Effect of dietary buriti oil on the quality, fatty acid profile and sensorial attributes of lamb meat.

Forty crossbred (Santa Ines × Dorper) male lambs were used to investigate the effect of dietary buriti oil (BOIL) intake on meat quality, fatty acid (FA) composition and sensory attributes. A completely randomized design with five BOIL intake levels (0, 12, 24, 36 and 48 g/kg DM) was used. Increasing dietary BOIL intake linearly reduced the DMI, slaughter weight, cooking loss, shear force, yellowness, 16:0, c9-18:1 FA concentrations, and the delta-9-desaturase activity index computed using c9-16:1 and 16:0 as product and substrate pair, cis-MUFA and Æ©MUFA/Æ©SFA ratio of lamb meat (P ≤ 0.05); however, increasing dietary BOIL intake quadratically increased the energy intake, lipid content and linearly increased the total FA intake, chroma, collagen, total BI, 18:0, 20:3n-6 concentration and sensory attributes of lamb meat (P ≤ 0.05), without affecting health indexes regarding fat consumption. BOIL addition at levels above 24 g/kg DM in lamb diet reduces growth, however, improves tenderness, flavor and "goat" aroma intensity and slightly changes meat FA levels, promoting better acceptance by panelists.

Finite element modelling of the surgical procedure for placement of a straight electrode array: Mechanical and clinical consequences.

A cochlear implant is an electronic device implanted into the cochlea to directly stimulate the auditory nerve. Such device is used in patients with severe-to-profound hearing loss. The cochlear implant surgery is safe, but involves some risks, such as infections, device malfunction or damage of the facial nerve and it can result on a poor hearing outcome, due to the destruction of any present residual hearing. Future improvements in cochlear implant surgery will necessarily involve the decrease of the intra-cochlear damage. Several implant related variables, such as materials, geometrical design, processor and surgical techniques can be optimized in order for the patients to partially recover their hearing capacities The straight electrode is a type of cochlear implant that many authors indicate as being the less traumatic. From the finite element analysis conducted in this work, the influence of the insertion speed, the friction coefficient between the cochlear wall and the electrode array, and several configurations of the cochlear implant tip were studied. The numerical simulations of the implantation showed the same pattern of the insertion force against insertion depth, thus indicating the different phases of the insertion. Results demonstrated that lower insertion speeds, friction coefficients and tip stiffness, led to a reduction on the contact pressures and insertion force. It is expected that these improved configurations will allow to preserve the residual hearing while reducing surgical complications.

Supplementation of crossbred cows with maize and urea in tropical pasture / Suplementação de vacas mestiças com milho e ureia em pastagem tropical

This research article aims to evaluate the effect of total replacement of corn by soybean meal and urea on intake and apparent digestibility of dry matter and its components, production and composition of milk and feeding behavior of lactating cows grazing, with intermittent stocking Mombaça grass (Megathyrsus maximus cv. Mombaça). Twelve milking cows were distributed in a triple 4 x 4 latin square. Experimental treatments consisted of four soybean meal replacement levels by corn more urea (0; 33; 66; 100%). The grass has an average content of crude protein and neutral detergent fiber of 19% and 59%, respectively. The replacement of soybean meal by corn and urea did not affect the intake and digestibility of nutrients. A linear reduction of milk production was observed, but there was no change in milk production corrected to 4.0% of fat. The milk components (g/kg) of fat, protein, lactose, and total solids were not altered, as well as feeding behavior. Under the conditions of this study, the replacement of the diets is suitable for crossbred dairy cows in lactation third medium, producing in average of 12.5 kg/day-1 when kept in quality pastures.(AU)

Este artigo de pesquisa tem como objetivo avaliar o efeito da substituição total do milho pelo farelo de soja e ureia sobre o consumo e a digestibilidade aparente da matéria seca e de seus componentes, a produção e a composição do leite e o comportamento alimentar de vacas em lactação mantidas em pastos capim-mombaça (Megathyrsus maximus cv. Mombaça) manejado sob lotação intermitente. Doze vacas em lactação foram distribuídas em um quadrado latino triplo 4 x 4. Os tratamentos experimentais consistiram de quatro níveis de substituição do farelo de soja pelo milho mais ureia (0; 33; 66; 100%). O capim-mombaça apresentou um teor médio de proteína bruta e de fibra em detergente neutro de 19% e 59%, respectivamente. A substituição do farelo de soja por milho e ureia não afetou o consumo e a digestibilidade dos nutrientes. Observou-se redução linear da produção de leite, mas não houve alteração na produção de leite corrigida para 4,0% de gordura. Os componentes do leite (g/kg): gordura, proteína, lactose e sólidos totais não foram alterados, assim como o comportamento ingestivo dos animais. Nas condições deste estudo, a substituição total do milho pelo farelo de soja e ureia nas dietas é adequada para vacas mestiças leiteiras em terço médio de lactação, produzindo, em média, 12,5 kg / dia-1 quando mantidas em pastagens de qualidade.(AU)

