Extent of laminin-5 assembly and secretion effect junctional epidermolysis bullosa phenotype.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin blistering disease with both lethal and nonlethal forms, with most patients shown to have defects in laminin-5. We analyzed the location of mutations, gene expression levels, and protein chain assembly of the laminin-5 heterotrimer in six JEB patients to determine how the type of genetic lesion influences the pathophysiology of JEB. Mutations within laminin-5 genes were diversely located, with the most severe forms of JEB correlating best with premature termination codons, rather than mapping to any particular protein domain. In all six JEB patients, the laminin-5 assembly intermediates we observed were as predicted by our previous work indicating that the alpha3beta3gamma2 heterotrimer assembles intracellularly via a beta3gamma2 heterodimer intermediate. Since assembly precedes secretion, mutations that disrupt protein-protein interactions needed for assembly are predicted to limit the secretion of laminin-5, and likely to interfere with function. However, our data indicate that typically the most severe mutations diminish mRNA stability, and serve as functional null alleles that block chain assembly by resulting in either a deficiency (in the nonlethal mitis variety) or a complete absence (in lethal Herlitz-JEB) of one of the chains needed for laminin-5 heterotrimer assembly.

Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent.

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Gamma 2 chain of laminin-5 is recognized by monoclonal antibody GB3.

Herlitz junctional epidermolysis bullosa is an autosomal recessive disorder characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane. The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patient skin. The antigen recognized by GB3 has been identified as laminin-5, a glycoprotein consisting of three subunits (alpha 3, beta 3 and gamma 2). To identify the laminin-5 protein chain that contains the epitope recognized by GB3 and to determine if chain assembly is required for antibody recognition, we expressed a gamma 2 protein constructed from a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclonal antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion experiments, each antibody removed most of the protein that was reactive with the other antibody. The epitope recognized by GB3 is present only when the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immunoblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells (gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluorescence staining, this epitope may be dependent upon a less stable conformation of gamma 2. We conclude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form of the gamma 2 chain.

Handling and culture of bovine embryos: survey of media used by 26 embryo transfer companies in the USA.

Embryo transfer has evolved from a few highly centralized clinics to a widespread number of clinics offering both clinic and on-the-farm services. Practitioners in the field have made very few changes in the handling and culture of embryos. A short review of general handling techniques, plus a survey of media used by 26 companies in the USA, is presented here. Currently, phosphate-bufferend saline (PBS) plus two other commercially available media (EMCARE and ViGro) are used by these companies. Fourteen different combinations of these products were reported and only 11 of the 26 consistently used the same medium for flushing, culture and freezing.

Estrus synchronization with an oral progestogen prior to superovulation of postpartum beef cows.

Ovarian follicular dynamics and steroid secretion patterns were monitored in postpartum beef cows that were synchronized for estrus with melengestrol acetate (MGA) or prostaglandin F(2alpha) (PGF) prior to superovulation. Twenty-four muhiparous Angus cows were stratified by number of days postpartum to an MGA or PGF treatment prior to superovulation. Cows in the MGA group were fed 0.5 mg MGA/d for 14 d in a grain carrier. Superstitnulatory treatments began 14 d after withdrawal of MGA from feed or 11 d after administering a single injection of 500 microg cloprostenol (PGF). Supersthnulatory treatments (FSH) were administered twice daily in decreasing doses (7.5, 5, 5, 2.5 mg) over 4 d. Sixty and 72 h after initiating the superstimulatory treatments, all cows were treated with 750 microg and 500 microg PGF, respectively Cows were inseminated at 0, 12, and 24 h from the onset of standing estrus with semen from 2 proven sires. Cows within treatment were inseminated with 1, 2 and 1 (single) or 2, 4 and 2 units (double) of semen at the designated insemination times. Blood sampling and transrectal ultrasonography of ovaries were performed daily beginning 2 d prior to the initiation of FSH treatment and were continued through embryo recovery. Ovaries were examined daily to determine the number and size of follicles. Plasma samples were analyzed for progesterone and estradiol. Follicles were counted and categorized based on a 5 to 9 mm range or >/= 10 mm. At the end of superovulatory treatment there were more (P /= 10 mm among cows that were estrus synchronized with MGA (75 +/- 1.2) than with PGF (3.9 +/- 1.2) These differences were reflected in higher (P

Quantitation of vital bleaching by computer analysis of photographic images.

BACKGROUND: The authors investigated the use of computer processing of photographic images to monitor changes in tooth brightness after nightguard vital bleaching, or NGVB. METHODS: Photographs of shade guides and clinical cases (patients' teeth) were taken on 35-millimeter film with electronic flash illumination and processed commercially. A slide scanner was used to digitize images as red, green and blue, or RGB, files, with constant brightness, contrast and linearity settings; the images were then analyzed with commercial software. Relevant image components (that is, teeth or shade guide tabs) were separated, and histograms of various numerical color descriptors were generated for each image component. RESULTS: Analysis of shade tab images showed that the mean pixel intensity for the RGB blue channel, or MPIb, was the most satisfactory brightness descriptor, with clear sequential MPIb increments from lighter to darker shades in each series of colors (A through D) and close correlation with the manufacturer's brightness scale (r = .83). Mathematical analysis of MPIb data for shade tabs in the same image yielded a brightness index that was reproducible and correlated well with the manufacturer's brightness scale. Sequential measurements of this index in three subjects whose teeth were bleached with carbamide peroxide for 14 days correlated well with assessments made by visual shade guide comparisons. CONCLUSIONS: The authors conclude that computer analysis of digitized photographic images with internal color controls provides an index of tooth brightness that is reproducible from image to image. CLINICAL IMPLICATIONS: A brightness index derived from computer analysis of digitized photographic images may be useful for monitoring the effectiveness of NGVB.

