Could X-ray microbeams inhibit angioplasty-induced restenosis in the rat carotid artery?

BACKGROUND: Parallel, thin (<100 microm) planes of synchrotron-generated X rays, have been shown to spare normal tissues and preferentially damage tumors in animal models. The aim of the present study was to assess the effect of such microbeams directed unidirectionally on angioplasted rat carotid arteries. METHODS AND MATERIALS: Three groups of Sprague-Dawley rats were studied: (a) rats with normal, untreated arteries, (b) rats treated by balloon angioplasty, but not irradiated, and (c) rats treated with balloon angioplasty and exposed to single fraction, unidirectional, parallel, microbeams an hour after angioplasty. The microbeam array, 15 mm widex7.6 mm high, consisting of 27-microm-wide beam slices, spaced 200 microm center-to-center laterally traversed the damaged artery. The in-depth in-beam dose was 150 Gy, the "valley" dose (dose midway between microbeams resulting mainly from X-ray scattering) was 4.5 Gy on average, and the "integrated" (averaged) dose was 26 Gy. RESULTS: Microbeam irradiation, as given in the present study, was tolerated, but was insufficient to significantly suppress the neointimal hyperplasia. DISCUSSION: The microbeam dose used is considered low. Dose escalation would be necessary to reach conclusive results regarding the X-ray microbeam efficacy to control restenosis.

Eosinophilic fasciitis occurring four weeks after the onset of dialysis in a renal failure patient.

BACKGROUND: Eosinophilic fasciitis is a rare, scleroderma-like disease that usually affects the extremities of young to middle-aged males. The disease may cause flexion contractures and limit joint mobility and is associated with peripheral eosinophilia. The fascia, by definition, is infiltrated with mononuclear cells and typically with eosinophils. Eosinophilic fasciitis may be separated from another sclerodermatous disorder, linear scleroderma, by its response to systemic corticosteroids. The etiology is unclear but eosinophilic fasciitis has numerous disease associations. However, it has not previously been associated with renal failure and hemodialysis. OBJECTIVE: This article reports a case of eosinophilic fasciitis occurring four weeks following the onset of hemodialysis. METHODS: The clinical and histologic features confirmed the diagnosis of eosinophilic fasciitis. He was treated with systemic corticosteroids with good response. CONCLUSION: This is the first reported patient who developed eosinophilic fasciitis in close temporal relationship with the start of hemodialysis. While eosinophilic fasciitis may be coincidental with a common disorder, namely, renal failure, it is interesting to note that hemodialysis patients often have immune-regulation abnormalities and peripheral eosinophilia.

Telemedicine: evolving technology in an e-health care world.

This article provides an overview of telemedicine and its expanding capabilities to deliver health care services. Clinical applications, including home care, continue to evolve and expand as the technology improves and the experience of telemedicine service providers grows. As an emerging technology in a changing health care environment, implementation of telemedicine is not without its challenges. The future of telemedicine will be impacted by the emerging emphasis on e-health care.

IgA1 is the major IgA subclass in cutaneous blood vessels in Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is characterized by palpable purpura predominantly involving the lower extremities. On direct immunofluorescence IgA can be seen deposited in the blood vessel walls of the superficial dermis. The subclass distribution of antibodies to this IgA was studied in the biopsies of 28 patients with HSP by direct immunofluorescence using anti-IgA1 and anti-IgA2 specific monoclonal antibodies. All 28 patients' biopsies demonstrated deposition of IgA1 while only one patient had IgA2 deposition. Positive and negative controls stained appropriately. This demonstrates that IgA1 is the dominant IgA subclass found in the skin in Henoch-Schönlein purpura.

IgA antibodies recognizing LABD97 are predominantly IgA1 subclass.

