Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

[Molecular and genetic aspects of idiopathic scoliosis. Blood test for idiopathic scoliosis]. / Molekulare und genetische Aspekte der idiopathischen Skoliose. Bluttest bei idiopathischer Skoliose.

Spinal deformities, and particularly scoliosis, are the most frequent forms of orthopedic deformities in children and adolescents. About 1-6% of the population has scoliosis. This disorder leads to severe spinal deformities and predominantly affects adolescent girls.Although the multifactorial origin of adolescent idiopathic scoliosis (AIS) is broadly recognized, the genetic causes of AIS are still largely unknown. Our previous studies suggested a generalized dysfunction of melatonin transduction (the hormone that is primarily produced in the brain and epiphysis). In the meantime we have demonstrated that such a defect of signal transduction is caused by chemical alterations, which inactivate the function of the inhibitory G protein-coupled melatonin receptors. This discovery has led to the development of the first blood test to detect children without symptoms who are at risk of developing scoliosis. Since a single function (cellular reaction to melatonin) is determined, the unique advantage of this test is that it can be performed without knowledge of mutations in defective genes that could provoke the onset of AIS.

The effect of pulsed electromagnetic fields on chondrocyte morphology.

Osteoarthritis is a debilitating joint disease where the articular cartilage surface degrades and is unable to repair itself through natural processes. Chondrocytes reside within the cartilage matrix and maintain its structure. We conducted in vitro experiments to investigate the morphological response of cultured human chondrocytes under different pulsed electromagnetic field (PEMF) conditions. In the control experiments, cultured chondrocytes attached to the bottom of a culture dish typically displayed either a stellate or spindle morphology with extended processes. Experimental chondrocyte cultures were placed in a Helmholtz coil to which a ramp waveform was applied. Exposure to PEMFs caused the chondrocytes to retract their processes, becoming spherical in shape. This change in morphology followed a progression from stellate to spindle to spherical. These morphological changes were reflected in an average reduction of 30% in the surface contact area of the chondrocytes to the culture dish. Understanding the mechanisms by which PEMFs affect the morphology of chondrocytes will help lead to new treatments for osteoarthritis.

Is it possible to identify a population in which the incidence of future development of AIS is greatly increased when compared to the normal population?

For future research of predictors of AIS, it would be advantageous to identify a general population in which the development of AIS is greatly increased when compared to the normal population. The probability of predicting future development of AIS among younger relatives of current patients based on the probability of AIS incidence was assessed from the research literature. Although there is considerable literature relating to familial relationships of the probability of developing AIS or having AIS, the probability is relatively low in most cases. Even with the best of predicted probabilities, the identification of patients with a high probability of developing AIS remained low. The identification of people among the general population who have a high probability of developing AIS based on the probabilities expressed in the literature is not possible.

The development of osteoblasts from stem cells to supplement fusion of the spine during surgery for AIS.

Surgical correction in severe cases of AIS is often hampered by insufficient autograft bone to facilitate the fusion. The development of other sources of bone generating cells would greatly enhance the surgical. Bone marrow derived stem cells were harvested from femoral reaming during total hip arthroplasty for the purpose of differentiating into osteoblasts. Stem cells were isolated from the marrow and successfully differentiated into three cell lines (osteoblasts, chondrocytes and adipocytes) to confirm multilineage potential. Osteoblasts were developed from the stem cells and demonstrated the ability to be cultured to possibly provide a source of bone generating cells to augment surgical fusions. It is anticipated that the addition of osteoblasts created from stem cells (combined with appropriate matrix) will have significant influence on the success of AIS surgery through improvement of bone fusion.

Transgenic mice overexpressing a dominant-negative mutant type II transforming growth factor beta receptor show enhanced tumorigenesis in the mammary gland and lung in response to the carcinogen 7,12-dimethylbenz-[a]-anthracene.

To test the hypothesis that the transforming growth factor-beta (TGF-beta) system has tumor suppressor activity in the mammary gland, we have generated transgenic mice overexpressing a dominant-negative mutant form of the type II TGF-beta receptor, under the control of the mouse mammary tumor virus-long terminal repeat. High-level expression of the transgene was observed in the mammary and salivary glands, with lower expression in the lung, spleen, and testis. Older nulliparous transgenic mice (9-17 months) showed a marked increase in the incidence and degree of lobulo-alveolar side-branching in the mammary glands when compared to wild-type littermates (24.8% of glands examined histologically versus 14.4%; P = 0.004), suggesting a role for endogenous TGF-betas in regulating development or maintenance of mammary alveoli. Spontaneous tumorigenesis was unchanged in the transgenic mice. However, following initiation with the carcinogen 7,12-dimethylbenz[a]anthracene, the transgenic group showed a significant increase in the incidence and multiplicity of mammary tumors when compared with wild-type littermates (40% incidence in transgenic mice versus 22% for wild-type, with 4 of 25 transgenics developing multiple mammary tumors versus 0 of 27 wild-type; P = 0.03). An early increase in the incidence of lung tumors was also observed in transgenic mice, but no difference between genotype groups was seen in the incidence of tumors in tissues in which the transgene is not expressed. The data show that the endogenous TGF-beta system has tumor suppressor activity in the mammary gland and lung.

