Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

BACKGROUND: Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. RESULTS: We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 µM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. CONCLUSION: Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Nephrogenic systemic fibrosis with a spectrum of clinical and histopathological presentation: a disorder of aberrant dermal remodeling.

BACKGROUND: Nephrogenic fibrosing dermopathy (NFD) has emerged as a clinicopathologic entity since 1997 and recently renamed as nephrogenic systemic fibrosis (NSF). The etiology and pathogenesis remain uncertain. Characteristic clinical presentation is described as diffuse thickening and hardening of the skin occurring in patients with renal insufficiency. Typical histological features include proliferation of CD34 positive fibrocytes, increased thick collagen bundles and mucin deposition, without significant inflammatory infiltrate. Variations in clinical presentations have been reported, including papular and plaque-like skin lesions, focal lesion only, as well as systemic involvement. Histological changes can be subtle and non-specific, overlapping with other disease processes and harboring features including calcification and osteoclast-like giant cells with osseous metaplasia. METHODS: We reviewed patients with NSF that presented to our dermatology clinic by chart review, clinical examination and histological examination. Skin biopsy specimens were obtained from all cases. Histopathology evaluations were carried out by three dermatopathologists (AD, BS and GK) independently and the features were compared among all the cases. Special stains and immunohistochemistry study were also performed to highlight the histological features. RESULTS: Seven cases of NSF presented with a spectrum of clinical manifestations, from classic diffuse hardening of the skin to localized linear plaques. On histological examination, proliferation of CD34-positive fibrocytes ranged from sparse to dense, collagen bundles ranged from thin to thick, and the interstitial dermal mucin accumulation ranged from scant-patchy to abundant. In addition, the lesion displayed various degrees of vascular proliferation, inflammatory infiltrates and intensities of CD68 and Factor XIIIa staining. Two cases showed extensive dermal calcification and ossification. CONCLUSION: NSF may present with a spectrum of clinical abnormalities, and exhibit overlapping histopathological features resembling cicatrix and other dermal reparative/regenerative processes. NSF may in fact to be a disorder of aberrant extracellular matrix remodeling in patients with renal insufficiency.

Microvesicating effects of sulfur mustard on an in vitro human skin model.

Bis-(beta-chloroethyl) sulfide (SM) is a potent skin vesicant previously used for chemical warfare. Progress in determination of the mechanistic basis of SM pathology, and development of prophylactic and/or therapeutic countermeasures to SM exposure has been hampered by lack of physiologically relevant models of human skin. The current work evaluated a newly developed tissue engineered full-thickness human skin model in a completely in vitro approach to investigation of SM-induced dermal pathology. The model was first characterized with regard to overall morphology, lipid composition, basement membrane (BM) composition and ultrastructural features that are important targets of SM pathologic activity. Well-developed BM ultrastructural features were observed at the dermal-epidermal junction (DEJ), thus demonstrating successful resolution of a primary deficiency of models previously evaluated for SM studies. Studies were then conducted to evaluate histopathological effects of SM on the model. Good replication of in vivo effects was observed, including apoptosis of basal keratinocytes (KC) and microblister formation at the DEJ. Tissue engineered skin models with well-developed basement membrane structures thus appear to be useful tools for in vitro mechanistic studies of SM vesicant activity and development of preventive/therapeutic approaches for SM pathology.

Activation of keratinocyte protein kinase C zeta in psoriasis plaques.

PKCzeta (protein kinase C-zeta), a member of protein kinase C family, plays an important role in cell proliferation, differentiation, and apoptosis. It acts as a downstream molecule for TNF-alpha (tumor necrosis factor) signal transduction and also regulates the expression of CD1d, an HLA-class I-like molecule. The interaction of CD1d with natural killer T (NKT) cells has been shown to be important in their Th1 cytokine production in psoriasis. In this study, we examined PKCzeta in psoriasis in order to define its role in the pathogenesis of the disease. We found that T-cell receptor (TCR) V alpha24+ V beta11+ NKT cells and CD1d molecules within psoriatic skin were increased. Moreover, there was an associated increase in PKCzeta mRNA and protein expression with membrane translocation in psoriasis lesions compared to uninvolved skin. Furthermore, cultured keratinocytes exhibited increased PKCzeta activity and membrane translocation upon stimulation by TNF-alpha, a cytokine known to play an important role in the pathogenesis of psoriasis. These results implied that PKCzeta is an important transduction molecule downstream of TNF-alpha signaling and is associated with increased expression of CD1d that may enhance CD1d-NKT cell interactions in psoriasis lesions. This makes PKCzeta a tempting target for possible pharmacological intervention in modifying the downstream effects of TNF-alpha in psoriasis.

Primary cutaneous Langerhans cell sarcoma without Birbeck granules: indeterminate cell sarcoma?

An 88-year-old white male presented with a rapidly growing skin nodule on the scalp. Clinically, the nodule did not appear unusual for an ordinary cutaneous neoplasm on sun-exposed skin of an elderly white male. Histopathological examination showed sheet-like epithelioid tumor cell growth with a vaguely nested pattern and frank malignant features, resembling malignant melanoma. However, the tumor cells possessed irregularly convoluted nuclei with nuclear groves, frequent multinucleation and fine vesicular cytoplasm, features highly suggestive of histiocytes. Immunohistochemistry studies showed that the tumor cells were diffusely positive for S-100 protein and CD1a and negative for HMB-45, Melan-A, cytokeratin and CD30. The provisional diagnosis of Langerhans cell sarcoma was thus favored. To confirm this diagnosis, electron microscopic examination was performed. Although classic features of histiocytes were readily identifiable, no Birbeck granules could be found upon a thorough search on repeated sections. These results are indicative of the indeterminate cell nature of the tumor. We propose a diagnosis of primary cutaneous indeterminate cell sarcoma for this unusual histiocytic neoplasm. Current classification of histiocytic neoplasms and differential diagnosis are reviewed.

