The effects of power toothbrushing on periodontal inflammation in a Canadian nursing home population: A randomized controlled trial.

OBJECTIVES: The aim of this study was to investigate whether twice-daily use of a rotating-oscillating power toothbrush (Oral-B Professional Care 1000™ ) in nursing home (NH) residents over a 6-week period, compared to usual care (UC), would reduce periodontal inflammation. METHODS: In this repeated measures single-blinded randomized controlled trial, 59 residents of one NH in Winnipeg, Canada, were randomized to receive either twice-daily tooth brushing with a rotating-oscillating power toothbrush (PB) or UC by caregivers. Consent was obtained from residents or their proxies. Participants had some natural teeth, periodontal inflammation, non-aggressive behaviour, no communicable diseases, were non-smokers and non-comatose. Outcomes were measured at baseline and 6 weeks, which included: inflammation (MGI, Lobene), bleeding (PBI, Loesche) and Plaque (Turesky). Comparisons of group changes in outcomes were analysed using an ANOVA with a repeated measure. RESULTS: Of 59 original study participants, one withdrew, one died prior to study commencement and three died before study completion. All oral parameters improved significantly for the remaining 54 residents over time (P<.0001), with no differences between groups. CONCLUSIONS: These results demonstrate that it is possible for caregivers to improve periodontal inflammation of residents over a 6-week period. Despite no significant group differences, periodontal inflammation of all study participants improved significantly, particularly in the reduction of bleeding, a direct measure of periodontal inflammation, which is a unique finding.

The effect of NIDCR R25 grant support on the curriculum and culture of a research non-intensive dental school.

Our objective was to evaluate changes in curriculum and culture within a research non-intensive dental school after implementation of programs supported by the NIH-NIDCR R25 Oral Health Research Curriculum Grant. We designed new curricular elements to foster an appreciation of research/discovery, an interest in academic/research careers, and application of biomedical/clinical advances to patient care. Funding was utilized to develop, implement, and assess a dedicated curricular track of continuous student research/scholarly activity throughout the four years of dental education. This track represented mandatory hours of didactic time exposing students to topics not traditionally included in dental curricula. Additionally, students were provided with customized flexible schedules to participate in elective "hands-on" mentored research/scholarly experiences at local, national, and international sites, including linkages to certificate, MS, and PhD programs. Funding was also used to support a wide array of faculty development activities that provided skill sets required to deliver integrated biomedical/clinical content, research-oriented evidence-based approaches to dental education, and translational case-based teaching methods emphasizing the application of new science/technologies to patient care. We measured changes in student, faculty, and institutional profiles/attitudes using traditional benchmarks, surveys, and focus groups. Comparisons were made between baseline data prior to R25 program initiation and data collected after years 3-4 of program implementation. Significant increases were demonstrated in: (1) student participation in research/scholarship, attendance at national meetings, research awards, publication of manuscripts, pursuit of advanced training/degrees, and expressions of interest in academic/research careers; (2) faculty participation in development activities, publication of manuscripts, and mentoring of students; and (3) increased institutional credibility within the university, supportive infrastructure for research/scholarship, and cultural expectations for academic excellence. Thus, we believe that the R25 programming changed the culture of our dental school, creating a supportive environment for research/scholarship, increasing academic productivity, and altering the attitudes of faculty/students.

Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28k gene.

To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates transcription of the rat renal calbindin-D28k gene, the rate of calbindin-D28k mRNA synthesis was measured directly in nuclei using the in vitro nuclear transcription assay. Nuclei were prepared from kidneys of vitamin D-deficient rats at various times after a single ip injection of 1,25-(OH)2D3, and transcription was allowed to proceed in vitro in the presence of [32P]UTP for 30 min at 29 C, at which time the incorporation of UTP into trichloroacetic acid-precipitable material was optimal. Incorporation of UTP was decreased by 64.6% by alpha-amanitin, which selectively inhibits polymerase II. Purified [32P]RNA was analyzed for newly synthesized calbindin-D-28k gene transcripts by hybridization to calbindin-D28k cDNA immobilized on nitrocellulose filters. Using this assay we found that the first significant increase in calbindin-D28k gene transcription occurred at 1 h, and the peak of transcriptional activity occurred at 2 h. Within 12 h of 1,25-(OH)2D3 treatment, calbindin-D28k gene transcription returned to control levels. Using Northern blot analysis, a significant increase in calbindin-D RNA was first observed 2 h after hormone administration, reaching a maximum at 12 h. Renal calbindin-D28k protein levels are significantly increased by 3 h and reach a maximum value 48 h after hormone administration. Our results suggest that the early increase in renal calbindin-D28k may be due to transcriptional regulation. The long time lag between transcription and the peak of calbindin mRNA and calbindin protein accumulation may reflect the involvement of post-transcriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Calbindin-D9K gene expression in human myometrium during pregnancy and labor.

