Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions.

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa.

In hypotrichous ciliated protozoa, genes are transcribed in the macronucleus where the genome consists of 'gene-sized' linear DNA molecules. We have isolated clones of actin, tubulin and H4 histone macronuclear genes from Oxytricha nova, Stylonychia lemnae and Euplotes crassus in an effort to determine if they possess molecules of similar size for a given coding function, and also to determine the size range of non-coding DNA present on these molecules. Our results indicate that while the length of their non-coding DNA can vary slightly, both between different hypotrichs and within the gene family of a single organism, actin and tubulin macronuclear molecules are similarly sized. The sizes observed for these molecules support the hypothesis that each macronuclear molecule encodes a single gene. However, the H4 histone macronuclear molecules show a much wider size range and generally are much longer than necessary to encode the H4 histone. We therefore sequenced a 1700-bp H4 histone macronuclear molecule from O. nova to determine if it might possibly encode additional gene products. Sequence data reveals the presence of nine open reading frames (ORFs) greater than 100 bp in length; however, Northern hybridization analysis of the products of this DNA molecule reveals only a single transcript.

Overamplification of macronuclear linear DNA molecules during prolonged vegetative growth of Oxytricha nova.

During prolonged vegetative growth of a clonal line of Oxytricha nova, several macronuclear linear DNA molecules increased greatly in copy number over the rest of the approx. 24,000 kinds of molecules comprising the macronuclear genome. One of the amplified sequences was the linear DNA molecule encoding rRNA (rDNA). We have cloned and sequenced the other, smaller, amplified molecules and found that they comprise a gene family, with different allelic versions of one of the family members being amplified. Thus, increased replication is a general property of the molecules comprising this gene family. To date, no function has been assigned to these genes; thus, whether the amplification of these sequences has functional significance is unknown. The rDNA molecule and the two small amplified sequences increased 11-, 24- and 107-fold, respectively, during clonal growth of this line, eventually comprising up to 15% of the macronuclear DNA molecules. Seven other macronuclear DNA molecules did not vary substantially in copy number at different times during the clonal growth of this strain. Analysis of cell-to-cell differences in copy numbers in this clonally aged strain indicated more extensive variation than is evident when large populations from different times are compared.

Passive transfer of adjuvant-induced arthritis into irradiated DA recipient rats.

Adjuvant-induced arthritis (AIA) can be passively transferred in Dark Agouti (DA) rats by spleen and lymph node cells after culture with Concanavalin A (Con A). A model not requiring in vitro Con A expansion and activation would be important in investigations of anti-rheumatic drugs in AIA. A new model using irradiated recipients fills this need. Donor DA rats treated with 0.1 ml complete Freund's adjuvant (CFA) containing 7.5 mg M. butyricum/ml were sacrificed 11 days after CFA injection, donor spleen cells harvested, and donor spleen cells injected intravenously into recipient DA rats previously irradiated with 5 Gy. Recipient rats developed arthritis 4-14 days after spleen cell transfer. This model can now be used to further define the effects of anti-rheumatic drugs in the passive transfer of AIA by eliminating the need for the in vitro Con A-induced expansion and/or activation of donor cells.

Identification of a cyanogen bromide fragment of porcine type II collagen capable of modulating collagen arthritis in B10.RIII (H-2r) mice.

Previous studies directed towards identifying epitopes on type II collagen (CII) important in collagen induced arthritis (CIA) in mice have focused primarily on responses mounted in susceptible H-2q strains. However, the nature of T and B cell responses against CII in susceptible H-2r strains remains ill-defined. In an effort to identify regions on CII important in CIA in H-2r mice, we examined the cellular and humoral response of susceptible B10.RIII (H-2r) mice against cyanogen bromide (CB)-cleaved fragments of porcine CII. Following immunization with native porcine CII, LNC from B10.RIII mice mounted proliferative responses predominantly to peptide CB10, while negligible proliferation was detected against fragment CB9, 7, CB8, CB11 or CB12. In contrast, sera from arthritic B10.RIII mice displayed a heterogeneous pattern of reactivity against porcine CII, with strong antibody binding measured against the major fragments CB11, CB8 and CB10. To determine the in vivo significance of the dominant cellular response to CB10, B10.RIII mice received an i.v. injection of soluble CB10 seven days before immunization with native porcine CII. Mice pretreated with CB10 were highly resistant to CIA compared to control animals. Interestingly, B10.RIII mice pretreated with fragment CB11, a region of CII implicated in H-2q restricted CIA, remained susceptible to arthritis induction. Collectively, our findings indicate that the CB10 region of porcine C11 bears determinants which may be important in the induction and/or regulation of CIA in the H-2r haplotype.