Linear IgA bullous dermatosis is a rare acquired subepidermal blistering disease of the skin. A recognized antigen in linear IgA bullous dermatosis is a 97-kDa basement membrane zone protein termed LABD97. Previous studies, using immunofluorescent techniques, have suggested that the IgA response is restricted to the IgA1 subclass. We studied the IgA antibody subclasses in the sera of 6 patients that contained circulating IgA antibodies reactive with LABD97. The methods used included direct and indirect immunofluorescence and Western immunoblot. All patients tested had IgA1 anti-LABD97 antibodies detected by all 3 methods. Two patients had IgA2 antibodies detected by direct immunofluorescence. Three patients had IgA2 antibodies on indirect immunofluorescence. Two of these also had anti-LABD97 IgA2 antibodies and 1 had secretory component containing anti-LABD IgA antibodies on Western immunoblot. We conclude that the predominant IgA antibody subclass reactive with LABD97 in LABD is IgA1, although the IgA2 subclass may be involved in some cases.

The immunoglobulin A antibody response in clinical subsets of mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) is an immunobullous disease. In MMP there is frequently a mixed antibody response with the presence of IgA and/or IgG antibodies directed toward basement membrane zone antigens. The IgG antibody response in MMP has been studied, but the antigens to which the IgA antibodies react have not been studied. OBJECTIVE: To determine the IgA autoantibody reactivity profiles in patients with MMP. METHODS: Patients who had both ocular and oral MMP were compared with patients who had ocular or oral MMP and with patients who had cutaneous linear IgA disease (LABD) by Western immunoblot studies. RESULTS: Five of 15 MMP patients and 1 of 5 LABD patients had IgA antibodies reactive with the 180-kD bullous pemphigoid antigen. Seven of 15 MMP patients had IgA antibodies reactive with the 97-kD LABD antigen. CONCLUSION: Major antigens in IgA MMP are the 180-kD bullous pemphigoid antigen and the 97-kD LABD antigen.

Characterization of the antibody response in oesophageal cicatricial pemphigoid.

Cicatricial pemphigoid (CP) is a subepidermal, autoimmune bullous dermatosis. It is classified as a clinical subset of bullous pemphigoid (BP). However, it differs from BP in some significant ways: (i) in CP mucosal involvement with clinical scarring is prominent; (ii) there is a prominent IgA class antibody response alone or in addition to the IgG class antibody response; and (iii) there is a heterogeneous antibody response in CP, whereas in BP the majority of the antibodies are directed against a 180-kDa hemidesmosomal protein, bullous pemphigoid antigen 2 (BPAg2). Oesophageal involvement in CP is a rare, but often devastating manifestation. In this study we examined the humoral autoimmune response in oesophageal CP, in an attempt to characterize the autoantibody reactivity profile. We used direct and indirect immunofluorescence and Western immunoblotting using normal human skin and oesophagus substrates. We studied patient sera over time in order to search for evidence of epitope spreading in these patients. All patients had positive direct immunofluorescence of perilesional oesophageal epithelium. All patients had positive circulating antibasement membrane zone autoantibody titres. There was a significant IgA class in addition to an IgG class autoantibody response. IgA and IgG antibodies demonstrated significant reactivity with BPAg2 and the 97 kDa linear IgA disease antigen on Western immunoblot suggesting intraprotein epitope spreading. There was no evidence of interprotein epitope spreading over time. Our findings suggest that there is a heterogeneous antibody response in oesophageal CP with the predominant antigen being BPAg2.

Biologic aspects of expression of stably integrated transgenes in cells of the skin in vitro and in vivo.

