of four out of six chronic leg ulcers degraded purified human tenascin-C in vitro, and degradation of tenascin-C correlated with high levels of functionally active leucocyte elastase and metalloproteinases in the wound fluid. To identify which proteinases were involved in tenascin-C degradation, we tested the effect of specific proteinase inhibitors. The addition of EDTA or E64 did not protect tenascin-C from degradation, suggesting that neither metalloproteinases nor cysteine proteinases are responsible for cleavage. Tenascin-C breakdown was inhibited by PMSF and SKALP/elafin, and we therefore conclude that leucocyte elastase and possibly other serine proteinases are the tenascin-C-degrading enzymes in ulcer exudate. Taking into account the possible effects of tenascin-C and tenascin-C fragments on cell behaviour, we hypothesize that degradation of tenascin-C could affect the healing process in chronic venous ulcers. Received: 8 January 1998     

Malignant progression and blockade of angiogenesis in a murine transgenic model of neuroblastoma

Targeted expression of MYCN to the neural crest [under control of the rat tyrosine hydroxylase (TH) promoter] causes neuroblastoma in transgenic mice (TH-MYCN) and is a well-established model for this disease. Because high levels of MYCN are associated with enhanced tumor angiogenesis and poor clinical outcome in neuroblastoma, we serially characterized malignant progression, angiogenesis, and sensitivity to angiogenic blockade in tumors from these animals. Tumor cells were proliferative, secreted high levels of the angiogenic ligand vascular endothelial growth factor (VEGF), and recruited a complex vasculature expressing the angiogenic markers VEGF-R2, alpha-SMA, and matrix metalloproteinases MMP-2 and MMP-9, all of which are also expressed in human disease. Treatment of established murine tumors with the angiogenesis inhibitor TNP-470 caused near-complete ablation, with reduced proliferation, enhanced apoptosis, and vasculature disruption. Because TNP-470 has been associated with neurotoxicity, we tested the recently described water-soluble HPMA copolymer-TNP-470 conjugate (caplostatin), which showed comparable efficacy and was well tolerated without weight loss or neurotoxicity as measured by rotarod testing. This study highlights the importance of angiogenesis inhibition in a spontaneous murine tumor with native tumor-microenvironment interactions, validates the use of mice transgenic for TH-MYCN as a model for therapy in this common pediatric tumor, and supports further clinical development of caplostatin as an antiangiogenic therapy in childhood neuroblastoma.     

Integrin beta3 overexpression suppresses tumor growth in a human model of gliomagenesis: implications for the role of beta3 overexpression in glioblastoma multiforme

alphaVbeta3 integrin complexes are overexpressed in the growing, invading margins of human glioblastoma multiforme (GBM) and in the GBM vasculature, suggesting a key role for alphaVbeta3 in GBM growth and invasion. The function of alphaVbeta3 complexes in tumor formation, however, has been challenged by studies showing that loss of alphaVbeta3 expression (via loss of beta3) in the host vasculature enhances, rather than suppresses, the growth of s.c. implanted carcinomas. To directly address the role of tumor-specific alphaVbeta3 overexpression in glioma formation, we increased alphaVbeta3 expression (via overexpression of a wild-type or constitutively activated beta3) in human astrocytes genetically modified to form anaplastic astrocytoma-like tumors (Ras cells) on intracranial injection in rats. Overexpression of beta3 selectively increased levels of alphaVbeta3 integrin complexes, but had no effect on anchorage-dependent or -independent growth in vitro. After intracranial injection, however, the Ras + beta3 cells formed fewer and smaller tumors than did Ras cells. Similarly, Ras-transformed mouse astrocytes that were derived from control animals formed smaller intracranial tumors than those derived from beta3 knockout animals. Although tumors formed by human Ras and Ras + beta3 cells were similar in blood vessel density, Ras + beta3 tumors had smaller, pericyte-depleted vessels and were significantly more hypoxic, suggesting a beta3-mediated vascular defect. The growth-suppressive actions of beta3, however, could be overcome by stimulation of pathways (Akt or vascular endothelial growth factor) commonly activated in GBM. These results show that tumor-specific alphaVbeta3 overexpression has growth-suppressive effects in gliomas, but that these deleterious effects are mitigated by alterations common to alphaVbeta3-overexpressing GBM.     

Comparison of a conventional cardiac-triggered dual spin-echo and a fast STIR sequence in detection of spinal cord lesions in multiple sclerosis

The current optimal imaging protocol in spinal cord MR imaging in patients with multiple sclerosis includes a long TR conventional spin-echo (CSE) sequence, requiring long acquisition times. Using short tau inversion recovery fast spin-echo (fast STIR) sequences both acquisition time can be shortened and sensitivity in the detection of multiple sclerosis (MS) abnormalities can be increased. This study compares both sequences for the potential to detect both focal and diffuse spinal abnormalities. Spinal cords of 5 volunteers and 20 MS patients were studied at 1.0 T. Magnetic resonance imaging included cardiac-gated sagittal dual-echo CSE and a cardiac-gated fast STIR sequence. Images were scored regarding number, size, and location of focal lesions, diffuse abnormalities and presence/hindrance of artifacts by two experienced radiologists. Examinations were scored as being definitely normal, indeterminate, or definitely abnormal. Interobserver agreement regarding focal lesions was higher for CSE (ϰ = 0.67) than for fast STIR (ϰ = 0.57) but did not differ significantly. Of all focal lesions scored in consensus, 47 % were scored on both sequences, 31 % were only detected by fast STIR, and 22 % only by dual-echo CSE (n. s.). Interobserver agreement for diffuse abnormalities was lower with fast STIR (ϰ = 0.48) than dual-echo CSE (ϰ = 0.65; n. s.). After consensus, fast STIR showed in 10 patients diffuse abnormalities and dual-echo CSE in 3. After consensus, in 19 of 20 patients dual-echo CSE scans were considered as definitely abnormal compared with 17 for fast STIR. The fast STIR sequence is a useful adjunct to dual-echo CSE in detecting focal abnormalities and is helpful in detecting diffuse MS abnormalities in the spinal cord. Due to the frequent occurrence of artifacts and the lower observer concordance, fast STIR cannot be used alone. Received: 9 September 1999; Revised: 14 December 1999; Accepted: 16 December 1999     