Effectiveness, side effects and long-term status of nightguard vital bleaching.

In this clinical trial of nightguard vital bleaching for six weeks, 92 percent of the patients experienced some lightening of treated teeth. About 97 percent of patients with teeth stained through aging, inherent discoloration, brown fluorosis or trauma experienced lightening, as did 75 percent with tetracycline-stained teeth. Sixty-six percent experienced side effects, which resolved in 24 to 48 hours. Earliest re-treatment was done after one year in less time. Minimal color change occurred for 74 percent after 1 1/2 years and 62 percent after three years.

Efficacy of foam liner in 10% carbamide peroxide bleaching technique.

The purpose of this study was to determine if the use of a foam liner in a tray during the nightguard vital bleaching technique would cause a difference in the rate of bleaching or the final shade of the bleached teeth. Ten subjects were selected who had previously successfully bleached their maxillary arch by using a 10% carbamide peroxide solution. Vacuum-formed guards were fabricated for the mandibular arch so that one quadrant was covered with a conventional-style guard, and the other quadrant was covered with a foam-lined guard. All patients were successful in bleaching their mandibular arch. No patient reported any difference between the two quadrants in the rate of bleaching or in the final shade, nor was any difference apparent to the operators clinically or in photographic evaluation. It appears that the addition of a foam insert does not noticeably alter the clinical result of home bleaching. If bleaching is successful in one arch, it can be expected to be effective in the other arch.

Laboratory evaluations of a toothbrush with diamond-shaped filaments for stiffness and efficacy.

The purpose of these studies was to evaluate four toothbrushes with a similar flat profile for stiffness and efficacy in removing artificial plaque deposits from interproximal areas, or depth of removal from broad tooth surfaces, and to relate these findings to bristle stiffness. The test toothbrush had 5-mil diameter, elongated diamond-shaped bristles (D). Three toothbrushes contained 5-mil (5), 6-mil (6), or 7-mil (7) diameter round bristles. Stiffness studies were performed using the ISO 8627 method. In the efficacy studies, toothbrushing was conducted with a vertical or horizontal brushing motion on simulated anterior or posterior teeth using a brushing weight of 250 g. The bristles were placed at a 90 degrees angle to the tooth surface, and brushing was performed for 15 seconds at two strokes per second with 50 mm strokes. Each of the toothbrush designs was tested 24 times. Interproximal access efficacy (IAE) was recorded as the maximum width of simulated plaque deposit removed. Depth of deposit removal (DDR) was determined using a color comparative scale, where 0 = no deposit change and 4 = maximum removal. The averages of wet and dry stiffness measurements were found to be 7 > D > 6 > 5; all values were in the range accepted as soft texture. The means for total IAE were ranked D > 5 > 6 > 7, and total DDR mean rankings were D > 7 > 6 > 5. For the round bristles, stiffness was inversely related to IAE and paralleled the DDR rankings. Using ANOVA, the D and 5-mil toothbrushes had significantly higher (p < 0.05) IAE means compared to the 6- or 7-mil products. The D toothbrush had significantly higher (p < 0.05) DDR means than the 5-mil product. The diamond-shaped bristles in these flat profile products resulted in improved depth of deposit removal compared to the same design with 5-mil round bristles.

Families need help too: group treatment for families of nursing home residents.

Despite long-standing interest in the family as the focal point of nursing care, there are few published reports of clinical interventions with families after they institutionalize an elderly relative. Family members experience loss, guilt, and grief, and report that these emotions continue throughout the time that their older member is in the nursing home. This paper presents the rationale for using a supportive, educational group approach with families of nursing home residents and describes a family group intervention, detailing the role of the geriatric mental health nurse specialist, group goals, and the focus of each session. Evaluations by families and staff are presented, and recommendations for practice are made.

The tension headache, migraine headache continuum: a hypothesis.

There is evidence in the literature that tension-type and migraine headaches do not exist as discrete pathophysiologic entities but form a continuum. The mechanisms for tension and migraine headaches are discussed and the evidence for this continuum, both basic scientific and clinical, is reviewed. A model for chronic headaches based on this evidence and based on clinical observations by the author is presented. Clinical and research implications of this model and means of testing the model are discussed.

Evaluation of two transport systems for gonorrhea cultures.

The ability of the Transgrow and JEMBEC systems to yield positive cultures when transported to a central laboratory was compared. There were no significant differences in the recovery rates of the two systems. Statistically significant decreases in recovery rate were noted when each system was compared with the traditional plate-candle jar technique.