The observation that transgenes can be stably integrated into the genome of fibroblasts using recombinant retroviruses enhanced interest in using these cells as a vector for gene therapy. This enthusiasm has lessened during the past 8 years, not because skin has lost the features that make it attractive for gene therapy, but rather because stable transgene expression in vivo has not been achieved. All investigators who have used genetically modified fibroblasts to study in vivo aspects of gene therapy have shown a decrease in transgene expression with time. This contrasts with transgene expression in similarly transduced fibroblasts in vitro, where expression is not lost or is lost very slowly. We have initiated an approach to bring further understanding to the biology of transgene expression by fibroblasts carrying stably integrated transgenes in an in vivo setting. Experiments described permit the following conclusions. Expression by and survival of genetically modified fibroblasts a) requires a persistent matrix scaffold in in vivo settings; b) is prolonged if the matrix is allowed to mature in vitro; c) is enhanced if the matrix is partially sequestered behind a coating of normal fibroblasts; and d) can be substantively prolonged in vivo by immortalizing the cells. These observations support the notion that prolonged expression of transgenes by fibroblasts can be achieved in vivo and that gene therapy utilizing fibroblasts and other cells of the skin has clinical utility.

Bullous pemphigoid sera that contain antibodies to BPAg2 also contain antibodies to LABD97 that recognize epitopes distal to the NC16A domain.

IgG antibodies from the sera of some patients with bullous pemphigoid (BP) react with a 180 kDa protein termed BPAg2. Antibodies in BP are directed to an extracellular noncollagenous domain of this protein termed NC16A. Our group has recently shown that a portion of the extracellular domain of BPAg2 is identical to LABD97 on the basis of amino acid sequencing. We evaluated sera from 33 patients with BP with circulating IgG antibodies on indirect immunofluorescence, which stained the epidermal side of split skin with titers ranging from 1:40 to 1:640. Immunoblotting was performed against (i) two preparations of proteins from epidermal extract, one containing BPAg2 and one containing LABD97, and (ii) the recombinant NC16A domain of the BPAg2 protein. Twelve sera reacted with the BPAg2 protein. Ten of these also reacted strongly with the NC16A domain. Nine of the 12 sera also reacted with the LABD97 antigen. Bound antibodies were eluted from the 97 kDa band and reapplied to split skin where they bound to the epidermal side. The eluted antibodies also reacted to the BPAg2 protein from the epidermal extract, but did not react with the NC16A domain on immunoblot. We conclude that these nine sera react with an epitope present within BPAg2 and LABD97 but not within the NC16A domain. This epitope is therefore distal to the previously described epitopes in BP. In BP, epitope spreading may occur and antibodies may be produced that recognize the distal portion of the BPAg2 molecule identical to LABD97 but that do not involve the NC16A domain.

Persistent transgene expression and normal differentiation of immortalized human keratinocytes in vivo.

Cells transduced ex vivo with transgenes encoded on retroviruses have constant and prolonged expression in vitro; however, in vivo expression is quickly lost. Much attention has been directed at methods to circumvent this problem. We have shown that loss of transgene expression does not occur when transduced immortalized 3T3 cells are transplanted to the in vivo setting of athymic mice. Ease of acquisition and potential for clinical application led us to assess the potential of using immortalized human keratinocytes for expression of transgenes in vivo. Human keratinocytes were immortalized with a HPV16-E6/E7 retrovirus, transduced with a lacZ retrovirus, cloned by limiting dilution, seeded onto a physiologic dermal substrate, and transplanted to athymic mice. Six weeks after transplantation, the immortalized transgene expressing keratinocytes had formed an epidermis that was indistinguishable from one formed by nonimmortalized keratinocytes; furthermore, there was no loss of expression of the lacZ gene. These observations show that methods to extend cell survival are an alternative approach to achieving stable and prolonged expression of transgenes in vivo and that HPV16-E6/ E7 immortalized keratinocytes generate an epidermis with normal morphology.

The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2.