Artificial intelligence and colon capsule endoscopy: development of an automated diagnostic system of protruding lesions in colon capsule endoscopy.

BACKGROUND: Colon capsule endoscopy (CCE) is a minimally invasive alternative for patients unwilling to undergo conventional colonoscopy, or for whom the latter exam is contraindicated. This is particularly important in the setting of colorectal cancer screening. Nevertheless, these exams produce large numbers of images, and reading them is a monotonous and time-consuming task, with the risk of overlooking important lesions. The development of automated tools based on artificial intelligence (AI) technology may improve some of the drawbacks of this diagnostic instrument. METHODS: A database of CCE images was used for development of a Convolutional Neural Network (CNN) model. This database included anonymized images of patients with protruding lesions in the colon or patients with normal colonic mucosa or with other pathologic findings. A total of 3,387,259 frames from 24 CCE exams were retrospectively reviewed. For CNN development, 3640 images (860 protruding lesions and 2780 with normal mucosa or other findings) were ultimately extracted. Training and validation datasets were constructed for the development and testing of the CNN. RESULTS: The CNN detected protruding lesions with a sensitivity, specificity, positive and negative predictive values of 90.7, 92.6, 79.2 and 96.9%, respectively. The area under the receiver operating characteristic curve for detection of protruding lesions was 0.97. CONCLUSIONS: The deep learning algorithm we developed is capable of accurately detecting protruding lesions. The application of AI technology to CCE may increase its diagnostic accuracy and acceptance for screening of colorectal neoplasia.

Effect of mesh anchoring technique in uterine prolapse repair surgery: A finite element analysis.

The female pelvic cavity involves muscles, ligaments, endopelvic fasciae and multiple organs where different pathologies may occur, namely the pelvic organ prolapse (POP). The synthetic implants are used for the reconstructive surgery of POP, but severe complications associated with their use have been reported, mainly related to their mechanical properties (e.g., implant stiffness) and microstructure. In this study, we mimicked a transvaginal reconstructive surgery to repair the apical ligaments (uterosacral ligaments (USLs) and cardinal ligaments (CLs)), by modeling, their impairment (90% and 50%) and/or total rupture. The implants to reinforce/replace these ligaments were built based on literature specifications and their mechanical properties were obtained through uniaxial tensile tests. The main aim of this study was to simulate the effect of mesh anchoring technique (simple stich and continuous stitch), and compare the displacement magnitude of the pelvic tissues, during Valsalva maneuver. The absence/presence of the synthetic implant was simulated when total rupture of the CLs and USLs occurs, causing a variation of the vaginal displacement (9% for the CLs and 27% for the USLs). Additionally, the simulations showed that there was a variation of the supero-inferior displacement of the vaginal wall between different anchoring techniques (simple stich and continuous stitch) being approximately of 10% for the simulation USLs and CLs implant. The computational simulation was able to mimic the biomechanical behavior of the USLs and CLs, in response to different anchoring techniques, which can be help improving the outcomes of the prolapse surgery.

Numerical simulation of lateral and transforaminal lumbar interbody fusion, two minimally invasive surgical approaches.

The present study aims to compare spinal stability after two different minimally invasive techniques, the lateral lumbar interbody fusion (LLIF) and the transforaminal lumbar interbody fusion (TLIF) approaches. Two nonlinear three-dimensional finite element (FE) models of the L4-L5 functional spinal unit (FSU) were subjected to the loads that usually act on the lumbar spine. Findings show that the LLIF approach yields better results for torsion load case, due to the larger surface area of the implant. For extension, flexion and lateral bending loads, the TLIF approach presents smaller displacements probably due to the anterior placement of the cage and to the smaller damaged area of the annulus fibrosus.