The 9-kilodalton vitamin D-dependent calcium-binding protein (CaBP9K) is expressed in the intestine and uterus of mammals. In this study, we demonstrated the intracellular location of CaBP9K and quantified its expression in human myometrial tissues from nonpregnant and pregnant women (before and after the onset of labor). By Western blot analysis, we found that antiserum raised against bovine intestinal CaBP9K was specific for CaBP9K in human myometrium. By Northern blot analysis, with an oligodeoxynucleotide probe specific for human CaBP9K, we identified a single 0.7-kilobase messenger ribonucleic acid (mRNA) species in myometrial tissues from pregnant women before and after the onset of labor. CaBP9K mRNA and immunoreactive protein were localized within myometrial smooth muscle cells by in situ hybridization and immunohistochemistry. The highest levels of CaBP9K mRNA and immunoreactive protein were found in myometrial tissues obtained from pregnant women at term before the onset of labor. CaBP9K mRNA and immunoreactive levels of CaBP9K were decreased significantly in myometrial tissues obtained after the onset of labor (2- and 8-fold, respectively). These findings demonstrated for the first time that CaBP9K was present in human myometrium and suggested that it may play a role in regulating uterine smooth muscle function during pregnancy.

Calbindin-D28k in nerve cell nuclei.

Calbindin-D28k is a member of the large EF-hand family of calcium-binding proteins, that is believed to function, in part as a cytosolic calcium buffer. Recent studies have demonstrated that cells containing Calbindin-D28k are protected from degeneration caused by conditions that elevate intracellular calcium concentrations. Since its initial discovery in 1966, Calbindin-D28k has been localized in the cytoplasm of many neuronal populations, but its nuclear localization has been uncertain. Using light and electron microscopic immunohistochemistry, and nuclear fractionation methods, we demonstrate localization of Calbindin-D28k not only in the cytoplasm, but also in the nucleus of rodent midbrain dopaminergic neurons and cerebellar Purkinje cells. The Calbindin-D28k immunoreactive staining intensity in the nucleus was routinely equal or greater than that in the cytoplasm. Since calcium signals are propagated to the nucleus, where they can regulate gene expression, the existence of nuclear Calbindin-D28k has important implications for cellular function.

Quantitative measurement of neuronal calbindin-D28k by radioimmunocytochemistry.

The calcium-binding protein calbindin-D28k (CaBP) has been localized in high concentration in several neuronal populations within the CNS and is believed to act as an intracellular calcium buffer. There has been much interest and speculation concerning its potential neuroprotective function. A radioimmunocytochemistry (RIC) technique for the cellular quantitation of protein has been applied to quantitative measurement of neuronal CaBP in vivo and in vitro. The method permits cellular comparison of CaBP content within tissue sections or cells in culture. Through the use of specific primary antibody, 35S-labeled secondary antibody, and photographic emulsion, RIC combines the simplicity of standard immunocytochemical procedures with the sophistication and power of in situ hybridization, autoradiography, and image analysis. CaBP levels are expressed as mean +/- S.E.M. silver grains/cell. CaBP content has been measured and compared in mouse cerebellar Purkinje cells (56.5 +/- 6.9 grains/cell), granule cells of the hippocampal dentate gyrus (10.3 +/- 2.1 grains/cell), midline ventral tegmental neurons (11.6 +/- 2.9 grains/cell), and human SH-SY-5Y neuroblastoma cells in culture (5.1 +/- 0.9 grains/cell). As measured by RIC, mouse cerebellar Purkinje cells contain approximately 5-fold more CaBP than granule cells of the hippocampal dentate gyrus/midline ventral tegmental neurons and 10-fold more CaBP than cultured human SH-SY-5Y neuroblastoma cells. Assay reproducibility was demonstrated by comparison of adjacent sections which yielded a 3-9% intra-assay variability. Results were validated and confirmed by comparison to previous radioimmunoassay studies which indicated similar ratios of CaBP levels between brain regions/cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium binding protein (calbindin-D28k) gene expression in the developing and aging mouse cerebellum.