Atypical shoulder muscle activation in multidirectional instability.

OBJECTIVE: Surface and intramuscular electromyography was used to investigate shoulder muscle activity in subjects with multidirectional instability (MDI). METHODS: Subjects (seven MDI, 11 control) performed repetitive shoulder abduction/adduction, flexion/extension and internal/external rotation movements on an isokinetic dynamometer. The activity of the deltoid, infraspinatus, supraspinatus, latissimus dorsi, and pectoralis major muscles were recorded using double-differential surface and intramuscular fine-wire electrodes. A repeated measures analysis of variance evaluated group differences in the amplitude, onset, termination and duration of the muscle activity. RESULTS: Significant activation parameter differences for the supraspinatus, infraspinatus, posterior deltoid and pectoralis major muscles were found in the subjects with MDI. The rotator cuff and posterior deltoid muscles demonstrated abbreviated periods of activity when performing internal/external rotation, despite activation amplitudes that were similar to the controls. In contrast, the activation of the pectoralis major differed from the control group in both the amplitude and time domains when performing shoulder extension. CONCLUSIONS: MDI is associated with atypical patterns of muscle activity that occur even when highly constrained movements are used to elicit the activity. SIGNIFICANCE: In addition to glenohumeral hyperlaxity, the results suggest that dysfunctional neuromuscular control of the rotator cuff is also a contributing factor to the pathoetiology of MDI.

Examination of the failure properties of the anterior cruciate ligament during the estrous cycle.

The purpose of this study was to determine whether the mechanical properties of the rat anterior cruciate ligament (ACL) vary when tested in vitro at different stages of the estrous cycle. Sixty female rats were allocated to four groups according to their stage of the estrous cycle: diestrus (n=16), proestrus (n=17), estrus (n=13) and metestrus (n=14). Right hindlimbs were harvested for mechanical testing and left hindlimbs were harvested for immunohistochemical staining to confirm the presence of the estrogen receptor. Results from the first relaxation test showed a significant difference between the estrus and proestrus stage, which was not observed in a second subsequent relaxation test. Likewise, no significant differences were found when comparing failure load and stiffness between the different stages of the estrous cycle. These results suggest that normal physiological fluctuations in estrogen during the estrous cycle did not alter the failure properties of the rat ACL.

Development of scoliosis following pinealectomy in young chickens is not the result of an artifact of the surgical procedure.

Pinealectomy in young chickens consistently results in scoliosis, which has many characteristics similar to those seen in adolescent idiopathic scoliosis. The mechanism underlying this phenomenon remains a mystery and it is not yet entirely clear whether some unidentified aspect of the extensive surgery is the major factor rather than the removal of the pineal gland. Four different types of pinealectomy surgery were performed on young chickens as well as deliberate damage to the cerebral cortex which simulated the extreme of any accidental damage that might occur during surgery. Scoliosis was assessed from weekly radiographs. No differences in incidence of scoliosis, degree of severity, or pattern of curve development were observed for any of the experimental groups when compared with controls. In all groups approximately 55% of the chickens developed scoliosis that progressed rapidly. Different pinealectomy procedures and deliberate damage to the cerebral cortex produce scoliosis in young chickens with the same incidence and characteristics. This suggests strongly that the mechanism behind the phenomenon is due to the removal of the pineal gland and not some artifact of the extensive surgery. The pinealectomy model in young chickens is proving to be a good model for studying AIS in humans. An understanding of the mechanism underlying this phenomenon has the potential to provide further insights into the etiology of AIS and can lead to the development of novel treatment methods.

Structure of the primordial diaphragm and defects associated with nitrofen-induced CDH.