Minimally invasive transforaminal and anterior lumbar interbody fusion surgery at level L5-S1.

This paper presents a finite element analysis to investigate the biomechanical changes caused by transforaminal (TLIF) and anterior lumbar interbody fusion (ALIF) at the L5-S1 level, applying two different implants: T_PAL (TLIF) and SynFix (ALIF). The main objective is to determine which one is more stable for patients. Numerical simulations of segmental motion show that, in the early postoperative phase, displacements and rotation angles obtained in ALIF are greater than the corresponding ones obtained in TLIF, as well as the principal stress values on the ligaments. So, TLIF performed with T_PAL is more stable than ALIF, especially during the recovery phase.

Simulation of the uterine contractions and foetus expulsion using a chemo-mechanical constitutive model.

During vaginal delivery women sustain stretching of their pelvic floor, risking tissue injury and adverse outcomes. Since studies in pregnant women are limited with ethical constraints, computational models have become an interesting alternative to elucidate the pregnancy mechanisms. This research investigates the uterine contractions during foetus expulsion without an imposed trajectory. Such physical process is captured by means of a chemo-mechanical constitutive model, where the uterine contractions are triggered by chemical stimuli. The foetus descent, which includes both pushing and resting stages, has a descent rate within the physiological range. Moreover, the behaviour of the foetus and the uterus stretch agree well with clinical data presented in the literature. The follow-up of this study will be to obtain a complete childbirth simulation, considering also the pelvic floor muscles and its supporting structures. The simulation of a realistic rate of descent, including the pushing and resting stages, is of significant importance to study the pelvic floor muscles due to their viscoelastic nature.

The effect of physiotherapy on fatigue and physical functioning in chronic fatigue syndrome patients: A systematic review.

OBJECTIVE: The objectives of this work were to fill the gap in the scientific literature and to evaluate the results of physical therapy treatments in individuals affected by chronic fatigue syndrome, considering only studies that employed a randomized controlled trial. METHODS: A systematic review was carried out according to PRISMA guidelines. Three bibliographic databases were searched: MEDLINE, Cochrane Library, and PEDro. The minimum prerequisites for papers to be included in the systematic review were that they had to (a) employ a randomized controlled trial; (b) be published in English; and (c) be published during the last ten years (2007-2017). The studies were evaluated according to Jadad score. RESULTS: Four studies were included. This systematic review suggests that a treatment that is more effective than all the others cannot be defined. This conclusion is related to the low number of investigated studies; therefore, the collected results cannot be generalized. CONCLUSION: Chronic fatigue syndrome is not yet a well-understood pathology, and the physical mechanisms that influence the outcomes still need more study. Rehabilitation programs that promote physiotherapy techniques such as exercise, mobilization, and body awareness (e.g., MRT and GET) are the most effective in reducing medium and long-term fatigue severity in CFS patients.

Associação entre o óleo de soja e o óleo de peixe na dieta de cabras em lactação como estratégia para melhorar o perfil de ácidos graxos do leite / Association between soybean oil and fish oil in the diet of lactating goats as a strategy to improve milk fatty acids profile

Os objetivos neste experimento foram avaliar os efeitos da associação entre o óleo de soja e o óleo de peixe na dieta de cabras em lactação sobre o consumo de matéria seca e de nutrientes, a variação do peso corporal, a produção e composição do leite, assim como o perfil de ácidos graxos. As cabras foram alocadas em baias individuais, onde receberam dieta composta por 50% de feno de "coastcross" e 50% de concentrado. Foram utilizadas nove cabras mestiças Boer x Saanen multíparas, distribuídas em três quadrados latinos 3 X 3. O experimento teve duração de 51 dias, divididos em três períodos de 17 dias, sendo os 13 primeiros dias para adaptação dos animais às dietas e os 4 dias subsequentes para colheita de amostras e de dados. Os tratamentos experimentais foram: a) dieta controle (CT), sem adição de óleo; b) dieta contendo 3% de óleo de soja (OS); e c) dieta contendo 2,5% de óleo de soja + 0,5% de óleo de peixe (OS+P). A inclusão dos óleos reduziu (P<0,05) o consumo de matéria seca, no entanto aumentou (P<0,05) a eficiência alimentar dos animais, sem afetar (P>0,05) a produção de leite. Houve efeito (P<0,05) da dieta no perfil de ácidos graxos do leite, sendo que ambos os tratamentos com adição de óleo elevaram as concentrações de ácidos graxos de cadeia média e longa, reduzindo os de cadeia curta. O tratamento com a combinação do óleo de soja com o óleo de peixe foi o que promoveu os maiores aumentos na concentração de ácido vacênico (398%), rumênico (352%) e de CLA total (341%) no leite. Os resultados permitem concluir que a suplementação lipídica elevou a eficiência alimentar dos animais e que o fornecimento de óleo de soja em combinação ao óleo de peixe aumentou a concentração no leite dos ácidos graxos benéficos à saúde humana.