Calbindin-D28k (CaBP28k) protein and gene expression were examined in the mouse cerebellum during development and aging utilizing slot and Northern blot hybridization analyses for mRNA levels, Western blot analysis and radioimmunoassay (RIA) for protein levels, and by in situ studies using immunocytochemistry and hybridization cytochemistry on prepared tissue sections. Samples were obtained and analyzed from C57BL/6J mice aged day of birth and postnatal weeks 1, 2, 4, 8, and 120. A specific cDNA and antibody for CaBP28k were utilized in these studies. Analysis of mRNA levels showed a steady rise in CaBP28k mRNA from birth to a peak at postnatal week (3.4-fold increase) and then a decline to steady-state levels at postnatal weeks 4 and 8 (47% reduction of peak level) followed by a reduction of CaBP28k mRNA to birth levels at postnatal week 120. The specificity of the changes observed was tested by reprobing blots with beta-actin cDNA. Analysis of CaBP28k protein levels by both Western blot and RIA showed a similar pattern. In situ analysis of CaBP28k mRNA levels, based on hybridization signal (silver grains per cell), demonstrated a rise in cellular CaBP28k mRNA levels which peaked at postnatal week 2 (416.9 +/- 52.1) and then declined to steady-state levels by postnatal weeks 4 and 8 (267.4 +/- 35.8). Cellular CaBP28k mRNA levels exhibited a dramatic reduction in the aged cerebellum (postnatal week 120; 78.3 +/- 16.0). The levels of cellular CaBP28k mRNA corresponded to the intensity of immunoreactive CaBP28k localized by immunocytochemistry. The results are consistent with the hypothesis that CaBP28k may play a critical role in Purkinje cell maturation and maintenance.(ABSTRACT TRUNCATED AT 250 WORDS)

Calbindin-D28k buffers intracellular calcium and promotes resistance to degeneration in PC12 cells.

The calcium-binding protein calbindin-D28k (CB) has been hypothesized to function, in part, as a neuroprotective protein. CB is localized within nerve cells that are often less vulnerable to degeneration in patients with Alzheimer's disease and Parkinson's disease, and cells containing CB can buffer intracellular calcium concentrations ([Ca2+]i). The present study was designed to directly test the hypothesis that CB can protect cells from degeneration by reducing [Ca2+]i. PC12 cells, transfected to express different levels of CB, were found to be significantly less vulnerable to degeneration caused by serum withdrawal, glutamate, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). However, CB did not protect cells from degeneration caused by the calcium ionophore A23187. CB-transfected cells exhibited reduced elevations in [Ca2+]i following treatment with bradykinin, or ATP compared to non-CB-containing cells. These data indicate that CB can protect cells from degeneration caused by certain conditions, and it reduces elevations in [Ca2+]i caused by influx from extracellular sources.

The neurotoxin MPTP increases calbindin-D28k levels in mouse midbrain dopaminergic neurons.

The calcium-binding protein calbindin-D28k (CALB) has been localized in high concentrations in several neuronal populations within the central nervous system (CNS) and is believed to act as an intracellular calcium (Ca2+) buffer. There has been much interest and speculation concerning its potential neuroprotective function. However, there is little direct evidence linking CALB content of individual neurons to Ca2+ buffering ability, resistance to Ca(2+)-mediated excitotoxicity, or vulnerability to Ca(2+)-mediated degeneration. It is necessary to demonstrate these relationships on a cellular level so that more definitive conclusions can be made. We have utilized immunocytochemical and Western blot techniques to determine whether cellular CALB content is altered in the nucleus A10 dopaminergic region of the midbrain following administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our data demonstrate a significant increase in the CALB content of nucleus A10 neurons (up to 227 +/- 23% above control) 3 and 6 h after MPTP treatment. CALB elevation demonstrated both time and dosage dependence as 6-h groups exhibited larger increases than 3-h groups, and a 60 mg/kg dosage induced a larger increase than a 20 mg/kg dosage. These data support the hypothesis that MPTP is neurotoxic by causing increases in free intracellular Ca2+ and that increased CALB in the midbrain dopaminergic neurons is a protective response to elevated intracellular free Ca2+.

