透骨消痛胶囊及其拆方对退变软骨细胞Wnt/β-catenin信号通路的影响

BACKGROUND: Previous studies have showed that Tougu Xiaotong Capsule (TGXTC) exerts better effects on osteoarthritis, by regulating Rho/Rock signaling pathway, inhibiting signal transduction of chondrocyte mitochondrial apoptosis pathway, varying the rate and pattern of subchondral bone remodeling and improving the arrangement of subchondral bone collagen fibers and calcium-phosphate crystallization. OBJECTIVE: To observe the effects of the serum containing TGXTC and its disassembled recipes on chondrocyte degeneration of rats via Wnt/β-catenin signal pathway, and to explore the main therapeutic method for osteoarthritis in the TGXTC. METHODS:Forty Sprague-Dawley rats were randomly assigned to receive the treatment of TGXTC, Bushen Rougan (BSRG), Huoxue Qufeng (HXQF) and normal saline, respectively, according to the dose conversion methods of animal to animal and animal to human. Then various drug-containing serums were prepared for the following cellular experiment. After culture and passage, chondrocytes from Sprague-Dawley rats at passage 3 were divided into five groups: blank control, model, TGXTC, BSRG, HXQF groups. Cells in the latter four groups were cultured in appropriate drug-containing serums (normal saline serum for the model group) for 72 hours, following intervention with interleukin-1β for 24 hours. Cells in the blank control group were cultured in normal saline serum. Afterwards, cells in all the five groups were collected for detecting expression of Wnt 4, β-catenin and matrix metalloproteinase 13 at mRNA and protein levels using real-time PCR and western blot assay, respectively. RESULTS AND CONCLUSION: Compared with the blank control group, the expression of Wnt 4, β-catenin, matrix metalloproteinase 13 was significantly increased in the model group. Compared with the model group, the expression of Wnt 4, β-catenin, matrix metalloproteinase 13 in the TGXTC, BSRG and HXQF groups were decreased significantly, sequenced as TGXTC group < BSRG group < HXQF group. These results indicate that TGXTC plays a synergistic protection against interleukin-1β induced degeneration of chondrocytes. In addition, BSRG as a disassembled recipe of TGXTC is the main therapeutic method for osteoarthritis. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

神经细胞分泌因子促进骨骼肌细胞生长

BACKGROUND: There is no effective treatment for muscle atrophy caused by peripheral nerve damage. Skeletal muscle cells, a structural unit of muscle contraction, can be used for studies on muscle atrophy when cultured in vitro. OBJECTIVE: To investigate the promotion effect of neuron-secreted factors on the growth of skeletal muscle cells in vitro. METHODS: Skeletal muscle cells primary cultured in vitro were divided into two groups: experimental group with neuron-secreted factors, and control group with common culture medium, respectively. Afterwards, the number of skeletal muscle cells and expression level of alpha actin were detected by hematoxylin-eosin staining and immunohistochemical staining, respectively. RESULTS AND CONCLUSION:The number of skeletal muscle cells and expression level of alpha actin in the experimental group were significantly higher than those in the control group (P < 0.05). In conclusion, neuron-secreted factors have the ability of promoting the growth of skeletal muscle cells and may be helpful for denervated muscle atrophy. Subject headings: Tissue Engineering; Satellite Cells, Skeletal Muscle; Actinin; Neurons; Cells, Cultured 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