The aims in this experiment were to determine the effects of the association between soybean and fish oils on dry matter intake (DMI) and nutrient intake, body weight change, milk production and composition and milk fatty acid profile of dairy goats. The animals were housed in tie stalls and fed a 50% of coastcross hay and 50% concentrate diet. Nine multiparous crossbred Boer x Saanen goats were assigned in tree 3 X 3 Latin Squares. The experimental period lasted 51 days; divided into three periods of 17 days, being the first nine days used to adapt goats to diets and the 4 other days for data collection. Experimental diets were: a) control diet (CT) without oil; b) control diet supplemented with 3% of soybean oil (SO); and c) control diet supplemented with 2.5% of soybean oil plus 0.5% of fish oil (SO+F). DMI was negatively influenced by oil addition. However, feed efficiency was higher in diets with oils, maintaining similar milk production (P>0.05 for all comparisons). The supply of oils changed milk fatty acids profile (P<0.05), increasing the concentrations of medium and long-chain fatty acids and reducing short-chain. Milk from goats fed the SO+F diet had higher concentration of vaccenic (398%), rumenic acid (352%) and total CLA (341%). The results indicate that the oil supply increased the feed efficiency of goats, and that the combination of soybean and fish oils caused a higher elevation of fatty acids considered to have health benefits.

Long-term AAV vector gene and protein expression in mouse brain from a small pan-cellular promoter is similar to neural cell promoters.

The neurogenetic, lysosomal enzyme (LSE) deficiency diseases are characterized by storage lesions throughout the brain; therefore, gene transfer needs to provide widespread distribution of the normal enzyme. Adeno-associated virus (AAV) vectors can be effective in the brain despite limited transduction because LSEs are exported to neighboring cells (cross-correction) to reverse the metabolic deficit. The extent of correction is determined by a combination of the total amount of LSE produced by a vector and the spatial distribution of the vector within the brain. Neuron-specific promoters have been used in the brain because AAV predominantly transduces neurons. However, these promoters are large, using up a substantial amount of the limited cloning capacity of AAV vector genomes. A small promoter that is active in all cells, from the LSE beta-glucuronidase (GUSB), has been used for long-term expression in AAV vectors in the brain but the natural promoter is expressed at very low levels. The amount of LSE exported from a cell is proportional to the level of transcription, thus more active promoters would export more LSE for cross-correction, but direct comparisons have not been reported. In this study, we show that in long-term experiments (>6 months) the GUSB minimal promoter (hGBp) expresses the hGUSB enzyme in brain at similar levels as the neuron-specific enolase promoter or the promoter from the latency-associated transcript of herpes simplex virus. The hGBp minimal promoter thus may be useful for long-term expression in the central nervous system of large cDNAs, bicitronic transcription units, self-complimentary or other designs with size constraints in the AAV vector system.

Deformation of the pelvic floor muscles during a vaginal delivery.

Pelvic floor dysfunction is a hidden problem with a magnitude unknown to many. Statistics show that one in every ten women will have pelvic floor dysfunction so severe that it will require surgery. Several studies have shown that pelvic floor injuries during a vaginal delivery can be considered a significant factor in the development of urinary incontinence, fecal incontinence, and pelvic organ prolapse. The objective of the present work is to contribute to the clarification of the mechanisms behind pelvic floor disorders related to a vaginal delivery. For this purpose, a numerical simulation based on the finite element method was carried out. The finite element model intends to represent the effects that the passage of a fetal head can induce on the muscles of the pelvic floor, from a mechanical point of view. The model used for the simulation represents the pelvic bones, with the attached pelvic floor muscles and the fetus. In this work, we simulated the movements of the fetus during birth, in vertex position. We simulated the engagement, descent, flexion, internal rotation, and extension of the fetal head. Results for the pelvic floor stretch values obtained during the passage of the fetus head are presented; the deformation field is also shown. The results were obtained using the finite element method and a three-dimensional computer model of the pelvic floor and fetus. The maximum deformation obtained was 0.66 for a vertical displacement of the fetal head of approximately 60 mm.

