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R G Russell

Publications and source records attributed to R G Russell.

At least 73 records · Page 4Linked to original sources
PubMed1996

Measurement of bone specific alkaline phosphatase in the horse: a comparison of two techniques.

For many years total alkaline phosphatase (AP) activity in serum has been used to monitor bone metabolism in different species. However, total AP lacks bone specificity because the total activity in serum is made up of several isoenzymes, of which the liver and bone isoforms predominate. The aim of the present study was to evaluate an immunoradiometric assay for measuring bone specific alkaline phosphatase (BAP) in horses. BAP, a specific marker of bone formation, was measured in sera from thoroughbred horses by using a previously characterised wheat germ lectin (WGL) precipitation assay and an immunoradiometric assay. The levels of immunoreactive BAP (iBAP) and WGL precipitated BAP (wBAP) were related to the serum levels of total AP and another marker of bone formation, the carboxy-terminal propeptide of type 1 collagen (PICP). In horses over one year old, iBAP correlated at least as strongly with total AP as with wBAP, which suggests that the immunoradiometric assay may partially cross-react with liver alkaline phosphatase in horse serum. This possibility was supported by the observation that there was a weaker correlation between iBAP and PICP than between wBAP and PICP. These data indicate that WGL precipitation is currently the most specific method for measuring bone specific alkaline phosphatase in horses.

Aging↗
PubMed1996

Effects of growth factors and interleukin-1 alpha on proteoglycan and type II collagen turnover in bovine nasal and articular chondrocyte pellet cultures.

The aim of this study was to investigate the effects of insulin-like growth factor-I, transforming growth factor-beta (TGF-beta), and interluekin-1 alpha (IL-1 alpha) on the deposition and degradation of a cartilage-like matrix in high-density pellet cultures of adult bovine chondrocytes. Proteoglycan was determined by toluidine blue staining and colorimetric assay. Type II collagen was determined by immunohistochemical staining and its unwinding in situ by a recently developed immunoassay. Bovine nasal chondrocytes cultured as pellets deposited a well-organized extracellular matrix of proteoglycan and type II collagen. Insulin-like growth factor-I (2-10 ng/ml) increased the synthesis and incorporation into the matrix of both these proteins. TGF-beta (2-10 ng/ml) also increased proteoglycan synthesis. However it inhibited proteoglycan deposition, presumably through increased degradation of the molecule, as shown by increased release of aggrecan fragments into the tissue culture medium. TGF-beta had no effect on type II collagen deposition. In pellet cultures of bovine nasal or articular chondrocytes, 20 ng/ml IL-1 alpha induced a significant degradation of both proteoglycan and type II collagen. The effect on collagen clearly involved proteolytic cleavage of its triple helix because there was an increase in the proportion of unwound type II collagen in the matrix, as well as a loss of total type II collagen. In explant cultures of intact bovine articular cartilage, incubation with 50 ng/ml IL-1 alpha stimulated significant degradation of the proteoglycan but no degradation of the type II collagen. These results demonstrate that although the articular chondrocytes are capable of degrading type II collagen when isolated, they do not do so in situ, presumably because of some inherent property of the mature extracellular matrix. This study demonstrates the utility of pellet cultures when investigating chondrocyte-mediated turnover of cartilage matrix and its modulation by cytokines and growth factors.

Animals↗
PubMed1995

The response of the skeleton to physical training: a biochemical study in horses.

In this study we tested the hypothesis that exercise induces an adaptive response in the developing skeleton which may be monitored in vivo by measuring biochemical markers of bone metabolism. The effects of exercise on two biochemical markers of bone formation were determined; the carboxy-terminal propeptide of type I procollagen (PICP), and the bone-specific isoenzyme of alkaline phosphatase (BAP), and one putative marker of resorption, the pyridinoline crosslinked telopeptide domain of type I collagen (ICTP). All three markers were measured for a year in 2-year-old thoroughbred horses exercised three times a week on a treadmill, and values compared to a control group of age-matched animals. Levels of all three markers fell in both exercised and control groups over the 12-month period reflecting normal age changes. However, there were differences between groups in the pattern of this decrease. When expressed as a percentage of baseline values, BAP was higher (p < 0.05) at 2 months and both BAP and the PICP were higher at 4 months (p < 0.01 and p < 0.05, respectively) in the exercised group, reflecting an increase in bone turnover in this group in the early stages of training. PICP levels were also elevated in the exercised group at 10 months and this result indicates an increase in bone turnover at this time. The changes in ICTP were different; at 2 months, levels were higher in exercised animals than in controls, but there was no significant difference between the two groups at 4 and 6 months. After 8 months, ICTP levels in the exercised group increased returning to near baseline values at 10 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1995

Age related changes in biochemical markers of bone metabolism in horses.

