Effects of capsules on attachment of human periodontal ligament fibroblasts to root surface

The stability of cimetidine (Tagamet) was investigated in total parenteral nutrition (TPN) mixtures containing different amino acid sources. TPN mixtures were stored at 5 degrees C in ethylvinyl acetate bags for 28 days and analysed by stability-indicating high-pressure liquid chromatography. Results indicated that cimetidine was physically compatible and chemically stable (less than 5% degradation) for at least 28 days in TPN mixtures containing either Freamine III, Vamin 14 or Aminoplex 12 as the amino acid source.     

Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts

Periodontal ligament fibroblasts (PDLFs) are a heterogeneous population of cells that are involved in the normal maintenance, repair and regeneration of both the ligament and adjacent hard tissues. Additionally, the ability of these cells to respond to mechanical stimulation suggests that they have a central role in mediating the osseous remodeling that underlies physiological and orthodontic tooth movement. To characterize their role further in this process, the current study evaluated the effect of tensional stress on the biosynthesis of extracellular matrix (ECM) proteins by human PDLFs. Cell strains were established from extracted human premolars and third molars. Cells exposed to 5% biaxial deformation (strain) at a frequency of 30 times/min for 24 hr exhibited statistically significant increases in type I collagen and fibronectin synthesis, and a statistically significant decrease in tropoelastin production relative to unstretched controls. Cells exposed to 10% strain exhibited similar responses for fibronectin and tropoelastin while the amount of type I collagen synthesized by stretched cells did not differ from control levels. These results indicate that mechanical stimulation of PDLFs alters type I collagen, tropoelastin and fibronectin production and that these cells are differentially responsive to varying levels of mechanical stress. The ability of these cells to alter ECM protein synthesis in response to specific magnitudes of tensional stress may in part explain how PDLFs regulate ligament and hard tissue remodeling.     

Adhesiveness of the free surface of a human endometrial monolayer for trophoblast as related to actin cytoskeleton

We have previously shown that angiotensin II(3-7) [Ang II(3-7)] stimulates behavioural activity of rats similar to angiotensin II (Ang II). The involvement of AT1 angiotensin receptors in stimulating the behavioural activity of rats, using their selective ligand losartan (DUP 753), was examined. Ang II(3-7), given intracerebroventricularly (i.c.v.) at a dose of 1 nmol, significantly enhanced recall of a passive avoidance behaviour, object recognition, learning of conditioned avoidance responses (CARs) and apomorphine (1 mg kg-1, i.p.) stereotypy. Losartan (1 microgram, i.c.v.) did not alter any of the behaviours except for that measuring anxiety which was diminished both, in peptide treated and in control rats. On the other hand, losartan abolished Ang II(3-7) facilitation of recall of the passive avoidance, object recognition and the increase in apomorphine stereotypy. Losartan did not influence the increased rate of CARs acquisition after the peptide. None of the treatments significantly changed locomotor activity estimated in an open field. These data point to some involvement of AT1 angiotensin receptors in the behavioural activity of Ang II(3-7).     

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

The dietary effect of 1,3-biseicosapentaenoyl-2-gamma-linolenoyl glycerol (STG) on the fatty acid composition of guinea pigs was examined and compared with that of an eicosapentaenoic acid ethyl ester (EPA-E) and of a soybean oil (SBO) diet. In terms of content of plasma lipid, EPA-E had a greater hypolipidemic effect than STG. On the other hand, in terms of EPA incorporation, contents of EPA in liver lipid were almost the same in the STG and EPA-E groups. Considering that the amount of EPA administered in the EPA-E group was almost 1.5 times that of the STG group, EPA may be absorbed more effectively as the glycerol ester than as the ethyl ester in guinea pigs. In all the tissue lipids, the STG group had a higher unsaturation index (UI) than the EPA-E group even though there is a lower UI in the STG diet than the EPA-E diet. These results suggest that greater amounts of desaturase products as a whole were synthesized in the STG group than in the other two groups. The dihomo-gamma-linolenic acid/arachidonic acid (DGLA/AA) ratio in plasma total lipids in the STG group was 3.5 times that of SBO group, and the DGLA/AA ratio in the EPA-E group was half that of the SBO group. In liver lipid, the ratios of DGLA/AA and EPA/AA in the STG group were 0.687 and 0.488 (phosphatidylcholine fraction) and 0.237 and 0.752 (phosphatidylethanolamine fraction), respectively. The ratio of DGLA/AA as well as the high EPA/AA ratio obtained in the present study with the STG diet may lead to physiological alterations, including enhanced synthesis of 1- and 3-series eicosanoids.     

