A simple technique using skin implants to produce histocompatability (BoLA) typing sera*

We describe a technique to produce high-titered bovine lymphocytotoxic anti-sera using skin implants. The main advantage of this technique is that the skin does not need to be processed prior to implantation and no surgical skill is required. In addition, the skin can be stored for up to 2 weeks and can be shipped to other laboratories without special handling and without loss of immunogenicity.     

3D non-linear analysis of the acetabular construct following impaction grafting

The study investigates the short-term behaviour of the acetabular construct following revision hip arthroplasty, carried out using the Slooff–Ling impaction grafting technique; using 3D finite element analyses. An elasto-plastic material model is used to describe the constitutive behaviour of morsellised cortico-cancellous bone (MCB) graft, since it has been shown that MCB undergoes significant plastic deformation under normal physiological loads. Based on previous experimental studies carried out by the authors and others, MCB is modelled using non-linear elasticity and Drucker Prager Cap (DPC) plasticity. Loading associated with walking, sitting down, and standing up is applied to the acetabular cup through a femoral head using smooth sliding surfaces. The analyses yield distinctive patterns of migration and rotation due to different activities. These are found to be similar to those observed in the clinical setting.     

负压创面治疗技术对皮肤移植成活的临床观察及实验研究

目的：采用负压固定移植皮片方法,观察负压创面治疗技术（negative-pressure wound therapy,NPWT）对游离皮片成活的影响,初步探讨微血管形成与皮肤成活之间的关系。方法：采用回顾性研究的方法,对65例皮肤缺损的患者,根据皮肤移植术后皮片固定方法的不同,分为两组,其中Ⅰ组为NPWT治疗组,有35例患者,刃厚游离皮片移植术后行创面负压吸引治疗;Ⅱ组为常规治疗组。有30例患者,刃厚游离皮片移植术后用打包或加压包扎的方式固定。Balb／c小鼠20只,按皮片移植后不同固定加压方式,分为实验组：负压创面治疗技术使用组（10只）,对照组：打包加压组（10只）,于皮片移植术后第5天,大体观察移植皮片颜色、有无水疱、有无皮下积液及质地,计算并比较皮片成活率,以免疫组化染色标记毛细血管内皮,检测皮片中微血管情况。结果：临床观察表明：Ⅰ组术后皮片成活时间平均较Ⅱ组缩短,有统计学差异（P〈0．01）,Ⅰ组术后住院治疗时间平均较Ⅱ组缩短5天,有统计学差异（P〈0．01）,Ⅰ组术后抗生素费用、换药次数及换药费用较Ⅱ组减少,有统计学差异（P〈0．01）。动物实验结果表明：术后第5天,实验组小鼠移植皮片中微血管增生较对照组明显增多（P〈0．05）。结论：与常规打包或加压包扎固定皮片的方式相比,负压创面治疗技术的应用可以缩短皮片成活时间,缩短患者住院治疗时间,减少抗生素的使用及换药次数,促进移植皮片中毛细血管增生,提高皮片成活率。     

Seasonal variation in HSP70 expression and oxidative stress in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle under tropical climate

The study aimed to evaluate inducible HSP70 (HSP70.1 and HSP70.2) gene expression and oxidative stress status in skin of cattle during different seasons. Ten each of Tharparkar (zebu) and Karan Fries (crossbred) heifers were selected from NDRI herd, Karnal. Animals were maintained under standard managemental practices followed at the farm. Skin biopsies were aseptically collected from each animal during winter, spring, and summer. Real time PCR was performed to examine HSP70 expression. Reactive oxygen species (ROS) and antioxidant enzymes (SOD and CAT) were determined by ELISA. In both the breeds, significantly higher (p < 0.05) levels of HSP70 expression, ROS, caspases, and antioxidant enzymes were observed during summer followed by winter and spring. Breeds showed no significant difference during winter and spring. During summer, HSP70 expression, ROS, and antioxidant enzymes were higher (p < 0.05) in Karan Fries than Tharparkar, whereas caspases levels were higher in Tharparker than Karan Fries. The study concludes that levels of HSP70 expression, ROS, caspases, and antioxidant enzymes in skin of cattle were strongly affected by seasonal change in temperature. Differences exist in skin tissue thermotolerance of Tharparkar and Karan Fries cattle. This might be an efficient and centrally important mechanism for better adaptability of zebu cattle to heat stress.     

