Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs.

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Endochondral ossification: how cartilage is converted into bone in the developing skeleton.

Endochondral ossification is the process by which the embryonic cartilaginous model of most bones contributes to longitudinal growth and is gradually replaced by bone. During endochondral ossification, chondrocytes proliferate, undergo hypertrophy and die; the cartilage extracellular matrix they construct is then invaded by blood vessels, osteoclasts, bone marrow cells and osteoblasts, the last of which deposit bone on remnants of cartilage matrix. The sequential changes in chondrocyte behaviour are tightly regulated by both systemic factors and locally secreted factors, which act on receptors to effect intracellular signalling and activation of chondrocyte-selective transcription factors. Systemic factors that regulate the behaviour of chondrocytes in growth cartilage include growth hormone and thyroid hormone, and the local secreted factors include Indian hedgehog, parathyroid hormone-related peptide, fibroblast growth factors and components of the cartilage extracellular matrix. Transcription factors that play critical roles in regulation of chondrocyte gene expression under the control of these extracellular factors include Runx2, Sox9 and MEF2C. The invasion of cartilage matrix by the ossification front is dependent on its resorption by members of the matrix metalloproteinase family, as well as the presence of blood vessels and bone-resorbing osteoclasts. This review, which places an emphasis on recent advances and current areas of debate, discusses the complex interactions between cell types and signalling pathways that govern endochondral ossification.

Malakoplakia in the urinary bladder of a kitten.

Malakoplakia is a form of chronic granulomatous inflammation that in humans most commonly affects the urinary bladder of middle-aged women. Naturally occurring malakoplakia is rare in animals, having been described twice in the pig only. An 8-week-old kitten was diagnosed with malakoplakia of the urinary bladder after a 3-week history of dysuria. Post-mortem examination revealed a markedly enlarged bladder with a diffusely nodular mucosal surface. Microscopically, there was diffuse submucosal infiltration by histiocytes stained positively by periodic acid Schiff (PAS) and described in the human condition as "von Hansemann cells". Intracellular and extracellular "Michaelis-Gutman" inclusion bodies were seen on light and electron microscopical examination. These structures are considered pathognomonic for malakoplakia. The pathogenesis of malakoplakia is enigmatic. Defective function of phagolysosomes is currently suspected to underlie the abnormal accumulation of submucosal histiocytes; however the primary functional defect remains unknown.

Eimeria taggarti N. Sp., a Novel Coccidian (Apicomplexa: Eimeriorina) in the Prostate of an Antechinus flavipes.

A novel coccidian species was discovered in the prostate of an Antechinus flavipes (yellow-footed antechinus) in South Australia during the period of postmating male antechinus immunosuppression and mortality. This novel coccidian is unusual because it develops extraintestinally and sporulates endogenously within the prostate gland of its mammalian host. Histological examination of prostatic tissue revealed dense aggregations of spherical and thin-walled tetrasporocystic, dizoic, sporulated coccidian oocysts within tubular lumina, with unsporulated oocysts and gamogonic stages within the cytoplasm of glandular epithelial cells. This coccidian was observed occurring concurrently with dasyurid gammaherpesvirus 1 infection of the antechinus' prostate. Eimeria-specific 18S small-subunit ribosomal (r)DNA polymerase chain reaction amplification was used to obtain a partial 18S rDNA nucleotide sequence from the antechinus coccidian. Bayesian phylogenetic analysis based on 18S rDNA gene sequences revealed that the novel coccidian clusters with reptile-host coccidians, forming an ancestral basal lineage of the eimeriid clade. The species has been named Eimeria taggarti n. sp. on the basis of both sporulated oocyst morphology and molecular characterization. It is suspected that E. taggarti is sexually transmitted via excretion of sporulated oocysts or free sporocysts with prostatic secretions in semen.

Hypertrophy and physiological death of equine chondrocytes in vitro.

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.

Plasmodium infection in a Leadbeater's possum (Gymnobelideus leadbeateri).