The effect of the neuroprotective agent riluzole on MRI parameters in primary progressive multiple sclerosis: a pilot study

Progressive axonal loss is the most likely pathologic correlate of irreversible neurologic impairment in primary progressive multiple sclerosis. In a run-in versus treatment trial, we show that the neuroprotective agent riluzole seems to reduce the rate of cervical cord atrophy and the development of hypointense T1 brain lesions on magnetic resonance imaging.     

Tumor necrosis factor related apoptosis inducing ligand triggers apoptosis in dividing but not in differentiating human epidermal keratinocytes

Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis in transformed and tumor cell lines, but usually not in other cells. Here we present a study on the effect of TRAIL on cultured keratinocytes. It is shown that differentiated and undifferentiated keratinocytes undergo apoptosis after addition of TRAIL to the medium as determined by morphologic and biochemical criteria, such as cellular shrinkage and activation of caspases. The sensitivity for TRAIL differs greatly between undifferentiated and differentiating keratinocytes, however, with undifferentiated cells being much more susceptible to apoptosis. Commitment to terminal differentiation in the absence of TRAIL does not in itself induce apoptosis. In contrast to the promyelocytic cell line HL60, internucleosomal DNA fragmentation is not observed in keratinocytes, as assessed by flow cytometric analysis and agarose gel electrophoresis. Interestingly, the prime effector of DNA fragmentation, DNA fragmentation factor of 40 kDa (DFF40), is expressed in keratinocytes, yet internucleosomal cleavage fails to occur. Our data indicate that programmed cell death during keratinocyte differentiation is distinct from receptor-mediated apoptosis in response to a death ligand.     

The effects of oral liarozole on epidermal proliferation and differentiation in severe plaque psoriasis are comparable with those of acitretin

The imidazole derivative liarozole is a potent inhibitor of cytochrome P450-dependent 4-hydroxyla-tion of endogenous all- trans retinoic acid, thereby increasing the levels of all- trans retinoic acid in both plasma and skin. As part of a large, double-blind, randomized clinical study, we investigated the cell biological alterations in uninvolved and lesional skin of 20 patients with severe plaque psoriasis, who were treated with either liarozole or acitretin. The extent and severity of the skin lesions, as recorded by the Psoriasis Area and Severity Index score, was significantly reduced ( P 005) after 12 weeks of treatment in both the acitretin- and the liarozole-treated group. A significant decrease in the markers for inflammation (neutrophils), epidermal proliferation (Ki-67-positive cells), normal differentiation (transglutaminase) and abnormal differentiation [cytokeratin 16 and skin-derived antileucoproteinase (SKALP), also known as elafin] was seen in both groups. No significant differences were noted in clinical scores or cell biological scores between the liarozole- and acitretin-treated group. None of the markers returned to the levels seen in uninvolved skin or in normal human skin. The expression of epidermal fatty acid binding protein (E-FABP) was only minimally decreased after 12 weeks of treatment, a substantial part of the stratum spinosum remaining positive. SKALP levels in serum fell in both groups with similar kinetics and showed a statistically significant correlation with clinical scores. A remarkable finding in the uninvolved skin of patients treated with liarozole or acitretin was the distinct focal expression of SKALP in the granular layer and the expression of E-FABP in the spinous layers, which is not found in normal human skin. Although the mechanism of action differs fundamentally, liarozole and acitretin show similar effects with respect to clinical effects and cell biological changes in the lesional and non-lesional skin.     

Optimizing the association between disability and biological markers in MS

OBJECTIVE: Axonal damage is an important feature of MS pathology and the likely substrate of development of progressive disability. Brain volume measurement on MRI can be used as an overall marker of tissue damage and axonal loss. The authors studied the relation of brain volume measurements with the MS Functional Composite (MSFC) in an attempt to improve the clinico-radiologic association. METHODS: In 137 patients with MS (80 relapsing-remitting [RR], 36 secondary progressive [SP], and 21 primary progressive [PP]) and 12 healthy controls, a brain MRI scan was obtained. Patients also underwent MSFC and Expanded Disability Status Scale (EDSS) assessments. MRI analysis included determination of hypointense T1- and hyperintense T2-weighted lesion load, and two brain volume measurements: 1) the parenchymal fraction (PF): whole brain parenchyma/intracranial volume; and 2) the ventricular fraction (VF): ventricular volume/whole brain parenchyma. RESULTS: The median PF was smaller and the median VF larger in the patient group (0.81 for PF and 0.029 for VF) than in the control group (0.87 for PF, p < 0.001; and 0.013 for VF, p < 0.01). For the patient population, moderate correlations were found between brain volume measurements and MSFC (0.36 for PF and -0.40 for VF). Patients with short disease duration showed a correlation of MSFC with both brain and lesion volume measurements on MRI, whereas patients with long disease duration only showed a correlation with brain volume measurements. CONCLUSION: Brain volume measurements are correlated with disability as assessed by the MSFC. Although in the early phase of the disease the amount of focal demyelination is important, the residual brain volume seems to be more relevant in determining disability in later phases of the disease.