The goals of this study were to further our understanding of diaphragm embryogenesis and the pathogenesis of congenital diaphragmatic hernia (CDH). Past work suggests that the pleuroperitoneal fold (PPF) is the primary source of diaphragmatic musculature. Furthermore, defects associated with an animal model of CDH can be traced back to the formation of the PPF. This study was designed to elucidate the anatomic structure of the PPF and to determine which regions of the PPF malform in the well-established nitrofen model of CDH. This was achieved by producing three-dimensional renderings constructed from serial transverse sections of control and nitrofen-exposed rats at embryonic day 13.5. Renderings of left- and right-sided defects demonstrated that the malformations were always limited to the dorsolateral portions of the caudal regions of the PPF. These data provide an explanation of why the holes in diaphragmatic musculature associated with CDH are characteristically located in dorsolateral regions. Moreover, these data provide further evidence against the widely stated hypothesis that a failure of pleuroperitoneal canal closure underlies the pathogenesis of nitrofen-induced CDH.

Variability of histochemical and morphometric data from needle biopsy specimens of human quadriceps femoris muscle.

Duplicate needle biopsies from the lateral portion of quadriceps femoris muscle from 20 young, healthy males were investigated morphometrically and histochemically. Mean results showed both the size and occurrence of the three main fibre types present to be similar to values obtained from a survey of the literature. However, considerable variations in the proportions of fibre types (coefficients of variation 30-40%) and significant (P less than 0.001) differences in fibre size between individuals were common. Within individuals, comparisons of samples taken at a reference site in the right thigh with samples obtained from deeper, more proximal or contralateral sites also often showed significant differences in fibre size. These results suggest caution is necessary when interpreting apparent changes in such values derived from subsequent biopsies of individuals.

The effects of melatonin therapy on the development of scoliosis after pinealectomy in the chicken.

The mechanism underlying the development of scoliosis after pinealectomy in young chickens is unknown. However, since the main product of the pineal gland is melatonin, melatonin remains an obvious focus in studies designed to discover this mechanism. One confounding factor is that serum melatonin levels are close to zero after pinealectomy but scoliosis does not develop in all chickens that have had this procedure. Therefore, the role of melatonin in the development of scoliosis in chickens after pinealectomy remains controversial. In the current investigation, two pilot studies demonstrated that a physiological therapeutic dose of melatonin (2.5 milligrams per kilogram of body weight) restored the circadian rhythm of melatonin, as measured by serum assay. In the main study, this dose was administered daily starting either immediately after the pinealectomy or two weeks after it, when scoliosis had developed. Scoliosis was assessed on weekly radiographs, and the Cobb angle was determined for all chickens in which scoliosis developed. Overall, scoliosis developed in only 56 percent (fifty) of the eighty-nine chickens that had had a pinealectomy; this rate was consistent throughout all experimental groups. Scoliosis did not develop in any of the control chickens, which did not have a pinealectomy. On the basis of the average Cobb angles in the chickens in which scoliosis had developed, it was determined that neither the prevalence nor the pattern of the scoliosis was affected by the therapy in any of the experimental groups. It was thus concluded that melatonin therapy after pinealectomy in young chickens has no effect on the development or progression of scoliosis. These results raise doubts regarding the role of melatonin in the development of scoliosis after pinealectomy in the young chicken.

The migration and distribution of somite cells after labelling with the carbocyanine dye, Dil: the relationship of this distribution to segmentation in the vertebrate body.

The cells of individual somites in 2-day-old chick embryos were marked by injecting a fluorescent dye into the somitocoele. This procedure permanently marked the cells and allowed their subsequent development and distribution to be followed. The cells were found to remain in close association with each other within limited boundaries and did not mix to any great extent with similar cells from adjacent somites. Fluorescent cells from single somites were found in the intervertebral disc, connective tissue surrounding two adjacent neural arches, all the tissues between the neural arches, the dermatome, and the associated myotome. No fluorescent cells were found in the notochord or in any nervous tissue apart from accompanying connective tissue. Surprisingly, the vertebral bodies and neural arches did not contain any fluorescent cells apart from those in the connective tissue surrounding them, but this absence of fluorescent cells was thought to be due to the dilution of the fluorescence following cell proliferation. These results provide further experimental support for the theory of resegmentation in vertebral formation, and also provide evidence of a compartmental method of development along the rostrocaudal axis in vertebrates, similar to that already discovered in insects. On the basis of cell lineage criteria, the sclerotome might be considered as a developmental compartment.

The binding pattern of peanut lectin associated with sclerotome migration and the formation of the vertebral axis in the chick embryo.