Biochemical markers of bone metabolism were analysed in serum samples obtained from 60 horses with no history of orthopaedic disease (age 3 months-20 years). Serum levels of the carboxyterminal propeptide of type I procollagen (PICP), a marker of bone formation and the pyridinoline cross linked telopeptide domain of type I collagen (ICTP), a putative marker of bone resorption, were measured by radioimmunoassay (RIA). Serum levels of the bone specific isoenzyme of alkaline phosphatase (BALP), another marker of bone formation, were measured by a wheatgerm agglutinin affinity (WGA) method. Total alkaline phosphatase levels were also determined. Serum levels of PICP were significantly correlated with bone ALP (r = 0.78, P < 0.0001) and ICTP (r = 0.87, P < 0.0001). ICTP levels also correlated significantly with bone ALP (r = 0.81, P < 0.0001). However, total alkaline phosphatase did not correlate significantly with PICP, ICTP and BALP in horses over 1 year of age. There was an inverse correlation between serum levels of all biochemical markers and age of animals, with the most significant changes seen over the first 2 years. In animals less than 1 year of age, the reference ranges (mean +/- s.d. 1.96) were as follows: PICP 1216-2666 micrograms/l, ICTP 13.8-26.7 micrograms/l, bone ALP 134-288 u/l and total ALP 223-498 u/l. In 2-year-olds, the equivalent reference ranges were: PICP 550-1472 micrograms/l, ICTP 7.96-22.8 micrograms/l, bone ALP 32.7-125 u/l and total ALP 134-238 u/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1995

Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors of bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae.

The mechanisms by which bisphosphonate drugs inhibit osteoclast-mediated bone resorption are unclear. Effects of bisphosphonates on cellular enzymes, metabolic pathways, and osteoclast morphology have previously been described and could culminate in a generalized cytotoxic effect or a decreased capacity of osteoclasts to resorb bone. Recent studies of the structure-activity relationship for the bisphosphonate side chain indicate, however, that at least the newer generations of nitrogen-containing bisphosphonates probably act by binding to a specific target at a site that is complementary in structure to the bisphosphonate side chain. We have previously proposed that such a target for bisphosphonates is also present in amoebae of the cellular slime mold Dictyostelium discoideum, because growth of this microorganism is inhibited by a wide range of bisphosphonates in a manner that closely reflects the antiresorptive potencies of the bisphosphonates in vivo. We have added support for this view by examining the potency towards Dictyostelium of bisphosphonates in which slight changes in the structure of the side chain or conformational restrictions to the side chain have marked effects on antiresorptive potency. The changes in the side chain that affected the in vivo antiresorptive potency of the bisphosphonates consistently affected in a similar manner the potency of the bisphosphonates as inhibitors of the growth of Dictyostelium amoebae. These observations confirm that bisphosphonate drugs have a molecular target that is common to both Dictyostelium amoebae and osteoclasts.

Animals↗
PubMed1994

Stimulation of cartilage resorption by extracellular ATP acting at P2-purinoceptors.