健步虎潜丸含药血清对体外成骨样细胞增殖和功能的影响

BACKGROUND: More recently, the focus has been on searching for a compound Chinese medicine for reinforcing kidney, which cannot only inhibit bone absorption, but also promote osteogenesis to protect against osteoporosis. OBJECTIVE: To explore effects of drug serum of Jianbuhuqian pills on proliferation and function of osteoblast-like cells in vitro. METHODS: Twenty-four adult female Sprague-Dawley rats were randomly divided into low-dose, medium-dose, high-dose and normal saline groups, and given intragastric administration of 1.5, 3.0, and 6 g/kg Jianbuhuqian pills and equal volume of normal saline, respectively twice daily for 1 week. At 1 hour after final gavage, rats were decapitated to prepare drug sera used for culturing osteoblast-like cells. At 24, 48 and 72 hours of culture, the cellular morphology was observed, as well as the cell proliferation and alkaline phosphatase activity was detected by MTT assay and alkaline phosphatase staining, respectively. RESULTS AND CONCLUSION: Compared with the normal saline group, the cell density began to increase significantly in three Jianbuhuqian groups at 24 hours after culture, mitotic figures were easy to be observed, cells were in overlapping growth, much secretions and matrix accumulation appeared, especially in the high-dose group. The obsorbance values in Jianbuhuqian groups were significantly higher than that in the normal saline group. After 24 hours of culture, the obsorbance values in the medium-dose and high-dose groups were significantly increased compared with the low-dose group, and the values showed significant differences among three Jianbuhuqian groups after 48 and 72-hour culture. In addition, the alkaline phosphatase activity presented overt increase in the Jianbuhuqian groups compared with the normal saline group, and significant differences could be found among Jianbuhuqian groups. To conclude, the drug serum of Jianbuhuqian pills can promote the activity of osteoblast-like cells in a time- and concentration-dependent manner. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

立体定向脑内移植神经干细胞改善颅脑损伤大鼠的神经运动功能

BACKGROUND: Cell replacement therapy as an effective strategy for reconstruction of the central nervous system has very broad application prospects. OBJECTIVE: To investigate the effect of stereotactic transplantation of neural stem cells into the brain on the neuromotor function of craniocerebral trauma rats. METHODS: Twenty male Sprague-Dawley rats were equivalently randomized into study and control groups. Animal models of craniocerebral trauma were made using the improved free-fall method in the rats. Then, model rats in the study and control groups were given parenchymal transplantation of embryonic neural stem cells and the same volume of culture medium with no stem cells at 1 day after injury, respectively. Neuromotor function of rats was assessed based on the neurological severity scores. At 2 weeks after transplantation, brain tissues were taken for hematoxylin-eosin staining, anti-BrdU, glial fibrillary acidic protein, β-tubulin III and tyrosine hydroxylase immunohistochemistry staining. RESULTS AND CONCLUSION: The neurological severity scores in the study group were significantly lower than those in the control group at 1 and 2 weeks after injury (P < 0.05). In the study group, there were many BrdU-positive neural stem cells in the brain tissues, some of which were positive for glial fibrillary acidic protein, β-tubulin III and tyrosine hydroxylase; while in the control group, there was no BrdU-positive cell in the brain tissues. Experimental findings show that neural stem cells stereotactically transplanted into the brain can proliferate and differentiate in the brain lesion, and thereby notably improve the neuromotor function of rats with craniocerebral trauma.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Expression of IL-10 and TNF-α in Rats with Cerebral Infarction after Transplantation with Mesenchymal Stem Cells

We investigated the effects of bone marrow-derived mesenchymal stem cells （MSCs） transplantation on the recovery of neurological functions in rat＇s MCAO （middle cerebral artery occlusion） model and its mechanism. MSCs were isolated from bone marrow of male Sprague Dawley （SD） rats. Female adult SD rats were randomly assigned into 4 groups： sham-operated group, MCAO group, vehicle group and MCAO ＋ MSCs-treated group. MSCs were injected into the lateral ventricle of rats in the MSCs-treated group and the same volume of PBS was given to the vehicle group. The expressions of IL-10 and TNF-α were assayed by RT-PCR and ELISA detections at day 1 and 4 after MCAO. The infarction volume was measured by TTC-staining. All rats underwent behavioral tests before, as well as 1, 4, and 14 days after MCAO. MSCs significantly improved functional recovery compared with the control at day 14 after transplantation. Compared with the MCAO group and the vehicle group, the expression of IL-10 mRNA and its protein level in the MSCs group significantly upregulated. However, the expression of TNF-α at day 4 after MCAO in the MSCs group significantly decreased compared with that of the MCAO group and the vehicle group. As a result, transplantation with MSCs significantly decreased infarct volume at day 1 and 4. This study strongly suggested transplantation with MSCs could reduce neuronal injury post focal cerebral ischemia in rats partly by regulating the expressions of IL-10 and TNF-α in the brain.     

