The final pH of bacteria comprising the predominant flora on sound and carious human root and enamel surfaces

Acidogenesis at low pH appears to be an important bacterial cariogenic trait. However, most information in this regard pertains to only a few of the acidogenic dental plaque bacteria. Therefore, the 'final' pH in sugar broth was determined for a wide variety of oral bacteria. Their source was: (1) carious material from advanced root lesions (ARL), (2) plaque from sound root surfaces of root-caries-free subjects (SRS), (3) plaque from "white spot" coronal lesions and sound coronal surfaces of caries-active subjects, and (4) plaque from sound coronal surfaces of caries-free subjects. Strains from groups 1 and 2 (ARL, 389 strains; SRS, 358 strains) were previously identified (van Houte et al., 1994) to the genus/species level and belonged to the predominant cultivable flora (PCF). Strains from groups 3 and 4 also belonged to the PCF but were not identified. All strains were placed in one of 4 final pH categories: < 4.2, 4.2-4.4, 4.4-4.6, and > or = 4.6. The main findings were: (1) ARL samples contained many strains with a final pH < 4.2 (mean percentage of 25.7). They included all strains of Lactobacillus and mutans streptococci (MS), most Bifidobacterium strains and non-mutans streptococci (non-MS), and about 20% of the Actinomyces strains. By contrast, SRS samples contained far fewer strains with a final pH < 4.2 (mean percentage of 8.4) which were nearly all non-MS. (2) Organisms with a final pH < 4.4 constituted mean percentages of 41.5 and 32.1 for the ARL and SRS samples, respectively. (3) The final pH distribution of strains in samples from coronal surfaces showed a tendency relative to caries activity (group 3 vs. group 4) similar to that for groups 1 and 2. Our findings further support the concept that increased cariogenic conditions are associated with increased proportions of organisms capable of acidogenesis at a low pH and that this shift involves organisms other than the MS and lactobacilli.     

An in vitro model for acoustic overstimulation

BACKGROUND/AIMS: A sequence of 40 amino acids within the nonstructural protein 5A of hepatitis C virus (HCV) has been suggested to be an interferon sensitivity determining region (ISDR). The variations in the ISDR after 12-14 years of chronic infection and the correlation between ISDR and interferon response were studied in patients who were infected by the same HCV isolate. METHODS: We determined the HCV-ISDRs of 13 chronically infected patients by direct sequencing of polymerase chain reaction products. All patients were infected by isolate HCV-AD78, but differed with respect to their sensitivity to interferon. Four patients were complete responders, two patients were non-responders, and seven showed a partial response. RESULTS: The ISDR of HCV-AD78 differed from a prototypical HCV-1b sequence in one amino acid and was therefore classified as an intermediate type. Direct sequencing of the HCV-ISDRs of the patients 12-14 years after infection, but before interferon therapy, revealed a rate of 2.2x10(-3) nucleotide substitutions per site per year, resulting in only single intermediate type amino acid exchanges. All sequences ranked with the intermediate type. Moreover, during interferon treatment no selection to a wild type ISDR was observed in five partial responders. CONCLUSIONS: Within the homogeneous patient group examined here, no correlation was found between the ISDR and the interferon response. Recent studies found only a small number of mutant type ISDRs in Europe. Additionally, our results indicate that the ISDR is a stable sequence element. This provides an explanation for the divergent data relating to the importance of the ISDR in different geographical regions.     

An in vitro model to study mesenchymal-epithelial transformation

PURPOSE: To determine if the lower milliampere second setting and shorter acquisition time of subsecond spiral computed tomography (CT) affects the image quality of thoracic CT scans. MATERIALS AND METHODS: In 92 consecutive outpatients referred for thoracic CT, spiral CT (120 kV, 292 mA) was performed with 1-second (n = 45) or 0.75-second (n = 47) scanning time. An equal percentage of patients (70%) in each group received intravenous contrast medium. At six mediastinal and six lung zones, degradation due to motion and noise, respectively, were graded independently on a four-point scale by three blinded radiologists. Statistically significant differences were determine with a two-tailed test. RESULTS: Mediastinal image quality was significantly better on 0.75-second scans than on 1-seconds scans (P < .001). Regions with the greatest improvement in image quality were around the aortic root, cardiac ventricles, and aortic arch. Lung image quality was also better on 0.75-second scans than on 1-second scans (P = .04). On 0.75- and 1-second scans, respectively, motion-related artifacts were found to degrade image quality 6.2 and 8.7 times more than noise-related artifacts in the mediastinum and 2.6 and 3.9 times more in the lungs. CONCLUSION: Subsecond spiral CT is associated with improved clarity and diminished motion artifacts on mediastinal and pulmonary images when compared with 1-second spiral CT.     