Calbindin-D28K in the nucleus of nerve growth factor-treated PC12 cells.

Calbindin-D28K (CB) is a calcium-binding protein found within a subset of neurons, which has been proposed to buffer intracellular calcium and to protect cells from calcium-induced neurodegeneration. The rat pheochromocytoma PC12 cell line has been an invaluable tool for studying neuronal development and characterizing the molecular actions of nerve growth factor (NGF). Using Western and RNAse protection analyses and immunocytochemical and cell fractionation techniques, we now report that NGF induces the expression of CB not only in the cytoplasm and neurites, but also within the nuclei of PC12 cells. These data indicate that PC12 cells can serve as a useful model system in which to study the functions of CB and the role of nuclear CB.

Nerve growth factor increases calcium binding protein (calbindin-D28K) in rat olfactory bulb.

Calbindin-D28K (CaBP28K) is a soluble intracellular protein capable of sequestering micromolar concentrations of calcium. The in vivo regulation of CaBP28K by recombinant human nerve growth factor (rhNGF) was studied in adult, male rats. Via Alzet 2002 pumps, each rat received, for 14 days, a lateral ventricle infusion (i.c.v.; n = 5-6/group) of 12 microliters PBS/day containing 1.0 microgram cytochrome C (control) or an equal amount of rhNGF. Six other animals received a vehicle or rhNGF infusion into the central neostriatum. CaBP28K was elevated by 75% (P less than 0.01) in the olfactory bulb following i.c.v. rhNGF in each of two experiments and was not altered in the temporal cortex, hippocampus, olfactory tubercle, cerebellum, or neostriatum. Direct striatal injections of rhNGF did not alter CaBP28K in the neostriatum or other regions (including the olfactory bulb). The increases in olfactory bulb CaBP28K protein levels were verified via Western blot analysis. CaBP28K immunocytochemistry revealed that 33% of olfactory bulb neurons are immunoreactive for CaBP28K and that the number or proportion of immunoreactive neurons did not change with i.c.v. infusions of rhNGF, suggesting that exogenously delivered rhNGF augments the content of CaBP28K in olfactory bulb neurons that normally express the protein. Endogenous NGF may function as a neuroprotective factor by enhancing the ability of these cells to sequester cytoplasmic calcium and retard calcium-mediated neurodegeneration.

Phytoestrogens alter hypothalamic calbindin-D28k levels during prenatal development.

Calbindin-D28K (CALB) in the medial basal hypothalamic (MBH) and preoptic area (POA) of male and female fetuses from pregnant rats fed different phytoestrogens diets during gestation was examined by Western analysis. In animals fed a phytoestrogen containing diet (Phyto-200), males displayed significantly higher CALB levels compared to females. Whereas, in animals fed a phytoestrogen-free diet (Phyto-free), females exhibited significantly higher CALB levels compared to Phyto-200 female values. The present data have far reaching implications where phytoestrogen content in diets apparently influence MBH-POA CALB levels prenatally. The altered CALB levels may in turn modify sexually dimorphic brain structures during neuronal development by buffering Ca2+ that is associated with programmed cell death.

Platelet-derived growth factor levels in wounds of diabetic rats.

Delayed wound healing is a troublesome complication of Diabetes. Results from recent investigations concerning the potential cellular and molecular mechanisms responsible for diabetic wound healing deficiency are preliminary in nature. Some studies have demonstrated that direct application of certain growth factors/cytokines can facilitate wound healing in diabetic models. It is possible that refractory diabetic wounds are the result of deficiencies in growth factors/cytokines important for the normal wound healing process. Platelet-Derived Growth Factor (PDGF) levels were examined by radioimmunoassay in wound tissue of normal and diabetic rats (streptozotocin-induced diabetes). Immunohistochemical analysis was utilized to localize and characterize PDGF immunopositive cells at the wound site of normal and diabetic animals. At the wound site, normal animals demonstrated significantly elevated PDGF levels compared to diabetic animals at 5 days post-wounding (no differences were observed in the spleen or contralateral control tissue). There appeared to be a visible increase in PDGF immunopositive cells at the wound site in both experimental and control groups. By day 10 post-wounding, PDGF levels at the wound site in normal animals were reduced becoming similar to PDGF levels in diabetic animals. This corresponded to an apparent reduction of PDGF immunopositive cells in both groups (similar to baseline levels). PDGF levels in both groups remained stable until day 20 post-wounding when a significant elevation of wound site PDGF levels occurred in the diabetic group. The findings suggest that absence of an initial increase in PDGF may play an important role in poor wound healing observed in diabetic animals. The reduction in PDGF may be related to decreased cellular PDGF production rather than a lack of PDGF-producing cells. Perhaps the diabetic state inhibits cellular PDGF gene expression signaled by wounding or interferes with normal PDGF expression at the wound site.