Familial cutaneous collagenoma.

A 41-year-old woman presented to our dermatology clinic in February 2005 with a chief complaint of numerous flesh-colored nodules on her back and abdomen. She initially noticed the lesions at age 17 years. The plaques had increased in size and number over time, but remained asymptomatic. The patient reported multiple similar lesions on a maternal uncle and a cousin. Her family history was also notable for cardiomyopathy, resulting in the death of her mother. The patient's past medical history was notable for poorly controlled type I diabetes, currently managed with an insulin pump; and coronary artery disease. The patient had undergone multiple cardiac procedures before the age of 40 years, including quadruple coronary artery bypass grafting surgery and placement of 9 cardiac stents. Her ejection fraction on cardiac catheterization in November 2004 was 65% with no wall motion abnormalities. On physical examination, numerous spongy, discrete, flesh-colored plaques and nodules were seen concentrated across the upper part of her back between the scapulae as well as underneath the breasts and across the flanks (Figure 1). All lesions were asymptomatic. Prior workup of this patient had included plain films of the long bones and hands, which were within normal limits. A biopsy from lesional skin on the back highlighted by trichome stain showed an increased number of markedly thickened and eosinophilic dermal collagen bundles compared with adjacent normal skin. Immunohistochemical studies with anticollagen type I and type III antibodies confirmed that the increased collagen material consisted of type I collagen fibers, which is the same type of collagen found in normal dermis. The elastic fibers, highlighted by Verhoeff-van Gieson stain (Figure 2), were diminished and haphazardly arranged. No increased cellular component or inflammatory infiltrate was observed. These findings were consistent with a collagenoma. Further analysis of the lesional tissue by electron microscopy revealed that the ultrastructural appearance of the collagen fibers, including arrangement and diameters, were not significantly different from that of the normal tissue (Figure 3).

Characterization of the initial response of engineered human skin to sulfur mustard.

We have used a new approach to identify early events in sulfur mustard-induced, cutaneous injury by exposing human, bioengineered tissues that mimic human skin to this agent to determine the morphologic, apoptotic, inflammatory, ultrastructural, and basement membrane alterations that lead to dermal-epidermal separation. We found distinct prevesication and post-vesication phases of tissue damage that were identified 6 and 24 h after sulfur mustard (SM) exposure, respectively. Prevesication (6 h) injury was restricted to small groups of basal keratinocytes that underwent apoptotic cell death independent of SM dose. Immunoreactivity for basement membrane proteins was preserved and basement membrane ultrastructure was intact 6 h after exposure. Dermal-epidermal separation was seen by the presence of microvesicles 24 h after SM exposure. This change was accompanied by the dose-dependent induction of apoptosis, focal loss of basement membrane immunoreactivity, increase in acute inflammatory cell infiltration, and ultrastructural evidence of altered basement membrane integrity. These studies provide important proof of concept that bioengineered, human skin demonstrates many alterations previously found in animal models of cutaneous SM injury. These findings further our understanding of mechanisms of SM-induced damage and can help development of new countermeasures designed to limit the morbidity and mortality caused by this chemical agent.

Benign cutaneous pseudoglandular schwannoma: a case report.

Schwannoma is a common peripheral neural neoplasm that could present as a primary skin lesion. In addition to typical schwannoma with classic Antoni A and Antoni B areas, many variant types have been described, such as plexiform, cellular, epithelioid, and ancient schwannomas. Glandular schwannoma is a rare variant characterized by the presence of glands in an otherwise typical schwannoma. There are also a few reported cases in the literature of pseudoglandular schwannoma from central nervous system, eye, submandible, and shoulder, in which the gland-like structures were lined by Schwann cells. We report here a patient with a benign cutaneous schwannoma composed of predominantly gland-like spaces that contained mucinous material and were lined by Schwann cells confirmed by immunohistochemistry and ultrastructural studies. The tumor was well circumscribed and showed minimal cytologic atypia, indicating benignity. We report this unusual case of benign cutaneous pseudoglandular schwannoma to further awareness of the morphologic diversity of schwannoma.

Immunolocalization of MAP-2 in routinely formalin-fixed, paraffin-embedded guinea pig brain sections using microwave irradiation: a comparison of different combinations of antibody clones and antigen retrieval buffer solutions.

The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

Sulfur mustard-induced apoptosis in hairless guinea pig skin.

The present study was aimed to examine whether apoptosis is involved in the pathogenesis of sulfur mustard (SM)-induced basal cell death. Skin sites of the hairless guinea pig exposed to SM vapor for 8 minutes were harvested at 3, 6, 12, 24, and 48 hours postexposure. Immunohistochemical detection of basal cell apoptosis was performed using the ApopTag in situ apoptosis labeling kit. Only occasional apoptotic basal cells (BC)were observed in nonexposed and perilesional control sites. At lesional sites, apoptosis of BC was not detected at 3 hours postexposure. However, at 6 hours and 12 hours postexposure, 18% and 59% of BC were apoptotic, respectively. At 24 and 48 hours postexposure, individual apoptotic basal cells were not clearly recognizable due to necrosis. At the ultrastructural level, degenerating BC exhibited typical apoptotic morphology including nuclear condensation and chromatin margination. The results suggest that apoptotic cell death is a cytotoxic mechanism with the number of BC undergoing apoptosis significantly increasing from 6 to 12 hours postexposure. In addition, because necrosis is preferential at 24 hours postexposure, we believe that SM-induced cell death involves early apoptosis and late necrosis, which temporally overlap to produce a single cell death pathway along an apoptotic-necrotic continuum.