CASE REPORT: A wild-caught, adult female Leadbeater's possum (Gymnobelideus leadbeateri) died while in captivity after suffering from chronic ill-thrift that progressed to acute respiratory distress. On histopathological examination of tissues, the cause of death was determined to be severe acute pneumonia with pulmonary oedema associated with an intracellular protozoan parasite present within erythrocytes. Transmission electron microscopy was performed on lung tissues and organisms consistent for Plasmodium sp. were identified within numerous erythrocytes. Molecular characterisation of the parasite from DNA extracted from tissue blocks of fixed lung determined the organism to belong to the genus Plasmodium (100% similarity to Plasmodium species when a BLAST analysis was performed); however, speciation of the organism was not possible. CONCLUSION: This is the first report of Plasmodium sp. infection and subsequent disease in a native Australian mammal. The lifecycle of this parasite remains unknown. It is also unknown what effects haemoparasitism may have on the population dynamics of this endangered possum species.

Development of morphological changes and ileal glucagon gene expression in the small intestine of lambs infected with Trichostrongylus colubriformis.

To investigate the consequences of subclinical Trichostrongylus colubriformis infection on the intestinal mucosa and the associated changes in entero-glucagon gene expression, sheep were infected with 30000 larvae and killed 5, 10, 15 or 20 days after infection. Histological and cytological changes were examined. In the main site of infection, the upper duodenum, villous atrophy associated with crypt hyperplasia developed gradually. Cytological changes in the enterocytes appeared concurrently, characterized by a progressive reduction in brush border and in the number of ribosomes in the cytoplasm, changes in the internal structure of mitochondria, and enlargement of the intercellular spaces. Neither histological nor cytological modifications were found before day 15. At the same time, villous hypertrophy developed distally, beyond the main site of infection; this was interpreted as an adaptive response to parasitism. Enteroglucagon gene expression in the ileum was measured in parallel with the mucosal changes but did not reveal any difference between infected and control sheep. The results indicate that this gastrointestinal hormone does not have a major role in the response to nematode parasitism.

Acute renal failure following Bull ant mass envenomation in two dogs.

Acute renal failure was diagnosed in a German Short Haired Pointer bitch and a Kelpie cross-bred dog following envenomation by Bull ants. Both dogs had been tethered over a Bull ant nest and had experienced mass envenomation. There was local reaction at the envenomation sites and each dog had experienced vomiting that was poorly controlled by symptomatic therapy. Intensive treatment of renal failure was successful in the German Short Haired Pointer and the bitch remains well 19 months after envenomation. The Kelpie cross-bred deteriorated despite intensive treatment and was euthanased 36 hours after presentation. Necropsy examination revealed haemorrhage and necrosis of the small intestine and myocardium, bilateral nephrosis with tubular necrosis, and patchy haemorrhage of the lung alveoli, pancreas and adrenal cortices. Electron microscopy revealed necrosis of the small intestine and hydropic swelling of proximal renal tubules with necrosis of medullary tubules.

[Evaluation of combined toxic action of benzene and ethanol in the rat fetus]. / Ocena lacznego dzialania toksycznego benzenu i etanolu na plód szczura.

It has been well evidenced that benzene exerts toxic action on mammalian fetuses. This effect may be probably modified by several xenobiotics metabolized by mixed function oxidase system. In this study the effect of ethanol on benzene metabolism and benzene-induced fetotoxicity in rats was investigated. Pregnant female Wistar rats were exposed to benzene vapor for 6 h/day from Days 8 through 15 of gestation at concentrations of 1500 and 3000 mg/m3 or combination ethanol and benzene at the same concentrations. Ethanol was administered intragastrically at a dose of 2.5 g/kg directly before exposure to benzene or 18 hrs before. An air-exposed or ethanol treated control rats were also examined. At Day 20 of gestation, the pregnant females were sacrificed and examined for possible reproductive abnormalities. The fetuses were observed for skeletal and visceral changes. The maternal blood ethanol concentrations were 180 or 0 mg/100 ml one or 18 hrs after administration of this compound, respectively. Ethanol co-administration with benzene in principle had no effect on the metabolism of benzene, whereas it reduced the conversion of benzene to free and conjugated phenol when given 18 hrs before. Ethanol administered directly before exposure to benzene increased frequency of incidence of resorption, whereas it had no effect when was given 18 hrs before. Delay of ossification of fetuses after exposure to combination of benzene and ethanol was less pronounced than in animals exposed to benzene alone. The results indicate that the fetotoxicity of benzene may be modified by ethanol.