Trunk exercise combined with spinal manipulative or NSAID therapy for chronic low back pain: a randomized, observer-blinded clinical trial.

OBJECTIVES: To study the relative efficacy of three different treatment for chronic low back pain (CLBP). Two preplanned comparisons were made: (a) Spinal manipulative therapy (SMT) combined with trunk strengthening exercises (TSE) vs. SMT combined with trunk stretching exercises, and (b) SMT combined with TSE vs. nonsteroidal anti-inflammatory drug (NSAID) therapy combined with TSE. STUDY DESIGN: Interdisciplinary, prospective, observer-blinded, randomized clinical trial with a 1-yr follow-up period. The trial evaluated therapies in combination only and was not designed to test the individual treatment components. SETTING: Primary contact, college out-patient clinic. PATIENTS: In total, 174 patients aged 20-60 yr were admitted to the study. MAIN OUTCOME MEASURES: Patient-rated low back pain, disability, and functional health status at 5 and 11 wk. INTERVENTIONS: Five weeks of SMT or NSAID therapy in combination with supervised trunk exercise, followed by and additional 6 wk of supervised exercise alone. RESULTS: Individual group comparisons after 5 and 11 wk of intervention on all three main outcome measures did not reveal any clear clinically important or statistically significant differences. There seemed to be a sustained reduction in medication use at the 1-yr follow-up. in the SMT/TSE group. Continuance of exercise during the follow-up year, regardless of type, was associated with a better outcome. CONCLUSION: Each of the three therapeutic regimens was associated with similar and clinically important improvement over time that was considered superior to the expected natural history of long-standing CLBP. For the management of CLBP, trunk exercise in combination with SMT or NSAID therapy seemed to be beneficial and worthwhile. The magnitude of nonspecific therapeutic (placebo) effects, cost-effectiveness and relative risks of side effects associated with these types of therapy need to be addressed in future studies.

The assembly of laminin-5 subunits.

Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC-25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.

The efficacy of spinal manipulation, amitriptyline and the combination of both therapies for the prophylaxis of migraine headache.

BACKGROUND: Migraine headache affects approximately 11 million adults in the United States. Spinal manipulation is a common alternative therapy for headaches, but its efficacy compared with standard medical therapies is unknown. OBJECTIVE: To measure the relative efficacy of amitriptyline, spinal manipulation and the combination of both therapies for the prophylaxis of migraine headache. DESIGN: A prospective, randomized, parallel-group comparison. After a 4-wk baseline period, patients were randomly assigned to 8 wk of treatment, after which there was a 4-wk follow-up period. SETTING: Chiropractic college outpatient clinic. PARTICIPANTS: A total of 218 patients with the diagnosis of migraine headache. INTERVENTIONS: An 8-wk course of therapy with spinal manipulation, amitriptyline or a combination of the two treatments. MAIN OUTCOME MEASURES: A headache index score derived from a daily headache pain diary during the last 4 wk of treatment and during the 4-wk follow-up period. RESULTS: Clinically important improvement was observed in both primary and secondary outcomes in all three study groups over time. The reduction in headache index scores during treatment compared with baseline was 49% for amitriptyline, 40% for spinal manipulation and 41% for the combined group; p = .66. During the posttreatment follow-up period the reduction from baseline was 24% for amitriptyline, 42% for spinal manipulation and 25% for the combined group; p = .05. CONCLUSION: There was no advantage to combining amitriptyline and spinal manipulation for the treatment of migraine headache. Spinal manipulation seemed to be as effective as a well-established and efficacious treatment (amitriptyline), and on the basis of a benign side effects profile, it should be considered a treatment option for patients with frequent migraine headaches.

Expression of HSP70 in healing wounds of diabetic and nondiabetic mice.

BACKGROUND: Heat shock proteins (HSPs) stabilize intracellular processes of cells under stress. Little is known about the role of HSPs in wound healing, or whether their expression is altered by systemic disease. The focus of this study was to examine the local heat shock response to wounding in diabetic mice. METHODS: Congenitally diabetic and phenotypically normal mice underwent standardized full-thickness cutaneous wounding. Mice were sacrificed at sequential time points and the wound beds excised. Tissues underwent immunohistochemical (IHC) and RT-PCR analyses for inducible HSP70. RESULTS: HSP70 protein expression in the wound bed by IHC peaked at 24 h in the nondiabetic mice. Expression of HSP70 was delayed in the diabetic mice until Day 3, which correlates with the clinical delay in healing seen in this model. The protein was especially prominent in the epithelium and in inflammatory cells migrating into the granulation tissue matrix. RT-PCR demonstrated upregulation of HSP70 mRNA within 12 h after wounding, lasting until Day 3, and decreasing thereafter in both the nondiabetic and the diabetic animals. CONCLUSION: Cutaneous wounding produces a HSP response in inflammatory cells, and expression of inducible HSP70 is delayed in diabetic mice. This delay may be related to the impaired inflammatory response of diabetics, and may contribute to impaired wound healing. The wound may be a continuing source of the heat shock response in inflammatory cells after injury.