Net loss of the cartilage extracellular matrix occurs in all forms of arthritis, and it is important to identify the factors that initiate and maintain this process. Extracellular ATP can elicit biological responses via P2-purinoceptors, and we have obtained evidence for the presence of these receptors at the surface of cultured human articular chondrocytes. We have extended this work by examining whether exogenous ATP also promotes cartilage resorption. Cultured explants of bovine nasal cartilage were used, and breakdown of proteoglycans was monitored by measuring the release of glycosaminoglycans. ATP, GTP, CTP, UTP, ITP, 2-methylthioadenosine 5'-triphosphate, and adenosine 5'-O-(3-thiotriphosphate) all promoted release of glycosaminoglycans, whereas ADP, AMP, adenosine, and adenosine 5'-(alpha,beta-methylene)triphosphate were inactive. On lowering the concentration of foetal calf serum in the tissue culture medium from 10% (v/v) to 2.5% (v/v), the response to ATP was enhanced and the minimum effective concentration was reduced. The ATP-elicited release of glycosaminoglycans was also enhanced by interleukin 1 beta, tumour necrosis factor alpha, and transforming growth factor-beta, although only high concentrations of the latter were effective. These data provide further evidence for the presence of P2-purinoceptors in cartilage, and indicate that if ATP arises extracellularly, it could have potentially deleterious effects. The enhancement of the response to ATP by interleukin 1 beta and tumour necrosis factor alpha suggests an additional mechanism whereby these cytokines can promote cartilage resorption.

Adenine Nucleotides↗
PubMed1994

Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum.

Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.

Adenine Nucleotides↗
PubMed1994

Effects of transforming growth factor beta and interleukin-1 beta on [3H]thymidine incorporation by human articular chondrocytes in vitro.

This is a study of the regulation of human articular chondrocyte proliferation by transforming growth factor beta (TGF beta) and interleukin-1 beta (IL-1 beta) in vitro. Human articular chondrocytes were cultured at different cell densities on plastic and on a collagen substratum, in the presence and absence of serum. The effects of TGF beta and IL-1 beta on proliferation of chondrocytes, as determined by [3H]thymidine incorporation, under these conditions of culture were examined. TGF beta was found to have both stimulatory and inhibitory effects on chondrocytes in vitro. Interactions between TGF beta and growth factors present in serum influence the modulation of chondrocyte proliferation by TGF beta. IL-1 beta caused a significant reduction of the TGF beta-stimulated increase in chondrocyte proliferation. The complex inter-relationships between TGF beta and IL-1 beta on chondrocytes have implications for cartilage repair.

Cartilage, Articular↗
PubMed1994

Transforming growth factor beta increases ecto-nucleoside triphosphate pyrophosphatase activity of human bone-derived cells.

Inorganic pyrophosphate (PPi) may be involved in the regulation of mineralization. The cell surface enzyme, ecto-NTP pyrophosphatase, could be a major source of extracellular PPi in bone, and agents that influence its activity in osteoblasts may modulate bone mineralization. We studied the effects of serum on the ecto-NTP pyrophosphatase activity of cultured human osteoblast-like cells. Enzyme activity was lowered when the concentration of fetal calf serum (FCS) was reduced from 10 to 2.5% (vol/vol) for 48 h, and a further decrease in activity was observed after 96 h. Relative to enzyme activity in cells cultured in serum-free medium for 96 h, adult human platelet-poor plasma (HPPP; 2.5-10% vol/vol) induced a small increase, similar concentrations of adult human serum (HS) induced much larger increases, and charcoal-depleted FCS was ineffective. In an attempt to identify the factor(s) present in serum that influence ecto-NTP pyrophosphatase activity, we examined transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). PDGFs AA, AB, and BB (0.1-10 ng/ml) were ineffective, but both TGF-beta 1 and TGF-beta 2 increased enzyme activity. The increase was dose dependent between 0.001 and 10 ng/ml, was enhanced in the presence of 2% vol/vol FCS, and was not potentiated by PDGF or by 1,25-(OH)2D3. Furthermore, the increase was independent of cell density and was blocked by inhibitors of protein and RNA synthesis. Ecto-NTP pyrophosphatase of subject-matched human dermal fibroblasts was unaffected by TGF-beta (10 ng/ml), suggesting that modulation of activity by the growth factor may be tissue specific. Alkaline phosphatase (ALP) probably serves to hydrolyze extracellular PPi in bone. In contrast to effects on NTP pyrophosphatase activity is osteoblast-like cells, TGF-beta 1 and TGF-beta 2 (0.001-10 ng/ml) decreased ALP activity dose dependently after 72 h. By inducing opposing changes in ecto-NTP pyrophosphatase and ALP activities, TGF-beta may increase extracellular PPi concentrations in osseous tissues and consequently modulate bone mineral properties in vivo.