[Absorption of chromium through the skin and urinary excretion in rats]. / Wchlanianie chromu przez skóre i wydalanie z moczem u szczurów.

The aim of the experiment was to evaluate the diffusion rate in vitro and the skin absorption rate of trivalent and hexavalent chromium from aqueous solution. Excretion of chromium in urine was also measured. The experiment was carried out on 47 Wistar male rats, 4-5 months old. The solutions of chromium sulfate or potassium dichromate contained 2.5, 5.0 and 10.0 mg Cr per milliliter. Exposure time was 4 hrs. It was found that diffusion rate of Cr3+ was seven-time less than of Cr2O7(2-). Absorption rates of both ions through the skin were similar. Excretion rates of chromium in urine were not different in case exposure to both xenobiotics. It can be concluded that diffusion does not play an important role in absorption of chromium through the skin. It seems that oxidation grade of chromium has no influence on the excretion rate of this metal from the organism.

[Modification of metabolism and toxicity of benzene by ethanol in pregnant rats]. / Modyfikacja metabolizmu i toksycznosci benzenu przez etanol u ciezarnych szczurów.

In last decade it was suggested that the metabolism and toxicity of some xenobiotics may be modified by several compounds that alter the activities of microsomal oxidative and conjugating enzymes. In present study the effect of ethanol on benzene metabolism and benzene-induced hemotoxicity in pregnant rats was investigated. Pregnant female Wistar rats were exposed to benzene vapors for 6 hr/day from Days 8 through 15 of gestation at concentrations of 1500 and 3000 mg/m3 or combination ethanol and benzene at the same concentrations. Ethanol was administered intragastrically at a dose of 2.5 g/kg directly before exposure to benzene or 18 hrs before. An air-exposed or ethanol treated control rats were also studied. Before, during and after exposure blood samples for leukocyte and lymphocyte counts were obtained and urine for free phenol and conjugated phenol determinations was collected. Exposure to benzene alone decreased the maternal body weight and reduced leukocyte and lymphocyte counts in peripheral blood. Ethanol administered directly before exposure to benzene in principle had no effect on the metabolism and toxicity of benzene. It reduced the conversion of benzene to free and conjugated phenol, and protected animals from reduction of body weight and lymphopenia when was given 18 hrs before. Obtained results indicate that the early phases of benzene metabolism and its toxicity may be modified by ethanol.

[Creams protective against electrolytes--evaluation of protection in vitro]. / Kremy ochronne przeciw elektrolitom--ocena skutecznosci in vitro.

Diffusion-inhibiting effect of the barrier creams in case of some electrolytes i. e. alkalies, acids, salts of alkaline metals, and salts of heavy metals in two chambers diffusion apparatus have been determined. Protection efficiency of two own creams and two reference creams (silicone Goldschmidt's cream, Essen FRG, and "Anthydro" made by Phypro Laboratories 86270 La Roche, Posay, France) were compared. High protection efficiency of own creams, especially in case of sulfuric acid, hydrochloric acid, sodium nitrite, potassium bichromate and nickel nitrate was found. Diffusion of potassium hydroxide and sodium hydroxide did not exceed 2% of control values during 4 hrs period.

The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification.

Endochondral ossification is the process that results in both the replacement of the embryonic cartilaginous skeleton during organogenesis and the growth of long bones until adult height is achieved. Chondrocytes play a central role in this process, contributing to longitudinal growth through a combination of proliferation, extracellular matrix (ECM) secretion and hypertrophy. Terminally differentiated hypertrophic chondrocytes then die, allowing the invasion of a mixture of cells that collectively replace the cartilage tissue with bone tissue. The behaviour of growth plate chondrocytes is tightly regulated at all stages of endochondral ossification by a complex network of interactions between circulating hormones (including GH and thyroid hormone), locally produced growth factors (including Indian hedgehog, WNTs, bone morphogenetic proteins and fibroblast growth factors) and the components of the ECM secreted by the chondrocytes (including collagens, proteoglycans, thrombospondins and matrilins). In turn, chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand. This review discusses how the growth plate chondrocyte contributes to endochondral ossification, with some emphasis on recent advances.