负压创面治疗技术对皮肤移植成活的临床观察及实验研究

目的:采用负压固定移植皮片方法,观察负压创面治疗技术(negative-pressure wound therapy,NPWT)对游离皮片成活的影响,初步探讨微血管形成与皮肤成活之间的关系。方法:采用回顾性研究的方法,对65例皮肤缺损的患者,根据皮肤移植术后皮片固定方法的不同,分为两组,其中I组为NPWT治疗组,有35例患者,刃厚游离皮片移植术后行创面负压吸引治疗;II组为常规治疗组,有30例患者,刃厚游离皮片移植术后用打包或加压包扎的方式固定。Balb/c小鼠20只,按皮片移植后不同固定加压方式,分为实验组:负压创面治疗技术使用组(10只),对照组:打包加压组(10只),于皮片移植术后第5天,大体观察移植皮片颜色、有无水疱、有无皮下积液及质地,计算并比较皮片成活率,以免疫组化染色标记毛细血管内皮,检测皮片中微血管情况。结果:临床观察表明:I组术后皮片成活时间平均较II组缩短,有统计学差异(P<0.01),I组术后住院治疗时间平均较II组缩短5天,有统计学差异(P<0.01),I组术后抗生素费用、换药次数及换药费用较II组减少,有统计学差异(P<0.01)。动物实验结果表明:术后第5天,实验组小鼠移植皮片中微血管增生较对照组明显增多(P<0.05)。结论:与常规打包或加压包扎固定皮片的方式相比,负压创面治疗技术的应用可以缩短皮片成活时间,缩短患者住院治疗时间,减少抗生素的使用及换药次数,促进移植皮片中毛细血管增生,提高皮片成活率。     

The antitheilerial effects of Theileria parva parva reaction and recovery sera in vitro

Peripheral blood leucocytes (PBL) of cattle were infected in vitro with the sporozoites of Theileria parva spp. The transformed cell lines were adapted to grow in sera from the PBL donors. The cattle were then infected with T. p. parva stabilate and either treated with parvaquone or the disease allowed to run its course. Sera harvested during severe disease reaction or early recovery were substituted for pre infection sera and caused the intracellular degeneration of the Theileria macroschizonts. Cell lines passaged in these sera died out as the parasites were eliminated. The antiparasitic effects of sera were short lived and were neither host nor parasite isolate restricted.     

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3162 = 4.8 % ) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand.
Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1 %) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida.
Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.     

The effect of pregnancy on paternal skin allograft survival

Elucidation of maternal-fetal tolerance mechanisms clarifies the role of regulatory T cells (Treg) in transplant tolerance. This study aim to investigate the effect of pregnancy on paternal skin allograft survival. Flow cytometry techniques, mixed lymphocytes reaction (MLR), PCR, real-time PCR and skin transplantation were key methods. Treg increased significantly from 4.2% before pregnancy to peak at 6.8% day 8 after pregnancy. Both heme oxygenase-1 (HO-1) and indoleamine 2,3-dioxygenase (IDO) mRNA express high in placenta while low in spleen (P<0.05). Although Treg increased during pregnancy, and splenocytes from the pregnant mice showed lower MLR response toward the paternal stimulator, single time pregnancy showed no significant protective effect on paternal skin allograft survival in the tested condition.     