Animals↗
PubMed1994

Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime mold Dictyostelium discoideum.

Bisphosphonates are inhibitors of bone resorption and are used increasingly as therapeutic agents for treating clinical disorders of skeletal metabolism. Their mode of action is still not fully understood. The demonstration that methylenebisphosphonate, a simple methylene analog of pyrophosphate, inhibits the axenic growth of amoebae of the slime mold Dictyostelium discoideum and is incorporated into adenine nucleotides suggested that this organism might be useful in elucidating the cellular effects of bisphosphonates. We examined 24 bisphosphonates, including all those of clinical interest as inhibitors of osteoclast-mediated bone resorption in vivo, for their effects on D. discoideum. All the geminal bisphosphonates inhibited growth of Dictyostelium, although the effectiveness of individual compounds varied widely. When the bisphosphonates were ranked there was a remarkable similarity between the order of potency as inhibitors of growth of Dictyostelium and the order of potency as inhibitors of bone resorption. Thus, bisphosphonates with more complex side-chain structures, especially those containing a nitrogen group, were more potent than simple substituted bisphosphonates, some inhibiting Dictyostelium growth even at concentrations below 10 microM. It therefore appears that the mechanism by which bisphosphonates prevent Dictyostelium growth could be similar to the mechanism by which these compounds affect the activity of osteoclasts. Because the mechanisms of action of bisphosphonates on osteoclasts remains unclear, Dictyostelium may provide an additional model for studying the biochemical mode of action of bisphosphonates. Furthermore, these studies suggest that Dictyostelium may also be a convenient organism for rapid evaluation of potentially active bisphosphonates.

Animals↗
PubMed1994

Modulation of ecto-nucleoside triphosphate pyrophosphatase activity of human osteoblast-like bone cells by 1 alpha,25-dihydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, parathyroid hormone, and dexamethasone.

Extracellular inorganic pyrophosphate (PPi) is involved in the regulation of mineralization, and there is evidence that the cell surface enzyme, NTP pyrophosphatase, is a major source of this metabolite in bone. Osteotrophic agents that influence bone turnover may exert their effects, in part, by modulating the activity of ecto-NTP pyrophosphatase in bone cells. We investigated the effect of 1, 25(OH)2 D3, 24, 25(OH)2D3, dexamethasone, and parathyroid hormone (PTH) on the activity of this enzyme in cultured human trabecular bone-derived osteoblast-like cells. 1,25(OH)2D3 at 10(-11)-10(-9) M induced a dose- and time-dependent increase in activity (at 96 h; maximum 10(-9) M, p < 0.001), whereas higher concentrations (10(-8) and 10(-7) M) had no effect. In contrast, 24,25(OH)2D3 was effective only at 10(-8) and 10(-6) M (at 96 h; p < 0.01). Dexamethasone (10(-9)-10(-7) M) caused a dose-dependent decrease in ecto-NTP pyrophosphatase activity (10(-7) M, p < 0.001); concentrations higher than 10(-7) M did not evoke greater inhibition. This effect became apparent by 48 h and was significantly enhanced after 72 h. The response to dexamethasone was attenuated by cycloheximide, indicating a requirement for de novo protein synthesis. Interestingly, the stimulatory effect of 10(-9) M 1,25(OH)2D3 on ecto-NTP pyrophosphatase activity was significantly enhanced in the presence of dexamethasone (10(-9)-10(-7) M). Human PTH(1-34) and bovine PTH(1-34) in the range 10(-10)-10(-7) M had no effect on enzyme activity over a 72 h period.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitriol↗
PubMed1994

Diclofenac sodium inhibits bone resorption in postmenopausal women.