生物反应器中的力学刺激促进组织工程软骨再生

BACKGROUND: Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE: To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracellular matrix in the concentration of 1×106/L to prepare cell-scaffold composites. Cell-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured statically for 1 day as control group. RESULTS AND CONCLUSION: Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradually with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of collagen type I and collagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of collagen type I and collagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cell-scaffold composite can produce more extracellular matrix, such as collagen and proteoglycan, strengthen the mechanical properties to be more coincident with the in vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage will be achieved.   中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

胰岛素干预对糖尿病大鼠骨折愈合及骨痂内细胞因子的影响

BACKGROUND: Fracture healing in diabetic patients is usually unsatisfactory because of hormones and metabolic disorder, and an eventual multiple organ dysfunction resulting from high blood glucose. OBJECTIVE: To dynamically observe the changes of cytokines during the fracture healing process in diabetic rats before and after insulin treatment. METHODS:A total of 120 Sprague-Dawley rats were included in this study. Of them, 90 rats intravenously injected with 5% tetraoxypyrimidine to induce rat models of diabetes were randomized into insulin treatment and diabetes groups, respectively. The remaining 30 rats were intravenously injected with equal volume of saline and selected as control group. The next day, blood glucose was determined. Healing at 1, 4, and 8 weeks after fracture were observed by the X-ray film. Biomechanical strength of the injured right tibia was measured at 4, 6, and 8 weeks after modeling. Cytokines in the osteotylus were determined by immunohistochemical staining and in situ hybridization technique. RESULTS AND CONCLUSION: The X-ray films showed that the speed of fracture healing in the diabetes group was slower than insulin treatment and control groups. Biomechanical strength of the osteotylus in the diabetes group was significantly decreased compared with the insulin treatment and control groups. However, there were no significant differences in above-mentioned parameters between the control and insulin treatment groups. Bone morphogenetic protein 2, basic fibroblast growth factor, transforming growth factor-beta, and vascular endothelial growth factor were widely expressed in the osteotylus and their expressions in diabetes group were significantly lower and slower than those in the control and insulin treatment groups. There was no statistical difference between control and insulin treatment groups. These results indicate that osteotylus formation speed, biomechanical strength, and growth factor expressions at the fracture site in diabetes rats were decreased compared with normal rats. Insulin treatment can enhance cytokine levels at the fracture site, thereby promoting the osteoblast proliferation and fracture healing. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

实验性根尖周炎模型大鼠核因子?B的表达

BACKGROUND:mRNA expression level of nuclear factor-kappa B (NF-?B) in periapical periodontitis has been shown to be significantly higher than that in normal periapical tissue. OBJECTIVE:To observe the expression of NF-?B in rats with periapical periodontitis. METHODS:Twenty male Sprague-Dawley rats aged 6 weeks were enrolled to establish apical periodontitis model at the right mandibular first motar (experimental group), and the left mandibular first motar as normal control group. Five rats were randomly selected at postoperative 1, 2, 3, and 4 weeks to make frozen sections of the mandibular tissue. The morphology of the periapical tissue was observed through hematoxylin-eosin staining, and the expression level and distribution of NF-?B were detected by immunohistochemical staining. RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining showed that the apical periodontal tissue of the mandibular first molar at the right side had local inflammatory response and spread to peripheral tissues; while there was no pathological changes in the control group. (2) Immunohistochemistry results revealed that NF-?B expression increased from the 1st week to 2nd week after surgery, and then went down at the 3rd week and reached the lowest point at the 4th week; moreover, the expression level at each time exhibited significant differences (P < 0.05). The amount of NF-?B-positive cells in the experimental group was significantly higher than that in the control group (P < 0.05). (3) These findings suggest that the expression level of NF-?B holds close correlation with the process of periapical periodontitis indicating that NF-?B may participate in the inflammatory reaction of periapical periodontitis.  中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