Identification of light and dark hypertrophic chondrocytes in mouse and rat chondrocyte pellet cultures.

Hypertrophic "light" and "dark" chondrocytes have been reported as morphologically distinct cell types in growth cartilage during endochondral ossification in many species, but functional differences between the two cell types have not been described. The aim of the current study was to develop a pellet culture system using chondrocytes isolated from epiphyseal cartilage of neonatal mice and rats, for the study of functional differences between these two cell types. Hypertrophic chondrocytes resembling those described in vivo were observed by light and electron microscopy in sections of pellets treated with triiodothyronine, 1% fetal calf or mouse serum, 10% fetal calf serum or 1.7MPa centrifugal pressure at day 14, and in pellets cultured with insulin or 0.1% fetal calf or mouse serum at day 21. A mixed population of light and dark chondrocytes was found in all conditions leading to induction of chondrocyte hypertrophy. This rodent culture system allows the differentiation of light and dark chondrocytes under various conditions in vitro and will be useful for future studies on tissue engineering and mechanisms of chondrocyte hypertrophy.

Physiological death of hypertrophic chondrocytes.

OBJECTIVE: Post-proliferative chondrocytes in growth cartilage are present in two forms, light and dark cells. These cells undergo hypertrophy and die by a mechanism that is morphologically distinct from apoptosis, but has not been characterized. The aims of the current study were to document the ultrastructural appearance of dying hypertrophic chondrocytes, and to establish a culture system in which the mechanism of their death can be examined. DESIGN: Growth cartilage from fetal and growing postnatal horses was examined by electron microscopy. Chondrocytes were isolated from epiphyseal cartilage from fetal horses and grown in pellet culture, then examined by light and electron microscopy, and quantitative polymerase chain reaction. RESULTS: In tissue specimens, it was observed that dying dark chondrocytes underwent progressive extrusion of cytoplasm into the extracellular space, whereas light chondrocytes appeared to disintegrate within the cellular membrane. Pellets cultured in 0.1% fetal calf serum (FCS) contained dying light and dark chondrocytes similar to those seen in vivo. Transforming growth factor-beta1 or 10% FCS increased the proportion of dark cells and induced cell death. Triiodothyronine increased the differentiation of dark and light cells and induced their death. Dark cells were associated with higher levels of matrix metalloproteinase-13 expression than light cells, and light cells were associated with higher levels of type II collagen expression. CONCLUSIONS: Light and dark hypertrophic chondrocytes each undergo a distinctive series of non-apoptotic morphological changes as they die. Pellet culture can be used as a model of the two forms of physiological death of hypertrophic chondrocytes.

Histochemical analysis of muscle fibers in myotome of teleost fishes (Carassius auratus gibelio).

Qualitative histochemical studies were carried out on lateral musculature of Carassius auratus gibelio. On the basis of some metabolic enzymes (SDH, LDH) and myofibrillar ATP-ase activities, red, intermediate, white and small diameter muscle fibers were discerned. When SDH method was used, two other fiber types were distinguished. They built up a transition zone, which lay between intermediate and white muscle fiber complexes. The histochemical reaction for myofibrillar ATP-ase activity, after alkaline preincubation, revealed so called "displaced fibers" located at periphery of red as well as intermediate zones.

Leucocyte phenotypes in involuting and fully involuted mammary glandular tissues and secretions of sheep.