IgA autoantibodies from the sera of some patients with linear IgA bullous dermatosis (LABD) recognize a 97 kDa antigen (LABD97) located in the lamina lucida of the basement membrane zone. As LABD autoantibodies do not react with the 180 and 230 kDa proteins recognized by bullous pemphigoid autoantibodies, LABD97 has been thought to represent a separate lamina lucida protein. In this study, we purified LABD97 from the extract of human epidermis using a monoclonal antibody immunoaffinity column and analyzed the amino acid sequence of the N terminus of purified LABD97. This revealed a 16 amino acid sequence that was identical to a previously reported sequence of the 180 kDa antigen in bullous pemphigoid (BPAg2). The N terminus was located 41 amino acids downstream from the carboxyl end of the transmembrane domain of BPAg2 and 11 amino acids downstream from the MCW-1 domain, the predominant bullous pemphigoid epitope. Purified LABD97 was subsequently enzymatically digested with endoproteinase Arg C and separated by chromatography, which resulted in multiple peptide fractions. Fourteen of these fractions were subjected to amino acid sequencing. The amino acid sequence of the peptide fractions, totaling 205 amino acids, were identical to sequences contained within the extracellular domain of BPAg2. Whereas the predominant epitope identified with bullous pemphigoid sera is located in the noncollagenous region of this protein, the epitope recognized by LABD sera is either within or adjacent to the collagenous portion. We conclude that LABD97 represents a portion of the extracellular domain of BPAg2 and that the IgA autoantibodies are directed against an epitope within or adjacent to a collagenous domain.

Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo.

Because human fibroblasts are easily brought to tissue culture conditions and can be stably transduced with retroviral vectors encoding transgenes ex vivo, genetically modified fibroblasts are frequently considered in strategies to correct disease with gene therapy. This enthusiasm has been dampened by studies showing that transgene expression by genetically modified fibroblasts diminishes with time in vivo, but not in vitro, for reasons that are unclear. We elected to study this problem using cloned human fibroblasts that had been cloned by limiting dilution and stably transduced with a retroviral vector encoding lacZ ex vivo. These were seeded onto a nonbiodegradable nylon matrix that was transplanted to nude mice. Transgene expression was followed prospectively by histologic exam. Data show that human fibroblasts can withstand the pressure of cloning by limiting dilution. In addition, they can be passaged from 10 to > 20 times, and > 1 x 10(20) of genetically modified fibroblasts can be generated as progeny of one cell. Loss of transgene expression by the cloned genetically modified fibroblasts in vivo occurs in an orderly and progressive fashion, but is not complete by 4 months. Neither the loss nor the persistence of expression appear to be random. These observations are most compatible with the thesis that a major cause of the loss of transgene expression in vivo is secondary to apoptosis of the genetically modified fibroblast. Loss of expression of transgenes in senescent genetically modified fibroblasts occurs more rapidly than in their presenescent counterparts in the age-neutral, in vivo setting of the nude mouse.

Single-and dual-energy CT with monochromatic synchrotron x-rays.

We explored the potential for clinical research of computed tomography (CT) with monochromatic x-rays using the preclinical multiple energy computed tomography (MECT) system at the National Synchrotron Light Source. MECT has a fixed, horizontal fan beam with a subject apparatus rotating about a vertical axis; it will be used for imaging the human head and neck. Two CdWO4-photodiode array detectors with different spatial resolutions were used. A 10.5 cm diameter acrylic phantom was imaged with MECT at 43 keV and with a conventional CT (CCT) at 80 kVp: spatial resolution approximately equal to 6.5 line pairs (lp)/cm for both; slice height, 2.6 mm for MECT against 3.0 mm for CCT; surface dose, 3.1 cGy for MECT against 2.0 cGy for CCT. The resultant image noise was 1.5 HU for MECT against 3 HU for CCT. Computer simulations of the same images with more precisely matched spatial resolution, slice height and dose indicated an image-noise ratio of 1.4:1.0 for CCT against MECT. A 13.5 cm diameter acrylic phantom imaged with MECT at approximately 0.1 keV above the iodine K edge and with CCT showed, for a 240 micrograms I ml-1 solution, an image contrast of 26 HU for MECT and 13 and 9 HU for the 80 and 100 kVp CCT, respectively. The corresponding numbers from computer simulation of the same images were 26, 12, and 9 HU, respectively. MECT's potential for use in clinical research is discussed.