差速贴壁法原代培养椎间盘髓核细胞中的应用优势

BACKGROUND: As the main function cell of intervertebral disc, nucleus pulposus cells are the focus of studying the degenerative mechanism; thereby, it is crucial to maintaining the physiological function of nucleus pulposus cells in vitro and the stability of the cell phenotype. OBJECTIVE: To study the excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of rat intervertebral disc through comparison. METHODS: Twenty male Wistar rats aged 4 weeks were enrolled, and then nucleus pulposus cells of intervertebral disc were isolated and cultured in vitro; cell passage culture was performed in different groups when the primary cells were merged to 90%. Differential velocity adherent group cells adhered for 30 minutes, and non-adherent cells were aspirated and transferred to new culture dish after readjusting the concentration; the controls received no intervention. Passages 1 and 2 cells in the differential velocity adherent group were isolated and purified by the same procedure. The morphology of three generations of cells in the two groups was compared, the purity of the identification was detected by immunohistochemistry, the cell viability was detected by cell counting kit-8 and the cell growth curve was drawn. RESULTS AND CONCLUSION: Inverted phase contrast microscope and hematoxylin-eosin staining revealed that the cell homogeneity of the differential velocity adherent group was significantly higher than that of the control group, and there were more kinds of fibroblast-like cells in nucleus pulposus cells in the control group. Identification and purity analysis of collagen type II showed that the cytoplasm of two groups were both stained brown, indicating that they were the nucleus pulposus chrondrocytes. The positive rate of differential velocity adherent group was significantly higher than that of the control group (P < 0.05). The cell growth curve of cell counting kit-8 showed cells in the two groups all passed by the latency phase within 2 days, then to the logarithmic phase of 3 days, and entered the lag phase, while the growth rate of the control group was more rapid during the latency and the early logarithmic phases. These findings suggest that differential velocity adherence method is a practical and effective procedure for the isolation and purification of primary cultured rat nucleus pulposus cells. Through the primary culture, twice differential velocity adherence procedure, the passage 3 rat nucleus pulposus cells are metabolic exuberant, consistent with the phenotype, the cell purity is higher, and the logarithmic growth phase can be used as the optimal time for studying the mechanism of intervertebral disc cells. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

TOLL样受体4拮抗剂干预周围神经损伤后的瓦勒变性

BACKGROUND: The mechanism underlying Wallerian degeneration following peripheral nerve injury is complex. Immune regulation on Wallerian degeneration is beneficial for early repair of perpheral nerve injury. OBJECTIVE: To investigate the effects of Toll-like receptor 4 (TLR4) antagonist on Wallerian degeneration and axonal regeneration after early peripheral nerve injury in rats. METHODS: Fifty male Wistar rats were recruited and randomly divided into treatment group (n=20), model group (n=20) and sham group (n=10). The right sciatic nerves of rats in treatment and model groups were cut and sutured end-to-end, while the sciatic nerves of rats in sham group were only exposed. In the treatment group rats were intravenously injected with 0.15 mg/kg TAK-242 via tail vein 1 hour preoperatively and 7 days postoperatively, and the rats in the other two groups were given intravenous injection of the same volume of normal saline. The sciatic nerves were removed at 24 hours, 3, 4 and 7 days after surgery. RESULTS AND CONCLUSION: Real-time PCR indicated that the mRNA expressions of interleukin-1β and monocyte chemoattractant-1 were significantly increased in the model group compared with the sham group at 24 hours after surgery (both P < 0.001), while the expressions were significantly decreased after TAK-242 injection (both P < 0.001). Immunofluorescence showed that compared with the model group, down-regulated expression of CD68+ and iba1+ cells appeared in the treatment group at 3 days after surgery (P < 0.01, P < 0.05). Luxol fast blue staining revealed that demyelination at the sciatic nerve stump appeared in both model and treatment groups at postoperative 7 days, but myelin debris clearance in the treatment group was significantly reduced compared with the model group (P < 0.05). Hematoxylin-eosin staining showed that a lot of inflammatory cells, Schwann cells and regenerated nerve fibers at the sciatic nerve stump were found in the model group, while there were few inflammatory cells, Schwann cells and regenerated nerve fibers in the treatment group at 7 days after surgery. Immunohistochemistry found that the expression of growth-associated protein-43 in the treatment group was significantly lower than that in the model group at 4 days postoperatively (P < 0.05). Besides, compared with the model group, a significantly decreased sciatic functional index was found in the treatment group at 20, 30 and 40 days after surgery (P < 0.05). These results show that TLR4 antagonists delay early nerve regeneration in rats after sciatic nerve injury probably by inhibiting the TLR4 signaling pathway. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.