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Biomedical subjects

G J Pinero

Publications and source records attributed to G J Pinero.

At least 19 recordsLinked to original sources
PubMed1999

Osteopontin (OPN) distribution in premalignant and malignant lesions of oral epithelium and expression in cell lines derived from squamous cell carcinoma of the oral cavity.

The objectives of this study were to assess the immunolocalization of human osteopontin (OPN) in oral lesions and to identify human cell lines of oral squamous cell carcinoma (OSCC) origin that express OPN mRNA. OPN was localized using immunohistochemistry in the following oral specimens: normal epithelium (n=6), epithelial hyperplasia (n=4), epithelial dysplasia (n=28), carcinoma in situ (n=11) and squamous cell carcinoma (n=43). Cell lines UMSCC-1, MDA TU 138, MDA 686LN, SCC4, SCC9, SCC25, CAL 27 and MDA 1483 were characterized for OPN mRNA expression using Northern blotting. OPN was not detected in normal oral epithelium. Intracellular and intercellular immunoreactivity was seen in 75% of hyperplasias, 57% of dysplasias, 54% of carcinoma in situ and 67% of squamous cell carcinomas. UMSCC-1 expressed high levels of OPN mRNA. We conclude that OPN protein is detectable in premalignant and malignant lesions arising from oral epithelium. UMSCC-1 may be a useful cell line in which to conduct in vitro studies designed to clarify the role of OPN in OSCC.

Adolescent↗
PubMed1997

Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.

Calcification of the extracellular matrix (ECM) can be physiological or pathological. Physiological calcification occurs in bone when the soft ECM is converted into a rigid material capable of sustaining mechanical force; pathological calcification can occur in arteries and cartilage and other soft tissues. No molecular determinant regulating ECM calcification has yet been identified. A candidate molecule is matrix GLA protein (Mgp), a mineral-binding ECM protein synthesized by vascular smooth-muscle cells and chondrocytes, two cell types that produce an uncalcified ECM. Mice that lack Mgp develop to term but die within two months as a result of arterial calcification which leads to blood-vessel rupture. Chondrocytes that elaborate a typical cartilage matrix can be seen in the affected arteries. Mgp-deficient mice additionally exhibit inappropriate calcification of various cartilages, including the growth plate, which eventually leads to short stature, osteopenia and fractures. These results indicate that ECM calcification must be actively inhibited in soft tissues. To our knowledge, Mgp is the first inhibitor of calcification of arteries and cartilage to be characterized in vivo.

Animals↗
PubMed1997

Improved immunohistochemical staining of osteopontin (OPN) in paraffin-embedded archival bone specimens following antigen retrieval: anti-human OPN antibody recognizes multiple molecular forms.

Studies to assess osteopontin (OPN) localization in adult human bone using immunochemical techniques produce conflicting results due to variations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-characterized antibodies and improved antigen detection. An anti-osteopontin (alpha-OPN) antiserum was developed that recognizes various soluble molecular weight forms of human OPN, including monomeric, cleaved, and dimerized products. An affinity column of full length recombinant human OPN (rOPN) coupled to support was used to purify alpha-OPN antibodies. Western analysis showed that the affinity-purified antibodies recognized numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohistochemical techniques combined with an antigen retrieval protocol utilizing a newly developed antigen retrieval solution, Retriev-Alltrade mark (Bronco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in archival bone specimens prior to antigen retrieval produced no demonstrable immunostaining even at high concentrations of alpha-OPN. Use of the antigen retrieval protocol restored OPN immunoreactivity, with strong staining apparent in cement lines, osteoblasts, osteocytes, canaliculi, osteoid, and bone matrix. We conclude that antigen retrieval restores immunochemical recognition of OPN in archival specimens containing bone without increasing nonspecific binding.

Adult↗
PubMed1996

Integrated processes responsible for soft tissue healing.

Wounded soft tissues undergo repair through a complex series of interrelated events that involve both physical and chemical activities. These processes are currently undergoing extensive investigation as efforts are directed toward achieving augmented and accelerated healing. Early wound-healing research focused on expanding traditional histologic descriptions of tissue healing by attempting to characterize the environment and biologic mediators responsible for healing. These initial studies successfully identified a number of agents and physiochemical factors present in healing wounds, but their precise roles and importance remain largely unknown. This review article summarizes the current literature on soft tissue healing. An effort has been made to correlate the activities of the major growth factors and cytokines with the individual reparative processes including the inflammatory response, hemostasis, fibroplasia, angiogenesis, and remodeling. Explanations and characteristics of growth factor function as well as brief descriptions of several major factors and their spectrum of activity are also provided.