Diabetes-induced impairment of macrophage cytokine release in a rat model: potential role of serum lipids.

Diabetes (type I and type II) affects approximately 13 million people in the United States. Delayed and incomplete healing of wounds can be a major problem for diabetic patients. Macrophages are an important cell in the complex process of wound repair representing the major source of cytokines throughout the wound healing process. Cytokines mediate many of the cellular responses critical to timely wound repair. It has been suggested that diabetes impairs wound healing through disruption of local cytokine production. We previously demonstrated that platelet-derived growth factor B chain (PDGF-B) levels are deficient at the wound site of diabetic rats. In the present study, we measured the levels of several marker cytokines released from cultured peritoneal macrophages of diabetic, nondiabetic hyperlipidemic, and normal rats. The diabetic condition was associated with a generalized reduction of macrophage cytokine release. Nondiabetic hyperlipidemic animals demonstrated similar cytokine reduction supporting the hypothesis that elevated serum lipids are the primary determinants of diabetes-induced reductions in macrophage cytokine release. Thus, manipulation of serum lipids may be a therapeutically useful modality for controlling macrophage cytokine release in the inflammatory and/or wound environment.

Myofibroblasts in phenytoin-induced hyperplastic connective tissue in the rat and in human gingival overgrowth.

Phenytoin is a commonly used anticonvulsant drug for the prevention of seizures. A common side effect of phenytoin (PHT) therapy is connective tissue hyperplasia, particularly in the oral cavity manifesting as gingival overgrowth. Our previous studies concerning the molecular mechanisms of drug-induced gingival overgrowth have demonstrated that PHT alters the normal tissue turnover/wound healing signal by causing changes in macrophage phenotype, resulting in the upregulation of essential polypeptide growth factors such as platelet-derived growth factor (PDGF). The cellular consequences of this elevation in growth factor have not been investigated. The present light and electron microscopic study of rat hyperplastic connective tissue and human gingival overgrowth induced by PHT treatment revealed the presence of numerous myofibroblasts. Cells identified as myofibroblasts were evident in all PHT-treated tissue samples and were characterized by an elongated fusiform cell shape, abundant cytoplasmic rough endoplasmic reticulum/polyribosomes, and accumulations of sub-plasmalemmal microfilaments containing spindle densities. These cells were never observed in control tissues. Myofibroblasts are associated with the later stages of tissue turnover, specifically with the transition from the granulation to the remodeling phases of the wound healing process. The presence of myofibroblasts in hyperplastic connective and gingival tissues induced by PHT treatment suggests that PHT exacerbates the normal tissue turnover/wound healing signals responsible for the appearance of myofibroblasts.

Pathophysiological relationships between periodontitis and systemic disease: recent concepts involving serum lipids.

Periodontitis has been traditionally regarded as a chronic inflammatory oral infection. However, recent studies indicate that this oral disease may have profound effects on systemic health. The search for cellular/molecular mechanisms linking periodontitis to changes in systemic health and systemic physiology has resulted in the evolution of a new area of lipid research establishing linkages between existing multidisciplinary biomedical literature, recent observations concerning the effects of serum lipids on immune cell phenotype/function, and a heightened interest in systemic responses to chronic localized infections. There appears to be more than a casual relationship between serum lipid levels and systemic health (particularly cardiovascular disease, diabetes, tissue repair capacity, and immune cell function), susceptibility to periodontitis, and serum levels of pro-inflammatory cytokines. In terms of the potential relationship between periodontitis and systemic disease, it is possible that periodontitis-induced changes in immune cell function cause metabolic dysregulation of lipid metabolism through mechanisms involving proinflammatory cytokines. Sustained elevations of serum lipids and/or pro-inflammatory cytokines may have a serious negative impact on systemic health. The purpose of this paper is to present the background, supporting data, and hypotheses related to this concept. As active participants in this emerging and exciting area of investigation, we hope to stimulate interest and awareness among biomedical scientists and practitioners.