Ultraviolet A irradiation upregulates type VII collagen expression in human dermal fibroblasts.

Type VII collagen, a major component of skin-anchoring fibrils, is synthesized by both fibroblasts and keratinocytes, the two principal cell types in the skin. In this study, we examined the effects of ultraviolet A (UVA) irradiation on the expression of type VII collagen in human fibroblasts. UVA irradiation (0-15 J/cm2) caused a dose-dependent increase (5- to 10-fold) in type VII collagen mRNA levels as detected by northern blot analysis. The UVA-induced enhancement of type VII collagen gene expression correlated with an increase in its protein level by immunoblot analysis of proteins secreted into the conditioned medium. The effect of UVA was observed at 12 h and reached its maximum by 18 h. Under these conditions, however, the expression of fibronectin, a major dermal matrix protein, remained unchanged, suggesting that the induction of type VII collagen expression was selective. Actinomycin D, a transcription inhibitor, blocked the UVA-mediated induction of type VII collagen gene expression, whereas cycloheximide, a protein synthesis inhibitor, superinduced the expression of type VII collagen, suggesting that de novo protein synthesis was not required for the action of UVA. Interestingly, in contrast to the increased type VII collagen expression in fibroblasts in response to UVA, a slight decrease in type VII collagen mRNA level was observed in the UVA-irradiated keratinocytes, suggesting that the effect of UVA on the type VII collagen expression is cell type specific.

Characteristics of vascular wall cells subjected to dynamic cyclic strain and fluid shear conditions in vitro.

We recently developed an in vitro silicone rubber tubular apparatus, the vascular simulating device (VSD), which simulates pressure, flow, and strain characteristics of peripheral arteries (Benbrahim et al., 1994, J. Vasc. Surg. 20, 184-194). In this report, we tested the ability of silicone rubber surfaces to support the growth and differentiation of endothelial cells (EC) and smooth muscle cells (SMC) and studied the effects of arterial levels of pressure, flow, and strain on these properties. Human umbilical and saphenous vein EC and bovine aortic EC and SMC were cultured on coated and uncoated silicone rubber in flat and tubular configurations (6 mm inner diameter) and on tissue culture plastic (TCP). Attachment, growth, and differentiation were compared on these surfaces. In addition, the effects of arterial pressure, flow, and strain conditions on adhesion and subsequent growth and differentiation were studied in the tubular configuration. Attachment and growth of vascular wall cells on fibronectin-coated silicone rubber was similar to that obtained on TCP. Application of arterial levels of pressure, flow, and strain did not alter adhesion of the cells to the tubes. Subsequent passage of these cells demonstrated that attachment, growth, and differentiation (uptake of LDL and expression of factor VIII-related antigen by EC and expression of muscle-specific actin by SMC) were similar in cells derived from experimental and control tubes which were not subjected to arterial conditions. Finally, mRNA expression of specific "housekeeping" genes was similar in cells isolated from experimental and control tubes. We conclude that the VSD supports the culture of viable and differentiated EC and SMC. These experiments demonstrate that it is possible to evaluate the effects of arterial strain and fluid shear on vascular wall cells in vitro, in a configuration similar to the blood vessel wall.

Deposition of granular IgA relative to clinical lesions in dermatitis herpetiformis.