Blood Coagulation↗
PubMed1995

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase.

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.

Alkaline Phosphatase↗
PubMed1995

Molecular analysis of rat dentin sialoprotein.

Dentin sialoprotein (DSP) is a noncollagenous protein originally isolated from rat dentin. Because it is made by odontoblasts that are actively synthesizing dentin. DSP may play an important role in dentinogenesis. We have isolated a full length DSP cDNA from a rat odontoblast/dental pulp cDNA library (Ritchie et al. [1994] J. Biol. Chem. 269:3698-3702) which codes for a 17 residue signal peptide and a 366 residue, 53 kDa mature protein. In situ hybridization revealed DSP mRNA expression by odontoblasts, but no other cells, in jaws from newborn rat. Northern analysis of various rat tissues demonstrated the presence of DSP transcripts in newborn tooth germs and 21 day old rat incisors. Moreover, multiple transcripts of 4.6 kb and 1.5 kb were found in these two tissues. To better understand the origin of these DSP mRNA multiple transcripts, we have isolated two rat genomic clones. Digestion of each clone with EcoRI followed by Southern analysis revealed that DSP cDNA hybridized to a 4 kb fragment in a lambda dash clone and to a 6 kb fragment in a cosmid clone. Since DSP cDNA hybridized to a 6 kb EcoRI fragment and a 4 kb EcoRI fragment obtained from a rat liver genomic cDNA digested with EcoRI, the multiple DSP mRNA transcripts are most likely derived from two related DSP genes which coexist in the rat genome.

Animals↗
PubMed1994

Continuously infused calcium hydroxide: its influence on hard tissue repair.

To study the ability of calcium hydroxide to promote hard tissue repair, Alza Alzet Osmotic Pumps, implanted in Sprague-Dawley rats, were used to deliver either calcium hydroxide and glycerol, barium hydroxide and glycerol, tetracycline and glycerol, or glycerol only to a standardized round bur defect in a rat femur. The pumps infused one of the reagents into the defects continuously over a 4-wk experimental period. The effects of each reagent on the healing of the bony defects were compared by histological evaluation. The Alza Osmotic Pump proved to be an effective method to deliver an agent to an experimental site. Our preliminary findings from a limited sample size indicated that calcium hydroxide contributed to a more complete osseous repair than either barium hydroxide or tetracycline. Barium hydroxide with a sustained pH equivalent to calcium hydroxide showed no greater healing than the controls. Tetracycline results were also similar to controls.

Animals↗
PubMed1994

Normal long bone growth and development in type X collagen-null mice.

To investigate the role of type X collagen in skeletal development, we have generated type X collagen-null mice. Surprisingly, mice without type X collagen were viable and fertile and had no gross abnormalities in long bone growth or development. No differences were detected between the type X collagen-null mice and controls when growth plates of both newborn and 3-week old mice were examined by histology and by immunostaining for extracellular matrix components of bone including osteopontin, osteocalcin and type II collagen. Our results suggest that type X collagen is not required for long bone development. However, mice and humans with dominant acting type X collagen mutations have bone abnormalities, suggesting that only the presence of abnormal type X collagen can modify bone growth and development.

Animals↗
PubMed1994

Differential effect of TGF-beta 1 and PDGF on proliferation of periodontal ligament cells and gingival fibroblasts.

Regeneration of periodontal tissues requires orchestration of several cell types. Two cell types, gingival fibroblastic cells (gingival fibroblasts) and cells from the periodontal ligament (PDL cells), were studied to compare the effects of supplemental addition of TGF-beta 1 and PDGF on proliferation. Cells obtained from healthy donors were cultured in 10% FBS supplemented with either 10 ng/ml TGF-beta 1, 20 ng/ml PDGF, or both. Thymidine incorporation was measured after 24, 48, or 72 hours. Data from PDL (analyzed by ANOVA) showed the following relations: at 24 hours TGF beta 1/PDGF = PDGF > TGF-beta 1 = control; at 48 hours TGF beta 1/PDGF > TGF-beta 1 > PDGF > control; at 72 hours TGF beta 1/PDGF > TGF-beta 1 > PDGF = control. Gingival fibroblast cultures showed the following relations: at 24 and 48 hours TGF beta 1/PDGF = PDGF > TGF-beta 1 = control; at 72 hours, TGF beta 1/PDGF = PDGF > control with TGF beta 1 not different from control or factor combinations. Both TGF-beta 1 and TGF-beta 1/PDGF showed a significantly greater increase in proliferation of PDL cells than in gingival fibroblasts at 48 and 72 hours (Student t test P < 0.05). In contrast, PDGF stimulated proliferation of gingival fibroblasts was significantly greater than PDL cells at 72 hours (P < 0.05). Thus, supplementation of complete cultures (containing 10% FBS) with TGF-beta 1 alone or combined with PDGF stimulates proliferation of PDL cells to a significantly greater extent than proliferation of gingival fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