Diabetes prevents periodontitis-induced increases in gingival platelet derived growth factor-B and interleukin 1-beta in a rat model.

Periodontitis is a chronic inflammatory disease characterized by a progression that is very much dependent on host response. The gingiva can be considered to be in a constant state of wounding (pathologic wounding by bacterial plaque) and a constant state of maintenance/repair. In this context, any metabolic disturbance in the host which compromises tissue repair/wound healing will exacerbate the progression of periodontitis. Diabetes presents an interesting example because two major complications of diabetes are delayed wound healing and periodontitis. Our previous studies indicate that delayed wound healing and periodontitis may be manifestations of a general systemic deficit in diabetes involving alteration of macrophage cytokine gene expression. The present study was designed to determine whether: 1) diabetes-induced metabolic alterations affect gingival cytokine levels; and 2) diabetes-induced metabolic alterations modify the gingival cytokine profile in periodontitis. Sprague-Dawley rats (N=12/group) were injected with streptozotocin (65 mg/kg) into the tail vein to induce diabetes (defined by blood glucose levels > 250 mg/dl) or received the injection vehicle or no treatment as controls. Periodontitis was induced in additional groups of diabetic and control rats by gavage with Porphyromonas gingivalis A7436. After 90 days, serum glucose was analyzed to document diabetes; alveolar bone level was measured to document severity of periodontitis; gingiva was harvested circumferentially from the first and second molars; and cytokines in gingival homogenates were assayed by ELISA using commercial kits. Cytokine levels were expressed as mean+/-SEM pg/microg protein. Diabetes alone did not alter the gingival cytokine profile for platelet-derived growth factor B (PDGF-B), interleukin 1-beta (IL-1beta), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha). Periodontitis alone demonstrated a significant increase (P < 0.05) in levels of PDGF-B and IL-1beta. Diabetes superimposed on periodontitis prevented these increases. Thus, diabetes-induced metabolic alterations do not affect gingival cytokine levels per se; however, they do alter the normal host response to periodontitis through blockage of periodontitis-induced increases in PDGF-B and IL-1beta.

Heightened gingival inflammation and attachment loss in type 2 diabetics with hyperlipidemia.

BACKGROUND: Our previous studies in diabetic (DB) rats suggest that hyperlipidemia may cause a dysregulation of the cellular and local cytokine response to periodontitis (AP). The objective of the present study was to determine if diabetes has a similar dysregulatory effect on the gingival response to AP in humans. METHODS: Peripheral blood, as well as gingival tissue (GT) and gingival crevicular fluid (GCF), was obtained from a total of 35 patients who were categorized into the following groups based on level of diabetic (type 2) control and presence or absence of adult periodontitis (AP): group 1, systemically and periodontally healthy (n = 6); group 2, systemically healthy with adult periodontitis (n = 7); group 3, well-controlled diabetes and periodontally healthy (n = 6); group 4, well-controlled diabetes with adult periodontitis (n = 5); group 5, poorly controlled diabetes and periodontally healthy (n = 5); group 6, poorly controlled diabetes and adult periodontitis (n = 6). All subjects were given a thorough periodontal examination, including probing depths (PD), clinical attachment levels (CAL), gingival index (GI), plaque index (PI), and vertical bitewing radiographs. Blood studies included levels of glycated hemoglobin (HbA1c), triglycerides (TG), cholesterol (CHL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). The levels of interleukin-1 beta (IL-1beta) in GCF and GT, interleukin-6 (IL-6), and platelet-derived growth factor AB (PDGF-AB) in GT from patients in each experimental group were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Our results indicate that all clinical indices except PI were significantly elevated in the poorly controlled and well-controlled diabetics, compared to systemically healthy patients, but only in the subjects without preexisiting AP (Tukey's multiple comparisons, P <0.05). Pairwise linear regression analysis revealed significant (P <0.01) positive associations between periodontal inflammation (PD, CAL, PI, GI) and levels of GCF IL-1beta, GT IL- 1beta GT IL-6, but not GT PDGF; moreover, GT IL-6 levels were significantly associated (P<0.05) with GT IL-1beta. As TG levels increased in the non-AP patients (group 1 < group 3 < group 5), there was a trend, not significant, for increased GCF IL-1beta levels and increased gingival inflammation. Interestingly, periodontitis resulted in increased PDGF-AB levels in the gingiva of systemically healthy and well-controlled diabetes patients, but this increase was obtunded in poorly controlled diabetes patients. CONCLUSIONS: This confirms our earlier work in the diabetic rat model. These studies indicate that decreased metabolic control in type 2 diabetics results in increased serum triglycerides and has a negative influence on all clinical measures of periodontal health, particularly in patients without preexisting periodontitis. Levels of the cytokine IL- 1beta showed a trend for increasing as diabetic control diminished. In contrast, levels of the growth factor PDGF, which normally increase in periodontitis, decreased in poorly controlled diabetics with periodontitis. These studies suggest a possible dysregulation of the normal cytokine/growth factor signaling axis in poorly controlled type 2 diabetics that may contribute to periodontal breakdown/diminished repair.