BACKGROUND: The results of experimental studies with animals indicated that prostaglandins stimulate bone resorption, that skeletal production of prostaglandin E2 is enhanced by ovariectomy and is diminished by 17 beta-estradiol, and that the nonsteroidal anti-inflammatory drug (NSAID) naproxyn prevents bone loss after ovariectomy. Studies were carried out to investigate the effects of the NSAID diclofenac sodium on bone and mineral metabolism in premenopausal women and to compare the effects of diclofenac sodium and conjugated estrogens on bone and mineral metabolism in postmenopausal women. PATIENTS AND METHODS: Ten healthy premenopausal women and 17 healthy postmenopausal women were studied while not being treated and again after 4 weeks of treatment with diclofenac sodium, 150 mg per day in divided doses (both groups), and conjugated estrogens, 0.625 mg per day (postmenopausal women). Cross-linked N-telopeptides of type I collagen were measured in the urine as an index of bone resorption. The postmenopausal women were separated into two groups, responders and nonresponders, based on their response to conjugated estrogens as assessed by linear discriminant analysis for groups. Conjugated estrogens lowered urinary N-telopeptides of type I collagen in responders, but not in nonresponders. RESULTS: Urinary cross-linked N-telopeptides were higher in the eight postmenopausal women responders than in the nine postmenopausal nonresponders or in the premenopausal women, and were not altered by diclofenac sodium in premenopausal women. In the eight postmenopausal women with higher rates of bone resorption, diclofenac sodium and conjugated estrogens significantly lowered both urinary calcium concentration and urinary cross-linked N-telopeptides. The effects of the two drugs were comparable. CONCLUSION: The preliminary results demonstrate that, at the dose used, diclofenac sodium is almost as effective as conjugated estrogens for decreasing bone loss in postmenopausal women. Further studies will be needed to determine whether diclofenac sodium can prevent postmenopausal bone loss.

Adult↗
PubMed1994

Studies on type II collagen and aggrecan production in human articular chondrocytes in vitro and effects of transforming growth factor-beta and interleukin-1beta.

Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.

Aggrecans↗
PubMed1994

Binding to and killing of human renal epithelial cells by hemolytic P-fimbriated E. coli.

Acute pyelonephritis is a common invasive infection frequently caused by E. coli that possess P-fimbriae and secrete hemolysin. We have examined the role of P fimbriae and hemolysin in the killing of putative target cells of acute pyelonephritis, that is, human renal epithelial cells (HRPTEC). Cultures of HRPTEC were overlaid with (1) a prototypic pyelonephritogenic E. coli (CFT073) which expresses both P fimbriae and hemolysin; (2) its hemolysin-negative isogenic mutant (CFT073hlyD::TnphoA); or (3) a prototypic nonpyelonephritogenic fecal E. coli (FN414) which is negative for both P fimbriae and hemolysin. CFT073 and CFT073hlyD::TnphoA but not FN414 adhered to HRPTEC, as demonstrated by electron microscopy and direct counting. Adherence was diminished by antisera directed against P fimbriae and by a monoclonal antibody recognizing the epithelial receptor for P fimbriae. CFT073 was significantly more cytolethal for HRPTEC than its hemolysin-negative mutant. The bacteria-free filtrate of CFT073 was both hemolytic and cytolethal whereas that of CFT073hyD::TnphoA was not hemolytic and was significantly less cytolethal. Finally, we demonstrated that CFT073 passed through monolayers of HRPTEC at a higher rate than CFT073hlyD::TnphoA, indicating that hemolysin damages HRPTEC, facilitating passage of bacteria through the epithelial barrier. With HRPTEC and a pyelonephritogenic strain of E. coli we have reproduced in vitro bacterial attachment and toxin delivery by P fimbriae and hemolysin, factors epidemiologically associated with acute pyelonephritis in patients.