Molecular analysis of the prevalent microbiota of human male and female forehead skin compared to forearm skin and the influence of make-up

Aims: To compare the bacterial diversity of two different ecological regions including human forehead, human forearm and to estimate the influence of make‐up. Methods and Results: Twenty‐two swab‐scraped skin samples were analysed by profiling bacterial 16S rRNA genes using PCR‐based sequencing of randomly selected clones. Of the 1056 clones analysed, 67 genera and 133 species‐level operational taxonomic units (SLOTUs) belonging to eight phyla were identified. A core set of bacterial taxa was found in all samples, including Actinobacteria, Firmicutes, and Proteobacteria, but pronounced intra‐ and interpersonal variation in bacterial community composition was observed. Only 4·48% of the genera and 1·50% of the SLOTUs were found in all 11 subjects. In contrast to the highly diverse microbiota of the forearm skin, the forehead skin microbiota represented a small‐scale ecosystem with a few genera found in all individuals. The use of make‐up, including foundation and powder, significantly enlarged the community diversity on the forehead skin. Conclusions: Our study confirmed the presence of a highly diverse microbiota of the human skin as described recently. In contrast to forearm skin, gender does not seem to have much influence on the microbial community of the forehead skin. However, the use of make‐up was associated with a remarkable increase in the bacterial diversity. Significance and Impact of the Study: This study enhances our knowledge about the highly complex microbiota of the human skin and demonstrates for the first time the significant effect of make‐up on the bacterial diversity of the forehead skin.     

大面积皮肤坏死患者的临床分析

目的探讨大面积皮肤坏死的诱因、治疗和危害。方法对1998年1月至2008年1月收治的36例患者大面积皮肤坏死的情况进行调查分析。结果大面积皮肤坏死引起肢体功能障碍15例,部分功能障碍19例,无功能障碍2例。结论大面积皮肤坏死与皮肤撕脱,厌氧菌和需氧菌混合感染,其他继发疾患引起有关,应采取积极治疗措施,提高患者生存质量。     

Polymorphism of the GHR gene in cattle and relationships with meat production and quality

Growth hormone (GH) exerts its effects on growth and metabolism by interacting with a specific receptor (GHR) on the surface of the target cells. Therefore, GHR has been suggested as candidate gene for traits related to meat production in cattle. The aim of the study was to analyse the polymorphism at position 257 in exon 10 of the GHR gene and investigate relationships with 14 in vivo traits and four meat characteristics in Piemontese animals. The biallelic polymorphism already described was detected using a new PCR procedure. The statistical analysis did not show significant gene substitution effects on growth, size and meat conformation traits. As for meat characteristics, a significant gene substitution of GHR(A) over GHR(G) was observed for drip losses at day 3, with the allele GHR(A) associated with higher values. A significant dominance effect was also observed for this trait. Further investigations in other breeds will be useful for better understanding information on the effect of this GHR polymorphism.     

Histological and immunological reaction of cattle skin to first-instar larvae of Dermatobia hominis

Abstract. Six cattle that had earlier exposure to Dermatobia hominis were infested experimentally with first-instar larvae of the parasite. Skin biopsies taken at intervals were studied in wax and in plastic sections. The avidin-biotin-peroxidase method was used to detect the presence and localization of host immunoglobulins (Igs) G and M and antigens of first and second instar larvae of Dermatobia hominis. The larvae penetrated actively through the skin and migrated towards the subcutaneous tissues. The great numbers of eosinophils suggest that they are the most important cell in mediating damage to D.hominis larvae. The immunoglobulins bound only to dead or moulting larvae in which access to binding sites may have been altered. This could represent a morphological manifestation of a mechanism that protects larvae from the host immune response. Large amounts of soluble antigens detected along the fistulous tract may be important in the maintenance of this tract by disturbing the normal cicatrization process.     