OBJECTIVE: To compare the deposition of IgA and C3 in the skin of patients with active dermatitis herpetiformis relative to the sites of disease. DESIGN: In the phase 1 study, skin biopsy specimens were obtained from erythematous perilesional skin, nonerythematous perilesional skin, and never-involved skin. In the phase 2 study, specimens from the nonerythematous perilesional and uninvolved skin from the same anatomic region were sampled. SETTING: The Dermatology Clinic at the University of Utah Health Sciences Center, Salt Lake City. PATIENTS: Patients with known dermatitis herpetiformis: 19 patients in the phase 1 study and 15 patients in the phase 2 study. Suppressive medications were stopped for 48 to 72 hours after biopsy specimens were obtained. All patients had active disease at the time that biopsy specimens were taken. MAIN OUTCOME MEASURE: The intensity of IgA and C3 immunofluorescent staining in 6 sections from each skin biopsy specimen was graded by using a semiquantitative scale (0 to 3+) in a blinded fashion by a single observer. RESULTS: Deposition of IgA was more intense in noninflamed perilesional skin in 11 of 19 patients compared with that in erythematous skin (P < .05). Erythematous skin was negative for IgA in 16% (3/19) of the specimens. Noninflamed perilesional skin showed more intense IgA deposition in 18 of 19 specimens compared with that in never-involved skin (P < .01); C3 was more intense in erythematous skin (P < .01). In the phase 2 study, skin from the same anatomic region revealed greater deposition of IgA near lesions in 12 of 15 patients (P < .001). CONCLUSIONS: In patients with dermatitis herpetiformis, IgA is not uniformly distributed throughout the skin, and IgA is present in greater amounts near active lesions. The preferred biopsy site for the diagnosis of dermatitis herpetiformis is normal-appearing skin that is adjacent to an active lesion.

IgA antibodies in chronic bullous disease of childhood react with 97 kDa basement membrane zone protein.

Chronic bullous disease of childhood (CBDC) is an autoimmune blistering disease occurring in prepubertal children. Both CBDC and its adult counter-part, linear IgA bullous dermatosis (LABD), are characterized by linear deposition of IgA along the cutaneous basement membrane zone (BMZ). Circulating IgA antibody in LABD has been found to bind to a 97-kDa BMZ antigen, whereas the antigen in CBDC has not been well characterized. The purpose of this study was to evaluate the immunoreactivity of BMZ IgA antibodies in a series of CBDC patients. We evaluated 12 sera from patients with CBDC with circulating IgA anti-BMZ antibodies on indirect immunofluorescence (IIF), which stained the epidermal side of split skin with titers ranging from 1:20 to 1:640. Immunoblotting was performed against two preparations of BMZ proteins: one enriched with the two bullous pemphigoid antigens (BP230, BP180) and one enriched with the LABD antigen (LABD97). Eight of the twelve sera reacted with a 97-kDa protein that co-migrated with the protein detected in many LABD sera. The intensity of the reaction on immunoblot correlated with serum antibody titers. There was no consistent pattern of reactivity of the IgA anti-BMZ antibodies with either the BP230 or BP180 antigens, although two sera reacted with several higher molecular mass proteins (160-200 kDa). The significance of this reactivity was examined with immunoblotting using BMZ-affinity-purified antibodies, and ELF using nitrocellulose-eluted antibodies. One serum also contained anti-BMZ IgA antibodies that reacted with a 180-kDa protein, corresponding to BP180. We conclude that IgA antibodies in CBDC sera recognize a 97-kDa BMZ antigen present on the epidermal side of BMZ split skin that co-migrates with the antigen previously identified in LABD. These findings suggest that CBDC and LABD are the immunologically related disorders occurring in different age groups.

Recurrent aphthous stomatitis: primary care management.

Recurrent aphthous stomatitis (RAS), often referred to as canker sore, is a chronic inflammatory disease. Frequently seen in the primary care setting, RAS affects over 50% of the population and may be observed in an otherwise apparently healthy individual. An unknown pathogenesis and absence of curative treatment make RAS a challenge to manage. Treating RAS should be patient specific and focus on reduction of pain and lesions. An awareness of literature suggesting etiologies, precipitating, and predisposing factors can assist the practitioner and patient in controlling RAS episodes. Contained in this review is a suggested course of management for patients with RAS, including present recommendations for pharmacological interventions.