A new model to assess staphylococcal adhesion to intraocular lenses under in vitro flow conditions

Adhesion of staphylococcal cells to intraocular lenses coated with heparin was studied under in vitro flow conditions (280 microl min(-1)) at 37 degrees C. The intraocular lenses were incubated with human cerebrospinal fluid for 1 h or with cerebrospinal fluid including 0.50% plasma for 12 h, prior to bacterial challenge. Two strains of Staphylococcus epidermidis selected for this study, were isolated from biomaterial-associated infections. Bacterial adhesion was quantitated by bioluminescence and visualized by fluorescence microscopy of acridine orange stained bacteria. Surface coating with heparin significantly decreased bacterial adhesion of both strains after incubation with cerebrospinal fluid including 0.50% plasma for 12 h (p = 0.0209). However, no difference in bacterial adhesion was obtained between intraocular lenses with and without heparin, after incubation with cerebrospinal fluid for 1 h (p = 0.327). Microscopy showed that more bacteria were present on intraocular lenses without heparin than on intraocular lenses with heparin. The results show that preincubation with a proteinaceous fluid influences subsequent bacterial adhesion to the polymer surface. The results suggest that IOLs with heparin coating may be less prone to bacterial adhesion under perfusion conditions after surface conditioning in human CSF with 0.50% plasma and a preincubation period of 12 h. Heparin coating might be a valuable tool to decrease implant-associated bacterial endophthalmitis.     

Quantitation of bacterial adhesion to polymer surfaces by bioluminescence

Quantitation of microbes adhering to a surface is commonly used in studies of microbial adhesion to different surfaces. We have quantified different staphylococcal strains adhering to polymer surfaces by measuring bacterial ATP (adenosine triphosphate) by bioluminescence. The method is sensitive, having a detection limit of 10(4) bacterial cells. Viable counting of bacterial cells may yield falsely low results due to the presence of "dormant" and adherent bacteria. By using bioluminescence, this can be avoided. Cells of different bacterial species and cells of strains of the same species were shown to differ significantly in their basal ATP content (8.7 x 10(-13) - 5.2 x 10(-22) MATP). The size of adherent and planktonic bacteria decreased with time (0.7 micron-->0.3 micron, 20 days). During incubation in nutrient-poor buffer ("starvation"), the ATP content of adherent bacteria decreased after 24-96 h whereas that of planktonic bacteria was stable over 20 days. The presence of human serum or plasma did not interfere significantly with the test results. Since the ATP concentration of bacterial strains of different species varies and is also influenced by the growth conditions of bacteria (solid or liquid culture medium), a species-specific standard curve has to be established for bacteria grown under the same culture conditions. We conclude that the method is a sensitive tool to quantify adherent bacteria during experiments lasting for less than 6 h and constitutes a valuable method to be used in conjunction with different microscopical techniques.     

An in vitro model for evaluating neural stimulating electrodes

A model for in vitro evaluation of materials for use as neural stimulation electrodes is developed. Critical areas of concern in developing an in vitro test model discussed include: selection of environment, choice of material, design of stimulating equipment, and analytical procedures used to evaluate materials response. A method of providing quantitative analysis of materials response to stimulation conditions is presented. Evaluation techniques involve the use of scanning electron microscopy, x-ray spectroscopy, atomic absorption spectrometry, and potentiographic and dielectric analysis of the test electrodes. A diagnostics matrix is presented which assigns a scale factor to quantify the relative corrosion response of the candidate materials. The corrosion response of Pt electrodes is evaluated in terms of scale factors assigned through use of the diagnostics matrix.     

Comparison of an in vitro skin model to normal human skin for dermatological research

EpiDerm, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and the lactate production decreased through day 3. In conclusion, the EpiDerm HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds.     

An animal model for human melanoma

Experimental animal models that are directly relevant to human melanoma are lacking. We propose the Angora goat as a potentially useful field model with experimental potential and to this end have examined the prevalence and site distribution of all skin cancers in 28 Angora goat herds in Queensland, Australia. The prevalence of benign melanocytic lesions (lentigines) and their experimental induction by sunlight were also investigated. Among 1731 goats over 2 years of age, 139 malignant skin tumors were excised from 95 affected animals. The prevalence of squamous cell carcinoma (SCC) was 3.8% and of melanoma, 2.2%. Main site of occurrence of melanoma (83%) was the dorsal surface of the ear; in contrast SCC occurred mostly (84%) on the perineum. Lentigines were darker and more prevalent on the exposed compared with the unexposed surface of the ear in Angoras, analogous to the higher prevalence of nevi on the exposed compared with the less exposed inner surface of the arm in humans. Lentigines, which were also found on the perineum though lighter in color than on the dorsal ear, were absent in young animals under 3 months but were numerous in 1-3 year olds. Furthermore in an experimental substudy eight goats, having one flank repeatedly shorn and the contralateral flank left unshorn, revealed consistently more solar lentigines on the shorn flank (P < 0.05) when both sides were examined after 9 months. Histopathological examination of paired skin biopsies from five of these goats also showed more abundant pigmentation in skin from the exposed, as compared with the unexposed flank. These findings indicate that sunlight induces tumors and lentigines in goats in a highly site-specific manner. The Angora goat model may suggest paradigms for explaining the site differences observed for human melanoma and may also be useful in the future clarification of molecular changes following carcinogenic levels of sun exposure.     