The effects of the brown ear-tick, Rhipicephalus appendiculatus, on milk production of Sanga cattle

Abstract. Lactating Sanga cows of the Mashona breed from Zimbabwe, receiving either a low or a high level of nutritional supplement, were exposed to two levels of infestation of adults of the brown ear-tick, Rhipicephalus appendiculatus in the highveld of Zimbabwe. The effect of the ticks on the milk yield was measured over an 11-week period during the rainy season from January to April 1986. A technique in which calves were weighed before and after suckling was used to estimate milk yield. There were significant treatment effects of ticks (P < 0.05) on milk production but no significant differences in liveweight gain between calves from tick-free and tick-infested dams. The loss in milk production was poorly related to the number of female ticks that engorged, being 9 g (SEM 4) per tick. A Friesian x Hereford (Bos taurus) reference group of cattle carried 50% more ticks than the Mashona cows, illustrating a difference in resistance between the breeds. Thirteen screw-worm (Chrysomya bezziana) strikes were recorded amongst the thirty-two Mashona cows compared with twenty-one amongst the ten Friesian × Hereford animals between January and the end of March.     

piggyBac转座子在牛基因组的整合位点及特征分析

piggyBac(PB)转座子作为一种遗传工具被广泛应用于多个物种的转基因及插入突变研究, 目前PB转座子在牛中的相关研究还较少。为了获得PB转座子在牛基因组中的整合位点, 总结其转座特征, 文章构建了PB[CMV-EGFP]和pcDNA-PBase二元转座系统, 利用细胞核电转技术共转染牛耳组织成纤维细胞, 经G-418筛选, 获得了稳定转染EGFP的转基因细胞系; 提取细胞基因组DNA, 利用基因组步移技术扩增PB转座子5′ Bac区插入位置的DNA序列; 通过与牛基因组序列进行BLAST比对, 得到PB转座子在牛基因组中的插入位点。文章共获得了8个有效的整合位点, 但仅有5个位点定位到染色体1、2、11和X染色体上。序列分析表明：在牛基因组中, PB转座子可特异性的插入到“TTAA”位置, 并整合到基因间的非调控区; 分析整合位点“TTAA”相邻一侧的5个碱基组成, 发现PB转座子5′端倾向于插入到GC(62.5%)碱基富集区。该研究表明, PB转座子可以在牛基因组中发生转座, 获得的整合位点信息为利用PB转座子在牛上开展遗传学研究提供了理论参考。     

The effects of the brown ear-tick, Rhipicephalus appendiculatus, on milk production in dairy cattle

Abstract. The effect is reported of artificially controlled levels of infestation with adults of Rhipicephalus appendiculatus on the milk yield of twenty commercial Bos taurus dairy cattle on a high plane of nutrition and eighteen crossbed B.taurus × Sanga cattle on a lower plane of nutrition in the highveld of Zimbabwe. The results showed no significant effect on milk yield of infestations averaging twenty engorging ticks per animal per day, despite severe ear damage in some animals. They indicate that milk production of dairy cattle under commercial management is not sensitive to infestation with R.appendiculatus. The results have important implications for management of ticks in Africa, but need to be interpreted within the context of the control of tick-borne diseases.     

The anterior tooth development of cattle presented for slaughter: an analysis of age,sex and breed

In a cross-sectional study, data from records of cattle slaughtered over a 1-year period at a large abattoir in South West England were analysed using an ordered category response model to investigate the inter-relationships between age, sex and breed on development of the permanent anterior (PA) teeth. Using the model, transition points at which there was a 50% probability of membership of each category of paired PA teeth were identified. Data from ∼60 000 animals were initially analysed for age and sex effect. The age transition was found to be ∼23 months moving from zero to two teeth; 30 months for two to four teeth; 37 months for four to six teeth and 42 months for six to eight teeth. Males were found to develop, on average, ∼22 days earlier than females across all stages. A reduced data set of ∼23 000 animals registered as pure-bred only was used to compare breed and type interactions and to investigate sex effects within the sub-categories. Breeds were grouped into dairy and beef-type and beef breeds split into native and continental. It was found that dairy-types moved through the transition points earlier than beef-types across all stages (interval varying between ∼8 and 12 weeks) and that collectively, native beef breeds moved through the transition points by up to 3 weeks earlier than the continental beef breeds. Interestingly, in contrast to beef animals, dairy females matured before dairy males. However, the magnitude of the difference between dairy females and males diminished at the later stages of development. Differences were found between breeds. Across the first three stages, Ayrshires and Guernseys developed between 3 and 6 weeks later than Friesian/Holsteins and Simmental, Limousin and Blonde Aquitaine 6 and 8 weeks later than Aberdeen Angus. Herefords, Charolais and South Devon developed later but by a smaller interval and Red Devon and Galloway showed the largest individual effect with transition delayed by 8 to 12 weeks.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