CD40 mediated activation of gingival and periodontal ligament fibroblasts

Lipids and lipoproteins have been associated with breast cancer risk; however, published results have been inconsistent. To clarify these associations, we measured fasting lipids in women undergoing breast biopsies. A case-control study examined the association of fasting levels of lipids with histologically defined breast cancer risk. Four groups of premenopausal women were assembled on the basis of histological appearance of breast tissue: 1) no epithelial proliferation (n = 102), 2) proliferation without atypia (n = 53), 3) atypical hyperplasia or carcinoma in situ (n = 53), and 4) node-negative invasive cancer (n = 102). A postoperative fasting blood specimen was analyzed for cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Demographics, risk factors, diet, physical activity, fasting weight, and skin-fold thickness were measured. Triglyceride levels were significantly higher in women with node-negative invasive cancer (0.94 +/- 1.04 mg/ml) than in those with no epithelial proliferation (0.83 +/- 1.04 mg/ml, p = 0.03). This association persisted after adjustment for age, body size, lipids, reproductive and familial risk factors, and previous benign breast problems (p < 0.01), in keeping with an independent association of elevated triglycerides with breast cancer risk.     

Differential expression of laminin receptors in human hepatocellular carcinoma

Distortion-product otoacoustic emissions (DPOAEs) have been shown to be ideally sensitive to interruptions of the cochlear blood flow. However, a 15- to 30-second latency typically occurs between cessation of circulation and measurable DPOAE level changes. DPOAEs can also be characterized by phase measures. The aim of the present study was to determine in 10 rabbits the effects on DPOAE phase of repetitively compressing the internal auditory artery. In contrast to the delays measured by DPOAE level, phase changes were detected 1 to 5 seconds after internal auditory artery compression. These data suggest that the essentially "real time" monitoring of cochlear function with DPOAE phase can be used to ensure hearing preservation during surgery involving the porus acousticus and skull base.     

The chemotactic attraction of human fibroblasts to a lymphocyte-derived factor

A quantitative assay that measures fibroblast chemotaxis in vitro is described. Application of this technique has revealed that peripheral blood lymphocytes stimulated by antigen or mitogen in vitro produce a factor that is chemotactic for human dermal fibroblasts. This lymphocyte-derived chemotactic factor for fibroblasts (LDCF-F) is different from the lymphokine that is chemotactic for monocytes or macrophages. Macrophages are required for the generation of LDCF-F by T lymphocytes stimulated by phytohemagglutinin. The fibroblast chemotactic factor is heat stable (56 degrees C for 30 min), trypsin sensitive, and neuraminidase resistant. LDCF-F could function to attact connective tissue fibroblasts to sites at which cell-mediated immune reactions are occurring in vivo.     

Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.     

alpha-Ketoglutarate uptake in human fibroblasts

The effects of Corynebacterium parvum on host protection, tissue reaction and "in vivo" chemotaxis in Schistosoma mansoni infected mice were studied. The C. parvum was given intraperitoneally using a dose of 0.7 mg, twice a week (for 4 weeks), thirty days before (prophylactic treatment) or after infection (curative treatment). The host protection was evaluated through the recovery of adult worms by liver perfusion and was lower in the prophylactic group as compared to the control group (p = 0.018), resulting in 44% protection. The "in vivo" leukocyte response in both prophylactic and curative groups was higher as compared to the infected/non treated group (p = 0.009 and p = 0.003, respectively). Tissue reactions were described in the experimental and control groups, but there were not remarkable differences among them. The possible biological implications and relevance of the findings for the defensive response of the host and control of schistosomiasis are discussed.     

In vitro attachment of human gingival fibroblasts to endosseous implant materials

In recent years, the focus of dental implant research has been the nature of the bone-implant interface associated with osseointegration, yet the transgingival portion of endosseous dental implants has received little attention. The purpose of this study was to determine the attachment of human gingival fibroblasts to three different implant materials: commercially pure titanium, non-porous hydroxyapatite, and porous hydroxyapatite. Cell attachment was quantified by radiolabeling gingival fibroblasts with tritiated thymidine and counting attached cells by liquid scintillation following incubation for periods of 20, 40, and 60 minutes. Additional studies coating implant surfaces with fibronectin were also performed. The nature of the implant material itself appeared to affect the number of attached cells. Determined on a surface area basis, fibroblast attachment was greatest to titanium followed by non-porous hydroxyapatite. Porous hydroxyapatite demonstrated the least amount of fibroblast attachment. When incubated with fibronectin at a concentration of 50 micrograms/ml, no increase in the number of cells attached to the various implant materials was observed. A small but statistically significant increase in the number of fibroblasts attached to porous hydroxyapatite at 40 minutes was observed when implant materials were pre-treated with fibronectin.     