An in vitro model of human red blood cell production from hematopoietic progenitor cells

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.     

Metabolism of puerarin and daidzin by human intestinal bacteria and their relation to in vitro cytotoxicity

The steady state plasma concentrations of clozapine and its two major metabolites, norclozapine and clozapine N-oxide, were compared in patients with schizophrenia treated with clozapine in combination with phenobarbital (n=7), and in control patients treated with clozapine alone (n=15). Patients were matched for sex, age, body weight, and antipsychotic dosage. Patients comedicated with phenobarbital had significantly lower plasma clozapine levels than those of the controls (232+/-104 versus 356+/-138 ng/ml; mean, SD, p < 0.05). Plasma norclozapine levels did not differ between the two groups (195+/-91 versus 172+/-61 ng/ml, NS), whereas clozapine N-oxide levels were significantly higher in the phenobarbital group (115+/-49 versus 53+/-31 ng/ml, p < 0.01). Norclozapine/clozapine and clozapine N-oxide/ clozapine ratios were also significantly higher (p < 0.001) in patients comedicated with phenobarbital. These findings suggest that phenobarbital stimulates the metabolism of clozapine, probably by inducing its N-oxidation and demethylation pathways.     

Adhesion of mammalian cells to polymer surfaces: from physical chemistry of surfaces to selective adhesion on defined patterns

The study of the adsorption of type I collagen from a solution containing Pluronic F68 has shown that the latter prevents collagen adsorption on polystyrene and does not prevent it on surface-oxidized polystyrene. This explains the control of mammalian cell adhesion by substrate surface hydrophobicity and composition of pre-conditioning solution. On that basis, selective adhesion of different types of mammalian cells (PC12 pheochromocytoma, MSC80 schwannoma, Hep G2 hepatoblastoma, rat hepatocytes) on patterned surfaces was achieved. Therefore tracks (width in the range of a few tens of microm) of reduced hydrophobicity were produced on polystyrene by photolithography and oxygen plasma treatment. After conditioning by a solution containing both Pluronic F68 and extracellular matrix protein (collagen, fibronectin), the latter adsorbed selectively on these paths thus allowing selective adhesion of the cells.     

In vitro side-view imaging technique and analysis of human T-leukemic cell adhesion to ICAM-1 in shear flow

The objective of the present study is to apply a novel side-view imaging technique to investigate T-leukemic Jurkat cell adhesion to a surface-immobilized ICAM-1 in shear flow, a ligand for leukocyte LFA-1. Images have revealed that Jurkat cell adhesion on ICAM-1 under flow conditions in vitro is quasistatic. The cell-substrate contact length steadily increased with time during the initial cell attachment to the ICAM-1-coated surface and subsequently decreased with time as the trailing edge of the cell membrane peeled away from the substrate under the influence of fluid shear forces. Changes in flow shear stresses, cell deformability, or substrate ligand strength resulted in a significant change in the characteristic adhesion binding time and contact length. A 3-D flow field with shear stresses acting on an adherent cell was calculated by using finite element methods based on cell shapes obtained from the in vitro images. The maximum shear stress acting on an actual cell body was found to be 3-5 times greater than the upstream inlet wall shear stress and was influenced by the extent of cell deformation within the flow channel. Therefore, the application of such a side-view imaging technique has provided a practical assay to study the mechanics of cell-surface adhesion in 3-D. The elongation of cells in shear flow tempers hydrodynamic shear forces on the cell, which affects the transients in cell-surface adhesion.     