[Perceived pain and weather changes in rheumatic patients]. / A percepçao da dor e alteracoes climatéricas em doentes reumáticos.

INTRODUCTION: Rheumatic patients with chronic pain describe in a vivid way the influence of climate on pain and disease activity. Several studies seem to confirm this association. OBJECTIVES: To evaluate and compare in a population of rheumatic patients the perceived influence of weather changes on pain and disease activity METHODS: This is a retrospective cross-sectional study. For three weeks an assisted self-reported questionnaire with nine dimensions and a VAS pain scale was performed on consecutive out-patients in our clinic. RESULTS: 955 patients 787 female 168 male mean age 57.9 years with several rheumatologic diagnosis were evaluated. Overall 70 of the patients believed that the weather influenced their disease and 40 believed that the influence was high. Morning stiffness was influenced in 54 high influenced in 34 . Autumn and Winter were the most influential periods as well as humidity 67 and low temperatures 59 . CONCLUSION: In our study as well as in literature we found that a high percentage of patients 70 perceived that weather conditions influenced their pain and disease. Fibromyalgia patients seemed to be strongly influenced by weather changes. Our study confirms that patients perception on the influence of climate on pain and therefore their disease is an important clinical factor and it should be considered when evaluating rheumatic patients.

Elevated E-cadherin and alpha/beta-catenin expression after androgen deprivation therapy in prostate adenocarcinoma.

The histological patterns of anti-androgen-treated prostate adenocarcinoma mimic high grade tumors classified according to the widely used Gleason scoring system. However, the biological characteristics of anti-androgen treated carcinoma are largely unknown. E-cadherin, alpha-catenin, and beta-catenin adhesion molecules are down-regulated in pharmacologically untreated high grade prostate carcinoma. In this study, we used immunohistochemical techniques to investigate their expression in twenty acinar adenocarcinomas after anti-androgen therapy in prostatectomy specimens. After adrogen ablation therapy, expression of all these adhesion molecules was higher than that of pretreatment biopsies of the same patient group and high grade matched untreated controls. These results emphasize the inaccuracy of the Gleason score for anti-androgen-treated prostate adenocarcinoma and the more differentiated phenotype of prostate adenocarcinoma after anti-hormonal therapy.

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme.

Down-regulation of retroviral vector expression occurs in a number of cell types after transplantation. Although a number of vector elements have been shown to affect expression in specific experimental situations, the results can vary depending on the specific cDNA being expressed, the individual retroviral elements included in vectors, the promoter, or the inclusion of selectable markers. In previous experiments with the lysosomal enzyme beta-glucuronidase, silencing has occurred in more than 95% of transduced cells regardless of the position of the expression unit within the vector, whether a eukaryotic or viral promoter was used, whether a bacterial selectable marker gene was present or not, the target cell type, or the species of the host. It has been a consistent finding that a small number of continuously expressing cells persist for long periods after transplantation. In this study we found that deletion of all the transcriptional regulatory elements from the vector LTR, inclusion of a permissive primer binding site sequence, and use of a eukaryotic housekeeping promoter could greatly increase the number of expressing cells in fibroblast grafts in subcutaneous neo-organs and in the brain. Furthermore, the level of enzyme expression was increased five-fold on a per positive cell basis, indicating that the deleted regulatory elements were exerting a negative effect on expression in the few cells that were positive before modification of the vector. This resulted in more than a 50-fold increase in total activity compared with the previous highest expressing vector.

Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.

Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

Sustained production of beta-glucuronidase from localized sites after AAV vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice.