Immunolocalization of connexin 43 in the tooth germ of the neonatal rat.

Rabbit polyclonal antibodies to amino acids 346-360 of connexin 43, the 'heart' gap junction protein, were employed to immunolocalize connexin 43 gap junctions in the neonatal rat molar tooth germ. Connexin 43 appears early in the differentiation of both ectodermally derived and ectomesenchymally derived cells of the developing tooth. Connexin 43 immunoreactivity is present in the epithelial components of the enamel organ, including the area of the proximal and distal junctional complexes of the ameloblast layer, and the stratum intermedium, stellate reticulum and outer enamel epithelium. Secretory odontoblasts and developing alveolar bone also display a pattern of connexin 43 immunostaining. Both the epithelial and ectomesenchymally-derived components of the developing tooth acquire connexin 43 channels in a manner that correlates with cell differentiation. In addition, three regions can be defined by connexin 43 immunostaining: the epithelia of the enamel organ that are derived from the oral epithelium, the odontoblast layer derived from the ectomesenchyme, and the alveolar bone. The results suggest that connexin 43 may provide the mechanism for functional compartmentalization of the tissues associated with tooth formation. Compartmentalization suggested by connexin 43 expression could play important roles in the development and functions of these tissues.

Animals↗
PubMed1991

Identification of the rat bone 60K acidic glycoprotein as alpha 2HS-glycoprotein.

Previous reports have described an Mr 60,000-64,000 glycoprotein present in guanidium chloride (GdmCl)/EDTA extracts of bovine and rat bone. We have purified this protein from the long bones of rats and have raised polyclonal antibodies to the purified protein. The 60K glycoprotein has amino acid and carbohydrate compositions that are similar to those reported for the 60-64K protein(s). Several lines of evidence indicate that the 60K bone glycoprotein is the rat homologue of human alpha 2HS-glycoprotein. First, immunochemical data demonstrated that the 60K bone glycoprotein was present in serum as well as in EDTA/GdmCl extracts of bone. Second, immunolocalization and metabolic labelling experiments showed that the 60K protein is synthesized in liver and not in bone cells, although it is sequestered in vascularized regions of bone matrix. Finally, the NH2-terminal sequence for the rat 60K bone glycoprotein was highly similar to that of the human alpha 2HS-glycoprotein A chain. A surprising finding was that small amounts of contaminating 60K/alpha 2HS-glycoprotein were found in several protein fractions purified by ion-exchange chromatography of bone EDTA/GdmCl extracts. Because this protein was found to be highly immunogenic, the presence of anti-60K antibodies in anti-sera prepared against purified bone proteins should be considered as a potential problem.

Amino Acid Sequence↗
PubMed1989

An ultrastructural study of human epithelial rests of Malassez maintained in a differentiated state in vitro.

Healthy human periodontal ligaments (PDL), obtained from the extracted teeth (premolars and third molars), were cultivated for 1-35 days, using a multi-purposes culture chamber (MPCC) equipped with various transparent membranes. The resting state of the epithelial rests of Malassez (ERM), similar to their in vivo counterparts, appeared as small islands or strands with scant cytoplasm containing poorly developed organelles. This state was most effectively maintained in MPCC with a cellophane sheet. MPCC with a Sartorius membrane filter permitted proliferation and emigration of ERM. Proliferating ERM were characterized by more profiles of rough endoplasmic reticulum and free ribosomes, new formation of actin-containing microfilaments, less prominent tonofilaments and desmosomes and loss of gap junctions. Most of these ultrastructural changes are manifested in epithelial cells during wound healing. The emigrating ERM from PDL explants, as well as occasional proliferating ERM within explants, consisted of two cell types--outer basal-like cells, as described above, and inner tonofilament-rich prickle-like cells, suggesting a propensity for differentiation of ERM. The results show the possibility of controlling the growth and differentiation of ERM through the MPCC culture environment.