Increased susceptibility of late passage human diploid fibroblasts to oxidative stress

Three strains of human diploid fibroblasts, TIG-3, TIG-7, and MRC-5, were serially cultivated. The susceptibility of early-passage and late-passage cells at 20-30 and 60-70 population doubling levels, respectively, to hydrogen peroxide, the superoxide radical (exposure to the hypoxanthine-xanthine oxidase system), or linoleic acid hydroperoxide was examined for lactate dehydrogenase release. The susceptibility of late-passage cells to such oxidative stress was considerably enhanced compared with early-passage cells. The concentration of reduced glutathione in late-passage cells was lower by 24-44% on a per-cell-number basis and by 86.0-94.5% on a per-protein-quantity basis than in early-passage cells. In addition, the activity of catalase in late-passage cells was lower by 19-46% compared with early-passage cells. There was, however, no difference between the mRNA levels of catalase in early-passage and late-passage cells. The activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase in late-passage cells were all higher than in early-passage cells. These results suggest that late-passage cells are more susceptible to oxidative stress than early-passage cells presumably because of decreases in cellular reduced glutathione concentration and catalase activity, and that their primary defense against oxidative stress is reduced glutathione.     

Specific attachment and migration of human astrocytoma cells on human but not murine laminin

Attachment sites and biological functions of laminin isolated from murine EHS sarcoma have been well studied. Recently several variants of laminin including human placental laminin have been shown to be distinct from EHS-laminin. This study was undertaken to determine attachment, proliferation, and migration phenomena of human astrocytoma cell lines to human and murine sarcoma EHS-laminin. Using short-term attachment assays human placental laminin was shown to be the better substrate for cell adhesion. EHS-laminin mediated approximately 30-50% of the effect observed on human laminin. The astrocytoma cells expressed beta 1, beta 3, and beta 4 subunit mRNA as determined by RT-PCR. Anti-beta 1 antibodies blocked adhesion to EHS-laminin, but antibodies against beta 1, beta 4, and alpha v subunits were all ineffective in blocking adhesion to human laminin. A migration assay showed that astrocytoma cells on human laminin dispersed from a central seeding area, while cells on EHS-laminin remained where they were seeded. The pattern of dispersion could not be accounted for by changes in growth rates of astrocytoma cells on the different proteins, since both cell lines grew equally well on the two laminins. We conclude that unique epitopes on human laminin are recognized by novel receptors on human astrocytoma cells which confer a migratory phenotype to the cells.     

X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts

X-ray induced mutation to 6-thioguanine (6TG)-resistance was studied in early passage cultures of human diploid fibroblasts. The appearance of phenotypic induced mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings and to the duration of the growth period prior to mutant selection; the maximum yield of X-ray induced mutants was observed when cells surviving radiation had completed 3--4 douplings (6--7 days growth) in non-selective medium. The maximum induced mutation frequency was linearly related to X-ray dose and the mutation rate was estimated to be 3.1-10(-7) mutations per viable cell per rad. The data obtained for X-ray induced mutations in cultured human diploid fibroblasts were compared with (a) similar experimental data obtained with established cell cultures and (b) with theoretical predictions of X-ray mutation rates in human germ cells.     

Presence of laminin alpha5 chain and lack of laminin alpha1 chain during human muscle development and in muscular dystrophies

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin alpha1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin alpha1 chain. Nevertheless, there seems to be no expression of laminin alpha1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin alpha1 chain as a recombinant protein and made antibodies specific for human laminin alpha1 chain (anti-hLN-alpha1G4/G5). We also made antibodies to the human laminin alpha5 chain purified from placenta. In the present report we show that hLN-alpha1G4/G5 antibodies react with a 400-kDa laminin alpha1 chain and that 4C7 reacts with a 380-kDa laminin alpha5 chain. Immunohistochemistry with the hLN-alpha1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin alpha5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin alpha1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.     