The lysosomal storage disorders are a large group of inherited diseases that involve central nervous system degeneration. The disease in the brain has generally been refractory to treatment, which will require long-term correction of lesions dispersed throughout the central nervous system to be effective. A promising approach is somatic gene therapy but the methods have so far been inadequate because they have only achieved short-term or localized improvements. A potential approach to overcome these limitations is to obtain sustained high level expression and secretion of the missing normal enzyme from a small group of cells for export to neighboring diseased cells, which might allow the therapeutic protein to reach distal sites. We tested this in a mouse model of mucopolysaccharidosis VII (Sly disease) using an adeno-associated virus vector. After a single treatment the vector continuously produced the normal enzyme from infected cells at the injection sites. The secreted enzyme was disseminated along most of the neuraxis, resulting in widespread reversal of the hallmark pathology. An extensive sphere of correction surrounding the transduction sites was created, suggesting that a limited number of appropriately spaced sites of gene transfer may provide overlapping spheres of enzyme diffusion to cover a large volume of brain tissue.

Production of increased titer retrovirus vectors from stable producer cell lines by superinfection and concentration.

Retrovirus vector titers could be increased by more than 50-fold after multiple superinfections of packaging cells with vector virus expressing a complementing host-range envelope glycoprotein. The high level of expression was stable for at least 90 passages in culture. The optimum conditions for density of cell seeding, length of incubation, and temporary storage of virus-containing medium was determined. This vector preparation was further concentrated by 20-fold or more in commercially available ultrafilters, which can be used with standard laboratory centrifuges. Ultrafilters from three different manufactures, with molecular weight cutoffs of 30-1000 kiloDaltons, were tested for concentrating ability and for toxicity of the concentrate on target cells. These methods can be used to produce vector virus containing retrovirus envelope proteins in milliliter volumes at titers in excess of 10(8) c.f.u./ml and in liter volumes at greater than 10(6) c.f.u./ml.

High level expression and export of beta-glucuronidase from murine mucopolysaccharidosis VII cells corrected by a double-copy retrovirus vector.

Retrovirus vectors were constructed to transfer and express the cDNA of the human lysosomal acid hydrolase beta-glucuronidase (GUSB) under control of the human GUSB promoter. Expression of the transcription unit (minigene) was evaluated in a GUSB-negative cell line established from a mouse with the lysosomal storage disease mucopolysaccharidosis (MPS) type VII. A vector designed to transfer single copies of the minigene (N2H beta H) expressed normal levels of GUSB activity in the deficient cells. GUSB expression was increased to several times greater than normal by inserting the minigene into a double-copy vector (DCH beta H), which places one copy of the transcription unit upstream of the retrovirus promoter in both the 3' and 5' long terminal repeats (LTRs) of the integrated provirus. The specific activity of GUSB and a control normal lysosomal enzyme, alpha-galactosidase (GLA), were higher in normal and in vector-corrected cells from confluent cultures than in subconfluent dividing cells. The ratios of GUSB to GLA were similar at all phases of cell growth, but the level of GUSB expression from the double copy vector was several-fold higher than from the single copy vector. To determine if this effect was controlled by the GUSB promoter, a vector was constructed using the thymidine kinase (TK) promoter to drive the human GUSB cDNA (NTK beta H). The levels of GUSB in cells corrected with this vector exhibited the same cell density dependent pattern as when the GUSB promoter was used, indicating that the variation in enzymatic activity was not a function of the GUSB promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.

Epidermolysis bullosa acquisita (EBA) is an acquired blistering skin disease characterized by the presence of IgG autoantibodies that recognize type VII (anchoring fibril) collagen. In this study, we have mapped the antigenic epitopes within the type VII collagen alpha chain by Western immunoblotting analysis with sera from 19 patients with EBA, using bacterial collagenase- or pepsin-resistant portions of type VII collagen and a panel of 12 recombinant fusion proteins corresponding to approximately 80% of the primary sequence of the alpha 1 (VII) collagen polypeptide. These studies identified four major immunodominant epitopes localized within the amino-terminal, noncollagenous (NC-1) domain. In addition to EBA, sera from three patients with bullous systemic lupus erythematosus (BSLE) were tested. The pattern of epitopes recognized by these sera were similar to those noted with EBA, suggesting that the same epitopes could serve as autoantigens in both blistering conditions. In contrast, sera from healthy controls or from patients with unrelated blistering skin diseases did not react with type VII collagen epitopes. Collectively, the results indicate that the immunodominant epitopes in EBA and BSLE lie within the noncollagenous regions of type VII collagen. The precise role of the circulating autoantibodies in the pathogenesis of these blistering diseases remains to be elucidated. Conceivably, however, such antibodies could disrupt the assembly of type VII collagen into anchoring fibrils and/or interfere with their interactions with other extracellular matrix molecules within the cutaneous basement membrane zone.