Postinsemination treatment of primiparous and multiparous cows with cloprostenol failed to affect ovulation and pregnancy rate in dairy cattle

Previous studies have demonstrated the facilitating effect of a single cloprostenol treatment postinsemination on ovulation and pregnancy rate in dairy cattle and Italian buffalos. This study was conducted to test the effects of 500 μg cloprostenol given intramuscularly immediately postinsemination to 55 primiparous and 60 multiparous Holstein cows (Bos taurus; TR) of a typical dairy operation in East Germany and to compare them with 57 primiparous and 48 multiparous saline-treated cows (CON). Animals of the TR and CON groups did not differ or only differed marginally for age at treatment, interval calving–treatment, lactation number, milk production on the day of treatment, body condition score, and their peri- and postparturient case histories. All animals were clinically and reproductively healthy on the day of treatment. They were inseminated once 12 h after the onset of estrus, scanned 24 h after insemination to confirm ovulation, and tested for pregnancy by transrectal palpation between Days 42 and 48 postinsemination. Treatment did not affect the number of cows ovulating. The overall combined pregnancy rate (PR) was 47.3%. Pregnancy rate did not differ statistically between TR and CON (46.1% vs. 48.6%; P > 0.05). In conclusion, this study failed to demonstrate beneficial effects of a postinsemination treatment of primiparous and multiparous cows with cloprostenol on ovulation and PR in a typical dairy cattle operation in East Germany.     

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

The role of human immortal skin keratinocytes-acellular dermal matrix scaffold in skin repair and regeneration

In this study, we aimed to investigate the phenotypic characteristics of human immortal skin keratinocytes (HaCaT) cells and the role of acellular dermal matrix (ADM) in coculture system of HaCaT cells and ADM. Flow cytometry was used to examine the cluster of differentiation (CD) makers of HaCaT cells. Apoptosis analysis was applied to detect the apoptosis rate of HaCaT cells. Morphological observation of ADM isolated from the reticular layer of Sprague-Dawley rat dermis was utilized to evaluate the morphological structure of ADM. Methylthiazolyl tetrazolium (MTT) assay and morphological experiments were further used to confirm the scaffold role of ADM in HaCaT cells. A wound-healing mice model accompanied by HaCaT-ADM scaffold transplantation was performed to further verify the function of HaCaT-ADM scaffold. Our results showed that CD71, CD49f, K19, and CD29 were highly expressed in HaCaT cells, and the percentage of apoptosis cells was significantly increased, which represented that HaCaT cells had much stronger capacities of adhesion and proliferation than normal human keratinocytes. Additionally, the morphological structure of ADM presented many natural microbores, which made cells rapidly grow on ADM. The results exhibited that the HaCaT cells indeed promptly proliferate on ADM and easily grow into the microbores of ADM. Finally, an in vivo experiment further confirmed that the transplantation of the HaCaT-ADM scaffold into the dorsal skin of a wound-healing mice model could gradually repair the injured wound. Thus, these findings indicated that HaCaT cells might be as seed cells to develop skin tissue engineering and the HaCaT-ADM scaffold might be a better candidate to promote skin repair and regeneration.