Lectins have been used extensively to detect changes in carbohydrate moieties on the surfaces of embryonic cells during early development. Peanut agglutinin (PNA) in particular has been used to investigate changes related to cell differentiation. PNA has also been used to differentiate between the rostral and caudal sclerotome halves which have been shown to be functionally different, with neural crest cells and neurites traversing only the rostral half during their migration. In this study, we have sectioned and stained chick embryos between 3 and 8 days of age with PNA to examine the distribution of PNA binding sites associated with the vertebral column during this period and also to determine the fates of the rostral and caudal sclerotome halves. Ultrastructural localisation of PNA-gold conjugate showed that binding sites for this lectin were present intracellularly and extracellularly both on cell surfaces and in the matrix. At the light microscope level, a clear banding pattern emerged after staining with PNA which consisted of alternating light and dark staining along the entire length of the vertebral axis of the embryo. In the younger embryos, a simple banding pattern emerged where the rostral sclerotome half of each segment stained only lightly while the caudal half stained darkly. This banding pattern was present throughout the 6 day period of development and could be traced continuously but grew more complex as the sclerotome cells migrated to surround the notochord and neural tube and as the dorsal root ganglia developed. The rostral sclerotome half was found to contribute to the caudal part of one vertebral body and its neural arch, while the caudal sclerotome half was found to contribute to the intervertebral disc, the rostral half of the next caudal vertebra, and part of its neural arch.

Regeneration following somite removal in chick embryos.

A technique was developed for ensuring complete removal of single somites with minimal damage to surrounding tissues in 2-day-old chick embryos. Histological examination of the site of somite removal at various time intervals after operation revealed that a regeneration mechanism could be triggered. Replacement of the cells that had been removed could occur, but the extent of the replacement was dependent on the immediate fate of the gap created. If the gap was closed by enlargement of the adjacent somites, no replacement of the cells occurred. If the gap remained, then cells invaded the gap and were able to produce a normal sclerotome and dermomyotome. By labelling adjacent cells with the carbocyanine dye. DiI, it was shown that the replacement cells could come from the adjacent somites, as well as the intermediate mesoderm. Use of an antibody to HNK-1 established that the replacement cells did not come from the neural crest and that the neural crest cell distribution was little affected. Staining with peanut agglutinin showed that the replacement cells were able to adopt the characteristics associated with rostral and caudal halves of the normal sclerotome. These results provide possible explanations for the variety of vertebral anomalies produced by removal of somites and for the production of some congenital vertebral anomalies.

Increases in the number of cells in different areas of epithelial somites related to changes in morphology and development.

There are two distinct groups of cells in the epithelial somite: cells in the epithelial ball that form the periphery, and loose mesenchymal cells found in the central cavity (somitocoele). Recent work has produced evidence to show that these two groups of cells have significant differences (morphology, origin, fibronectin content, reaction to peanut lectin, communication properties) but the significance of these differences has yet to be established. It is not yet clear whether the epithelial somite stage of development is merely a time for cell proliferation, or whether it is a time when significant differences develop which have consequences in subsequent morphogenesis. Certainly, there are indications that the two groups of cells might form different structures related to the vertebral column based on their position in the subsequent sclerotome. In this study, we have examined the number of cells that are present in both the epithelial ball and the somitocoele at various stages of maturity. The results show that later-formed somites contain significantly more cells in both the epithelial ball and the somitocoele. Furthermore, while the density of cells in the epithelial ball remains constant (accounting for an increase in dimensions of the somite), there is a significant increase in density of cells in the somitocoele. This suggests that there is an important distinction being created between the cells of the epithelial ball and those in the somitocoele.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of somite removal on vertebral formation in the chick.

We have examined the effects on vertebral development of various combinations of somite removal in two day old chick embryos as shown by vertebral formation after a further seven days of incubation. Each combination produced one of a variety of results ranging from completely normal vertebral formation, through fusion of various vertebral elements, to the absence of complete vertebral halves and the formation of hemivertebrae. Assessment of our operating ability showed that we were removing at least 90% of the somitic material and therefore these results suggest that there is a regulating mechanism available to the embryo, at least with regards to vertebral development. When two consecutive somites were removed, vertebrae frequently developed that were lacking certain elements. This suggests that the somitic cells are already determined with regards to formation of specific vertebral elements. Experiments involving the removal of a bilateral pair of somites (a repetitive unit) also provided evidence of a counting mechanism which ensures that the correct number of total vertebrae are present.

Communication compartments in the axial mesoderm of the chick embryo.