Cyclosporine A upregulates interleukin-6 gene expression in human gingiva: possible mechanism for gingival overgrowth.

Cyclosporine A (CsA) is a widely used immunosuppressant for transplant patients and is also used for the treatment of a wide variety of systemic diseases with immunologic components. A prominent side effect of CsA administration is gingival overgrowth. It has been postulated that CsA alters fibroblast activity through effects on various cytokines such as the interleukins, however, as yet, data concerning the molecular mechanisms involved in connective tissue proliferation are still preliminary in nature. The purpose of this study was to evaluate interleukin-6 (IL-6) gene expression in gingival tissues of patients receiving CsA therapy and exhibiting gingival overgrowth. Radioimmunoassay (RIA) demonstrated a significant difference in tissue levels of IL-6 as mean +/- SEM. IL-6 content in CsA-stimulated tissue was 184.3 +/- 30.2 ng/mg total protein versus 23.3 +/- 6.5 ng/mg total protein in control tissue. In situ hybridization indicated that overgrown gingival tissues from patients taking CsA had a significantly higher content of IL-6 mRNA when compared to control tissues. Expressing IL-6 mRNA levels as silver grains/cell, CsA-stimulated tissue had 166.9 +/- 12.0 grains of IL-6 mRNA/cell while control tissue had 12.8 +/- 3.0 grains of IL-6 mRNA/cell. These results demonstrate that CsA therapy results in increased levels of IL-6 protein and IL-6 mRNA in overgrown human gingival tissues. This is the first report of CsA-upregulated IL-6 gene expression in vivo, and may explain in part the molecular mechanisms responsible for CsA-induced gingival overgrowth.

Phenytoin increases gene expression for platelet-derived growth factor B chain in macrophages and monocytes.

The mechanism by which phenytoin (PHT) induces gingival overgrowth remains unclear. We hypothesized that PHT increases macrophage production of platelet-derived growth factor (PDGF), an important cytokine in connective tissue growth and repair, and that excessive production PDGF in gingiva could lead to redundant growth. To test the hypothesis, rat peritoneal macrophages and human blood monocytes were cultured in the presence of PHT (5 to 20 micrograms/ml medium) or an equal volume of its solvent for 3 days and tested for expression of PDGF-B mRNA by in situ hybridization. Approximately 300 cells/culture well were examined (3 wells/drug level) for positive indication of PDGF-B mRNA. Data were compared by chi square test. All levels of PHT in both cell types induced a 2- to 8-fold increase in PDGF-B mRNA positive cells, significant in all cases at P < 0.001. Northern blot analysis of RNA from similarly cultured rat macrophages confirmed these findings. Cells treated with 10 micrograms PHT/ml medium or solvent revealed 2.2 +/- 0.3 and 1.0 +/- 0.2 (mean +/- SEM) arbitrary units PDGF mRNA respectively (t tests, P < 0.05). Additionally, rat macrophages were cultured in presence of 5 micrograms PHT/medium or its solvent and medium was analyzed for PDGF secretion by radioimmunoassay. Mean values (+/- SEM) were 1.28 +/- 0.49 and 0.78 +/- 0.07 ng/mg protein respectively (t test, P < 0.05). These data showed that PHT augmented the expression of c-sis, the gene for PDGF-B, and offered a possible explanation for PHT-induced gingival overgrowth.