Mammary glandular tissues and mammary secretions were obtained from sheep at 2-60 d after weaning to study the leucocyte phenotypes associated with mammary involution. From 2-4 d after weaning, neutrophils were the predominant leucocytes in the alveolar and ductal lumina. Lymphocytes were present in the alveolar and ductal epithelium, interalveolar and periductal areas. Most of the lymphocytes in the alveolar and ductal epithelium (IEL) were CD8+, some were CD45R+ and few were CD4+. In the periductal clusters and in the interalveolar areas most of the lymphocytes were CD4+. There was a significant increase (P < 0.05) in the percentages of CD45R+ granulated IEL from 2 to 7 d after weaning, and this paralleled the increase in the percentages of apoptotic cells in the glandular epithelium. By 7-60 d after weaning, most cells within the alveolar and ductal lumina were macrophages followed by predominantly CD8+ lymphocytes. CD8+ lymphocytes were still predominant in the alveolar and ductal epithelium while CD4+ cells were predominant in the interalveolar areas. Very few gammadelta+ T cells were observed at all the stages examined. The cells in the mammary secretions correlated with those observed in the alveolar and ductal lumina. At the early stages of involution, the neutrophils and macrophages were heavily laden with lipid droplets, casein and cellular debris. The most interesting feature was the presence of cells either with extensive cytoplasmic processes (LCA+MHC class II+) or cytoplasmic veils (LCA+MHC class II+CD1+), probably dendritic cells. It is concluded that the cellular constituents of the mammary gland at the latter part of involution may afford the mammary gland more resistance to infection than the lactating gland and the gland at early stages of involution. The CD45R+IEL may trigger apoptotic cell death in the mammary glandular epithelium during mammary involution.

Involution of the sheep mammary gland.

Changes in the ovine mammary gland epithelium during initiated involution were studied by light and electron microscopy. Apoptosis of the duct and alveolar epithelial cells was first identified at 2 d after weaning, reached a peak at 4 d and then progressed gradually thereafter. Apoptotic cells were phagocytosed by intraepithelial macrophages and alveolar epithelial cells. Occasional apoptotic epithelial cells were observed in the alveolar and duct lumina. The highly vacuolated cells in the alveolar and duct lumina were confirmed to be macrophages as they were CD45+, MHC class II+. Changes in myoepithelial cells involved shrinkage and extension of cytoplasmic processes into the underlying stroma and no apoptosis was observed. Regression of the blood capillaries was also by apoptosis. The resulting apoptotic bodies were either taken up by adjacent endothelial cells or were shed into the capillary lumen to be phagocytosed later by mural endothelial cells or blood monocytes. The mammary glands were completely involuted by 30 d after weaning. It was concluded that the mammary gland involutes by apoptosis, a process which allows deletion of cells without the loss of the basic architecture and the integrity of the epithelial lining of the gland.

Effect of cyclosporin A on the survival and ultrastructure of Echinococcus granulosus protoscoleces in vitro.

Surgical treatment of human hydatidsosis involves the use of various scolicidal agents to kill infective Echinococcus granulosus protoscoleces that may disseminate into the peritoneal cavity during surgery and potentially re-infect the patient. Currently, no scolicidal agent is completely effective in killing intracystic protoscoleces in humans. Cyclosporin A (CsA) has previously been found to be lethal for E. granulosus protoscoleces in vitro. In this study, we further assessed the effectiveness of CsA as a scolicidal agent by testing the toxic effect of CsA at higher doses over various time-periods. Experiments were performed on activated and unactivated protoscoleces cultured in nutrient medium or sheep hydatid cyst fluid. All activated protoscoleces were killed following culture in 100 microg/ml of CsA for 3 days and 50 or 20 microg/ml for 5 days. The lethal effect of CsA on unactivated protoscoleces varied but reached 100% over 15 days in culture with 100 or 50 microg/ml of CsA. Pulse treatment of protoscoleces with 50, 20 or 10 microg/ml of CsA for 5 min or 72 h killed all parasites by day 10 and day 5 respectively. Untreated protoscoleces remained greater than 95 % viable for the duration of experiments. Changes in protoscolex ultrastructure induced by treatment with 10 microg/ml of CsA over 10 days in in vitro culture was assessed by TEM. Protoscolex alterations observed in treated parasites included an increase in cellular vacuolization, swelling of mitochondria, rounding of cells, damage to the tegument, decrease in glycogen, a breakdown of the extracellular matrix and an increase in lipid globules. The untreated protoscoleces, by comparison, had few changes during the 10-day culture period with the exception of large amounts of extracellular glycogen observed in the protoscoleces at culture days 7 and 10. From these results, CsA is clearly an effective scolicidal agent in vitro that may have potential application as a new therapeutic agent in the treatment of human hydatid disease.