Recombinant human acid alpha-glucosidase corrects acid alpha-glucosidase-deficient human fibroblasts, quail fibroblasts, and quail myoblasts

Thirty-seven children with intrauterine growth retardation (IUGR) were enrolled in a 3-mo longitudinal study. Weight, length, and knee-heel length (by knemometry) were measured at birth and at 7, 14, 30, 60, and 90 d. GH, IGF-I, IGF binding protein (BP)-3, IGFBP-1, and C-peptide were measured at birth and at 2 mo. IGFBP-3 Western immunoblotting and proteolytic activity assay were also performed. Twenty-five newborns with birth weight appropriate for gestational age were chosen as controls. At birth IUGR newborns showed levels of GH and IGFBP-1 significantly higher, and IGF-I, IGFBP-3, and C-peptide significantly lower than control subjects. At 2 mo GH and IGFBP-1 levels decreased, whereas IGF-I, IGFBP-3, and C-peptide rose, attaining the concentrations found in control subjects at birth. Baseline peptide levels as well as their 2-mo variations did not correlate with the gain in weight, supine length, and knee-heel length recorded at 3 mo. Fourteen of nineteen IUGR cord blood samples showed the presence of the intact approximately 42-39-kD IGFBP-3 doublet and the major approximately 29-kD fragment. At 2 mo the IGFBP-3 band pattern was characterized by the predominance of a approximately 18-kD fragment in 6 of 19 tested IUGR infants. The incubation of 2-mo IUGR samples with normal adult serum induced the appearance of the approximately 18-kD band, which was not modified by the addition of EDTA. These results suggest that: 1) the IGF-related growth-promoting mechanism is impaired in IUGR children at birth but is fully restored at 2 mo; 2) the cord blood levels of GH, IGF-I, IGFBP-3, IGFBP-1, and C-peptide are not predictive of the weight and length gain during the first 3 mo of life; 3) IUGR children have at least two different IGFBP-3 proteases, one cation-dependent protease that is present at birth and able to yield the major approximately 29-kD IGFBP-3 fragment and a second one, with a different activation timing, which exhibits cation independence and induces the formation of a approximately 18-kD IGFBP-3 form.     

Aromatization of androstenedione by cultured human fibroblasts

The conversion of (1, 2, 6, 7-3H)androstenedione to (3H)estrone was measured in fibroblast monolayers grown from biopsies of genital and nongenital skins obtained from 15 control subjects, 9 males with developmental defects of the urogenital tract, and 8 patients with hereditary male pseudohermaphroditism. Under the standardized conditions utilized in this study, the rate of estrone formation in the fibroblasts from normal controls varied from less than 0.2 to 5.5 pmol/100 mg protein/h, and these rates were enhanced by incubation of intact monolayers with choleragen, theophylline, or dexamethasone. Rates of estrone formation were higher in some foreskin strains grown from subjects with developmental defects of the urogenital tract and in strains from scrotum and foreskin of patients with familial incomplete male pseudohermaphroditism, types 1 and 2 than in normal strains or strains from patients with testicular feminization. The meaning of these apparent high rates of estrone formation is unclear.     

Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.     

The postnatal development of F-actin in tension fibroblasts of the spiral ligament of the gerbil cochlea

The tension fibroblasts of the spiral ligament of the mammalian cochlea are thought to create radial tension on the basilar membrane. Their postnatal development was investigated in the gerbil (Meriones unguiculatus) with confocal fluorescence microscopy using phallotoxin as a specific marker for F-actin. In the adult cochlea, tension fibroblasts were restricted to the basal cochlear turn and were arranged in 2-4 rows in the marginal region of the spiral ligament. They contained intensely stained parallel bundles of F-actin. In upper cochlear turns, the marginal region of the spiral ligament was occupied by sparsely distributed, unobtrusively labeled fibrocytes, the bone lining cells. The spiral ligament of young postnatal stages (newborn--6 days after birth (DAB)) lacked F-actin labeling patterns that are characteristic for tension fibroblasts in the adult. Rather, the whole inner surface of the otic capsule throughout all cochlear turns was outlined by cell layers with distinct but diffuse cytoplasmic F-actin label. These cells may represent perichondrial fibrocytes. Around 9 DAB, the perichondrium revealed changes in morphology and F-actin patterns that indicate a further differentiation into tension fibroblasts (basal turn) or bone lining cells (more apical turns). At 12 DAB, around onset of hearing, adult-like bone lining cells were found in the marginal regions of the spiral ligament of upper cochlear turns. In the basal turn, tension fibroblasts were present, but their F-actin cytoskeleton was not fully developed. During the following days, F-actin label increased in tension fibroblasts and reached adult-like configuration at 17 DAB, coinciding with mature hearing characteristics. The role of tension fibroblasts in development of hearing characteristics is discussed.