Intracellular microinjection of the fluorescent tracer Lucifer Yellow into mesoderm cells along the rostrocaudal axis of the early chick embryo has revealed compartments where the intercellular diffusion of dye, presumably via gap junctions, is restricted at the borders between groups of cells. Cells in the segmental plate were dye-coupled, as were cells forming the epithelial somites. However, dye-coupling was not observed between different somites, nor was it observed between the outer epithelial cells and the cells in the somitocoele. On dispersal of the somite, dermatome cells were dye-coupled. However, sclerotome cells were found to be divided into rostral and caudal compartments separated by a group of cells bordering the intrasclerotomal fissure (of von Ebner) that also exhibited dye-coupling, restricted primarily to cells along the fissure. Some of these compartment borders can be accounted for by the presence of a morphological barrier which reduces cell-cell contact, but others are more difficult to explain, as there appears to be extensive cell-cell contact across the border. This would be analogous to some compartments found in insects. Some of the compartments also have borders similar to those described by cell lineage studies. The results also indicate that dye-coupling becomes restricted in a spatial and temporal manner as the mesodermal cells mature.

The immediate fate of cells in the epithelial somite of the chick embryo.

The precise origins and fates of cells forming the epithelial ball and those contained within the somitocoele at the centre of the somite in the chick embryo are unknown. In particular, it is not known whether the progeny of the proliferating cells in both the epithelial ball and the somitocoele simply mix with each other, or whether they represent two separate populations that remain isolated during proliferation. We tested whether cells found in both of these locations are the result of cell migration, cell division or both. To do this, we injected single cells in both the epithelial ball and the somitocoele with lysinated fluorescein dextran, a molecule that can only be transferred to other cells through cell division, and observed their development over the next 24 h. Control experiments were also performed to assess the incidence of error associated with the microinjection method and to provide reliable and valid comparative data. Our results showed that the progeny of the cells in the somitocoele, as well as any non-proliferating cells, appear to remain in the somitocoele. In contrast, the progeny of the cells in the epithelial ball either remain in the epithelial ball or migrate into the somitocoele to mix freely with the other cells present. These results suggest that the cells in the epithelial ball and the somitocoele develop as discrete groups, with the possibility of diverse fates accompanying further development.

Identification of the location, extent, and pathway of sensory neurologic feedback after mechanical stimulation of a lateral spinal ligament in chickens.

STUDY DESIGN: This study traced the location, extent, and pathway of sensory feedback after the mechanical stretching of a lateral spinal ligament in young chickens. The pathway was traced by locating the sites of Fos protein production in neuronal cell bodies at various sites in the nervous system. OBJECTIVES: To trace the location, extent, and pathway of sensory feedback after the mechanical stretching of a lateral spinal ligament in young chickens. SUMMARY OF BACKGROUND DATA: The innervation of ligaments is thought to form part of a protective feedback mechanism to provide stability for joints. The precise pathway and extent of the feedback for spinal ligaments is currently unknown. Such information would provide a clear focus for future studies, especially for diseases such as scoliosis where it has been suggested that there is abnormality in perception of sensory feedback. METHODS: The intertransverse ligament on the right side at T3-T4 in 4-week-old chickens was exposed by blunt dissection. After Fos production resulting from the surgery had been stopped, the ligament was stretched mechanically and repeatedly for 60 minutes using a 300-g weight. Various areas of the nervous system then were sectioned and processed immunohistochemically to identify areas of Fos production in nerve cell bodies. The presence of Fos indicated neurons that had been stimulated by the stretching the ligament, including interneurons along the feedback pathway. RESULTS: Fos protein was identified in nerve cell bodies in the dorsal root ganglia and intermediate gray matter of the spinal cord at the level of stimulation as well as at several spinal cord levels above and below the site of stimulation. Identification was made on the ipsilateral and the contralateral sides, although the extent of Fos production was less on the contralateral side. Fos presence also was identified in sympathetic ganglia at these sites. Nerve cell bodies in the combined nucleus cuneatus and gracilis in the medulla oblongata, the vestibular nuclei, and the thalamus also contained Fos-positive particles. CONCLUSIONS: Stretching a single lateral ligament of the spine produces a barrage of sensory feedback from several spinal cord levels on both sides of the spinal cord. This sensory information also is transferred to higher levels in the brain, including the nucleus gracilis and cuneatus, the vestibular nuclei, and the thalamus. These sites of Fos production suggest the locations of pathways for this sensory information, which include the dorsal columns and the spinocerebellar tracts. The information obtained from this study provides a clear focus for future studies in this area, particularly for diseases such as scoliosis where it is thought that incorrect perception of sensory information from the ligaments might be a major contributing factor.