Adolescent↗
PubMed1987

Ultrastructure of fibroblasts in cyclosporin A-induced gingival hyperplasia.

Specimens from 2 cases of gingival hyperplasia resulting from the treatment with the immunosuppressant cyclosporin A (CSGH) and 4 cases of inflamed gingiva (non-CSGH) were examined by electron microscopy with particular interest in the fibroblasts. In general, the fibroblasts in CSGH revealed the ultrastructural characteristics of active protein synthesis and secretion and less cytotoxic or degenerative changes. Some fibroblasts in the CSGH (23.8%) displayed cytological modifications comparable to so-called myofibroblasts, i.e., microfilament bands with semi-periodic dense nodes, nuclear indentations, and basal-lamina associated, cell-to-stromal junctions. Conversely, 5.9% of the fibroblasts in the non-CSGH showed these modifications. The modified fibroblasts were found most predominantly in the transitional area between inflamed and fibrous connective tissues. From these findings, it is suggested that the myofibroblastic modification in CSGH is regarded as a common morphological sign in actively proliferating fibrous tissue and may participate in the host-tissue response to the plaque-associated irritants.

Adult↗
PubMed1987

Microfilaments in human cementoblasts and periodontal fibroblasts.

An electron microscopic survey was carried out on the human periodontal ligament (PDL), including a part of the gingival connective tissue attached to extracted tooth roots (11 functioning premolars and 6 nonfunctioning third molars) in order to examine the characteristics of microfilaments (6 nm) in cementoblasts and PDL fibroblasts. Microfilaments which were grouped in bundles with semiperiodic dense nodes or in meshworks just beneath the cell membrane were seen predominantly in the cells characterized by their ultrastructurally immature appearance. These microfilaments were more commonly observed in third molar PDL than in premolar PDL and, in general, more conspicuous in cementoblasts than in fibroblasts. The significance of microfilaments in human PDL is discussed, particularly in relation to cell differentiation and morphogenesis.

Actin Cytoskeleton↗
PubMed1987

Ultrastructural and morphometric analyses of human cementoblasts and periodontal fibroblasts.

In order to elucidate the cytological characteristics of human cementoblasts which distinguish them from periodontal fibroblasts, the periodontal ligament and gingival connective tissue attached to 37 extracted teeth from 27 patients (ages 10-67) were analyzed by electron microscopy coupled with a morphometric procedure. The cementoblasts largely consisted of either immature or resting types of collagen-producing cells (CPC), both of which were poor in the rough endoplasmic reticulum and Golgi complex; these organelles were well-developed in the relatively less common active cementoblasts. The cementoblasts consistently revealed a higher mean-volume-density per tissue unit than the fibroblasts and sometimes were grouped into clusters with the formation of junctional apparatuses. The relative volume of glycogen particles per cytoplasm was significantly higher in the cementoblasts, whereas the rough endoplasmic reticulum was higher in the fibroblasts. From the present study, it is suggested that the cementoblasts are functionally less active CPC than the periodontal fibroblasts.

Actin Cytoskeleton↗
PubMed1984

Human gingival fibroblast production of interferon.

Three human gingival fibroblast cell lines were used to determine whether they could be induced by a synthetic RNA and superinduced by metabolic inhibitors to produce interferon (IFN-beta). When established procedures were followed for human fetal or newborn skin fibroblast cell lines, the adult gingival fibroblasts produced comparable amounts of IFN-beta. It was shown that the superinducers alone would not cause an IFN-beta production response, and that the absence of serum in the production medium also inhibited the production of IFN-beta. The effect of IFN-beta on cell growth was carried out in T-flasks seeded with 10(5) HEp-2 cells. After one and two wk, the cells of triplicate control flasks and triplicate flasks containing various dilutions of the production media were enumerated to determine a cell multiplication inhibition (CMI) value. A correlation between the IFN-beta content and the CMI effect, however, was not obtained, and it was concluded that other CMI agents, possibly more potent than the IFN-beta, were being produced by the stimulated human gingival fibroblasts. Cell protein assays which gave a high ng/cell protein content correlated with TEM micrographs which showed clusters of complex lysosomes, primarily in cells cultured in the IFN-beta-containing nutrient. However, since commercial IFN-beta initiated no such lysosomal response, it was further concluded that the complex lysosomes were due to CMI agent(s) other than IFN-beta.

Adult↗