An in vitro model for analysis of oxidative death in primary mouse astrocytes

Heart rate (HR) was monitored in 66 French pubertal boys (B, n = 28) and girls (G, n = 38) aged 11-16 years to evaluate habitual physical activity (HPA) over a 1-week period in the winter. The HR and the percentage of heart rate reserve (%HRR) were taken to be indexes of the metabolic activity for the whole day and for the different parts of the day. The HPA was evaluated from the time spent each day below 50%HRR, between 50%-70%HRR and above 70%HRR, which related to the time spent in no or low physical activity (NLPA), moderate physical activity (MPA) and vigorous physical activity (VPA), respectively. No sex differences were observed in the average %HRR each day [%HRRmean, [B 30 (SD 4)%; G 32 (SD 4)%]] or in NLPA [B 715 (SD 61) min, G 711 (SD 81) min] and VPA [B 19 (SD 16) min, G 21 (SD 21) min] throughout the week. During school days, daily %HRRmean was 7% smaller in 14-16 year olds compared to 11-13 year olds. This was linked to a decrease in MPA and a concomitant increase in NLPA (P < 0.05). Daily %HRRmean varied significantly during the week (range: 28-34% HRR). There were significant differences among the periods of the day (P < 0.05). The HR was the greatest during physical education lessons [128 (SD 11) beats x min(-1)], recreation [113 (SD 15) beats x min(-1)] and lunch break [108 (SD 12) beats x min(-1)] and the lowest during the evening [94 (SD 10) beats x min(-1)]. It was only during the lunch breaks that %HRRmean was greater (P < 0.05) on school days than on free days. Of all the teenagers studied 32% were considered active during the week.     

Specific inhibitors of bacterial adhesion: observations from the study of gram-positive bacteria that initiate biofilm formation on the tooth surface

Oral surfaces are bathed in secretory antibodies and other salivary macromolecules that are potential inhibitors of specific microbial adhesion. Indigenous Gram-positive bacteria that colonize teeth, including viridans streptococci and actinomyces, may avoid inhibition of adhesion by host secretory molecules through various strategies that involve the structural design and binding properties of bacterial adhesins and receptors. Further studies to define the interactions of these molecules within the host environment may suggest novel approaches for the control of oral biofilm formation.     

Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro

Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.     

Cellular responses to chemical and morphologic aspects of biomaterial surfaces. I. A novel in vitro model system

The clinical success of any implant is directly dependent upon the cellular behavior in the immediate vicinity of the interface established between the host tissue and the biomaterial(s) used to fabricate the device. All biomaterials have morphologic, chemical, and electrical surface characteristics that influence the cellular response to the implant. Quantitative measurement of specific aspects of this local host response to different but well-characterized biomaterial surfaces provides a crucial link in the understanding of the overall phenomenon of implant biocompatibility. A system has been devised for in vitro examination of responses of cells to controlled but independent changes in both the chemistry and morphology of polystyrene (PS) tissue culture surfaces. Micromachined silicon wafers were used as templates to solvent-cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either none, 0.5- or 5.0-microns-deep surface grooves arranged in a radial array. When all possible morphologies were combined with all possible polymers, nine model biomaterial surfaces (MBSs) were produced. The chemical characteristics of the MBSs were determined using electron spectroscopy for chemical analysis, secondary ion mass spectroscopy, and contact angle techniques and were found to be distinct. The types and amount of proteins that adsorb onto these surfaces from serum containing media were examined and found to consist of multiple molecular layers of relatively uniform composition. Self-contained tissue culture vessels formed from the MBSs were capable of supporting the growth of confluent cultures of rat calvarial cells. The model biomaterial system described here can be used to examine how simultaneous stimuli resulting from the chemical and morphological characteristics of a test material may influence biologic responses. Such multifactorial biocompatibility research is needed to properly document material-host interactions.     

An in vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation

A novel in vitro system was developed to examine the effects of traumatic mechanical loading on individual cells. The cell shearing injury device (CSID) is a parallel disk viscometer that applies fluid shear stress with variable onset rate. The CSID was used in conjunction with microscopy and biochemical techniques to obtain a quantitative expression of the deformation and functional response of neurons to injury. Analytical and numerical approximations of the shear stress at the bottom disk were compared to determine the contribution of secondary flows. A significant portion of the shear stress was directed in the r-direction during start-up, and therefore the full Navier-Stokes equation was necessary to accurately describe the transient shear stress. When shear stress was applied at a high rate (800 dyne cm-2 sec-1) to cultured neurons, a range of cell membrane strains (0.01 to 0.53) was obtained, suggesting inhomogeneity in cellular response. Functionally, cytosolic calcium and extracellular lactate dehydrogenase levels increased in response to high strain rate (> 1 sec-1) loading, compared with quasistatic (< 1 sec-1) loading. In addition, a subpopulation of the culture subjected to rapid deformation subsequently died. These strain rates are relevant to those shown to occur in traumatic injury, and, as such, the CSID is an appropriate model for studying the biomechanics and pathophysiology of neuronal injury.