Bacterial Adhesion↗
PubMed1994

Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a cause of acute pyelonephritis, produces at least four types of fimbriae, including MR/P (mannose-resistant/Proteus-like) fimbriae. To investigate the contribution of MR/P fimbriae to colonization of the urinary tract, we constructed an MR/P fimbrial mutant by allelic exchange. A 4.2-kb BamHI fragment carrying the mrpA gene was subcloned into a mobilizable plasmid, pSUP202. A 1.3-kb Kanr cassette was inserted into the mrpA open reading frame, and the construct was transferred to the parent P. mirabilis strain by conjugation. Following passage on nonselective medium, 1 of 500 transconjugants screened was found to have undergone allelic exchange as demonstrated by Southern blot. Colony immunoblot, Western immunoblot, and immunogold labeling with a monoclonal antibody to MR/P fimbriae revealed that MrpA was not expressed. Complementation with cloned mrpA restored MR/P expression as shown by hemagglutination, Western blot, and immunogold electron microscopy. To assess virulence, we challenged 40 CBA mice transurethrally with 10(7) CFU of wild-type or mutant strains. After 1 week, geometric means of log10 CFU per milliliter of urine or per gram of bladder or kidney for the wild-type and mutant strains were as follows: urine, 7.79 (wild type) versus 7.02 (mutant) (P = 0.035); bladder, 6.22 versus 4.78 (P = 0.019); left kidney, 5.02 versus 3.31 (P = 0.009); and right kidney, 5.28 versus 4.46 (P = 0.039). Mice challenged with the wild-type strain showed significantly more severe renal damage than did mice challenged with the MR/P-negative mutant (P = 0.007). We conclude that MR/P fimbriae contribute significantly to colonization of the urinary tract and increase the risk of development of acute pyelonephritis.

Animals↗
PubMed1994

Cell association and invasion of Caco-2 cells by Campylobacter jejuni.

Adherence and invasion studies were conducted in monolayers of Caco-2 cells. Three-day-old monolayers were inoculated with Campylobacter jejuni 81-176 at a bacterium/cell ratio of 1,000:1. Saturation studies demonstrated time- and dose-dependent saturation curves for C. jejuni cell association and invasion into Caco-2 cells. Electron microscopy revealed intracellular C. jejuni located within membrane-bound vacuoles. Cell association and invasion were inhibited by 0.3 and 0.5 M concentrations of various sugars, including D-glucose, D-mannose, and D-fucose. However, there was no inhibition with the corresponding L-sugars, indicating physiological specificity. The inhibition of cell association with phloridzin was less pronounced. There was no inhibition of bacterial entry with monodansylcadaverine or g-strophanthin, indicating that it was unlikely that coated-pit formation is important in the invasion of C. jejuni into Caco-2 cells. Furthermore, there was no inhibition with cytochalasin D, vincristine, or vinblastine. Inhibition of cell association was demonstrated at 4 degrees C. Significantly decreased cell association and invasion were seen in potassium-depleted cells. Treatment of cells with bromelain also caused reduction in the number of C. jejuni binding to cells. A nonmotile aflagellate variant of C. jejuni also showed reduced invasion. The results of this study are consistent with energy-dependent invasion mechanisms. The results do not support an endocytic method of invasion for C. jejuni into Caco-2 cells.

Campylobacter jejuni↗
PubMed1994

Cells cultured from the growing tip of red deer antler express alkaline phosphatase and proliferate in response to insulin-like growth factor-I.

Deer antler growth provides a unique natural model of rapid and complete bone regeneration. In this study, the distal antler tips of male red deer (Cervus elaphus) were collected post-mortem during the annual growth period (April-August), and an in vitro system established for the culture of cells from three regions; the inner layer of the perichondrium, the reserve mesenchyme and the cartilage zone. Alkaline phosphatase (ALP) expression by cultured cells, as demonstrated by enzyme histochemistry and biochemical assay, reflected the stage of cellular differentiation. ALP activity was highest in cells cultured from the hypertrophic cartilage region (3.6 +/- 0.2 mumol/micrograms cell protein/minute), and lowest in undifferentiated mesenchymal cells (0.3 +/- 0.01 mumol/microgram cell protein/minute). ALP expression was lost with passage in culture. Levels of ALP activity in cultured cells correlated with the pattern and extent of enzyme expression in tissue sections as demonstrated by histochemical staining. Insulin-like growth factor (IGF)-I (10(-9)M-10(-7)M) was found to be mitogenic for cultured cells from all three zones as shown by increased incorporation of [3H]thymidine into DNA. These results demonstrate that cells from three different regions of the antler tip can be maintained in culture, and that antler cells share certain phenotypic characteristics of growth plate chondrocytes. These data provide further evidence of a role for IGF-1 in the regulation of antler growth. Antler regrowth is a potentially useful model for the study of the factors that regulate bone formation.

Alkaline Phosphatase↗