T-cell receptors and collagen induced arthritis in H-2r mice.

Mouse strains B10, B10.RIII, RIIIS/J and the F1 and backcross progeny arising from them were tested for susceptibility to porcine type II collagen-induced arthritis (PII-CIA). The clinically severe arthritis of rapid onset that is characteristic of PII-immunized B10.RIII mice developed predominantly in hybrid offspring that had inherited at least one copy of wild type T cell receptor (TCR) genes (V beta b genotype) from the B10 or B10.RIII parent. The results indicate that, in the development of PII-CIA, mice expressing the H-2r/r haplotype preferentially utilize TCR V beta genes that are normally encoded within the TCR V beta genomic deletion region of RIIIS mice (V beta c). After aggressive immunization with PII, the use of alternative TCR V beta genes, encoded outside of the RIIIS deletion region, produced a high IgG antibody response that was cross-reactive with mouse type II collagen (MII) and equivalent to that of B10.RIII mice, but only a very mild, late onset arthritis of 56% (27/48) incidence in RIIIS male mice and 28% (10/35) incidence in RIIIS female mice. In comparison, B10.RIII mice routinely developed early onset of PII-CIA of significantly higher incidence (100%; p < 0.005) and four-fold greater severity, even after milder immunization protocols. The data are compatible with the proposal that the clinically weak CIA response of RIIIs mice may be primarily antibody driven while the severe CIA of B10.RIII mice reflects the added inflammatory effects of collagen-reactive effector-T cells in the joint.(ABSTRACT TRUNCATED AT 250 WORDS)

Exacerbation of collagen-induced arthritis in rats by rat cytomegalovirus is antigen-specific.

Collagen-Induced Arthritis (CIA) is an experimentally induced and genetically controlled animal model of chronic joint inflammation. In rats, there are informative strain differences in susceptibility to CIA. DA rats (RT1avl) develop severe CIA after immunization with bovine (BII), chick (CII), or homologous rat (RII) type II collagens. In contrast, the MHC-congenic DA. 1N(BN) and WF.1N(BN) rats (RT1n) are relatively resistant to CIA and develop moderate CIA in response to immunization with CII but not BII or RII. We previously found that simultaneous infection with rat cytomegalovirus (RCMV) greatly exacerbates the severity of arthritis that develops in BII-immunized DA rats. To examine the mechanism of RCMV amplification of CIA, the effect of simultaneous infection with RCMV on arthritis and autoimmunity to type II collagen was determined in WF.1N and DA.1N rats after immunization with BII, CII and RII. RCMV increased the incidence of CIA and the level of autoimmunity to type II collagen (skin-testing and IgG antibody titer) selectively in DA.1N and WF.1N rats immunized with CII, but not in littermates immunized with BII, although the transient reversal of CD4+/CD8+ mononuclear cell ratios in peripheral blood that is associated with RCMV infection occurred equally in both BII- and CII- immunized DA.1N rats. Likewise, RCMV infection moderately increased the levels of anti-RII autoimmunity and arthritis in DA rats sub-optimally immunized with RII but had no consistent effect on either anti-RII immunity or arthritis in RII-immunized DA.1N and WF.1n rats. The data show that RCMV augments arthritis only in rats that are genetically susceptible to CIA and that are appropriately immunized with a species of type II collagen that is arthritogenic for the MHC-haplotype being tested. Two possible mechanisms are suggested by these data: RCMV-associated increases in anti-RII autoimmunity in rats with CIA may result from amino acid sequence homologies between RCMV and type II collagen; alternatively, virus-induced pro-inflammatory cytokines may activate RII-reactive lymphocytes thereby potentiating autoimmunity and arthritis.

Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen.

DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.

Inhibition of acid production from oral bacteria by fluorapatite-derived fluoride.

The inhibitory effect of fluorapatite (FAP)-derived fluoride upon resting cell suspensions of Streptococcus mutans incubated at pH 4.5 and 6.5 was studied using lactic acid production from 0.1% sucrose as an indicator of fermentation activity. Cells incubated with FAP produced significantly less lactic acid than did cells incubated with hydroxyapatite (HAP). Addition of HAP to cell suspensions containing FAP reduced this inhibition, suggesting that dissolution of the FAP was necessary for inhibition. Incubation with low concentrations of NaF showed significant inhibition in cell suspensions incubated with as little as 0.45 micrograms/mL F at pH 5.0. These results provide further support to the hypothesis that fluoride levels in plaque and enamel, achievable through use of fluoridated water and/or fluoride dentifrices, may produce appreciable inhibition of glycolysis at the acidic pH levels which are readily achieved in plaque. Thus, bacterial acid production may activate plaque and enamel-bound fluoride, resulting in inhibition of further acid production, and thereby contribute substantially to the other cariostatic mechanisms of fluoride.

Effect of pH upon sucrose and glucose catabolism by the various genogroups of Streptococcus mutans.

Sucrose and glucose catabolism by seven strains of Streptococcus mutans belonging to six serotypes was assayed at pH's 6.5, 5.0, 4.5, and 4.0 with a radioisotopic tracer assay. The strains differed in their patterns of metabolic stimulation and inhibition at the different pH levels, falling into groups corresponding to the genetic groups described by Coykendall. The genogroup I (serotypes c and e) strains were the most acid-tolerant, having a pH optimum for lactic acid production at pH 5.0. These data furnish additional metabolic confirmation of the distinctiveness of these S. mutans subgroups.

Reduction of calculus accumulation in domestic ferrets with two dentifrices containing pyrophosphate.

Work performed by King et al. in the 1940's and 1950's, as well as recent studies by our group, have shown that the domestic ferret is a suitable model for the study of calculus formation, offering several advantages over the rodent and dog models in current use. We have demonstrated that mineral supplementation of a moist diet accelerates calculus accumulation, and that twice-daily application of a regular dentifrice slows the initial rate of calculus formation, but permits significant accumulation by the eighth week. The present study compared calculus accumulation in female ferrets receiving mineral-supplemented cat food and a twice-daily application of either Regular Crest toothpaste (Crest), Anti-tartar Crest (Crest-AT), or Anti-tartar Colgate Gold toothpaste (Colg-AT). Animals received an ultrasonic prophylaxis, then were fed once daily for eight weeks with moist canned cat food supplemented with sucrose and mineral salts, and were scored for area and extent of calculus accumulation at four and eight weeks after prophylaxis. The data show that the groups treated with the anti-calculus dentifrices produced significantly less calculus than the group treated with regular dentifrice; the Colg-AT group also exhibited lower scores than did the Crest-AT group, especially at four weeks. These results, similar to those seen in human studies, demonstrate that the ferret is a suitable model for the study of anti-calculus dentifrices.

Measurement of dietary and dentifrice effects upon calculus accumulation rates in the domestic ferret.

Most animal models for the study of calculus accumulation and control currently use rodents or dogs. In an effort to overcome limitations inherent in the use of these species, we investigated calculus formation in domestic ferrets, a species used by King et al. in the 1940's and 1950's. Ferrets are much smaller than dogs, and, unlike rodents, can be scored while alive. In this study, we examined the kinetics of calculus formation in female ferrets fed with moist canned cat food--either plain or supplemented with sucrose--and two combinations of mineral salts. An additional group given supplemented cat food was treated twice daily with regular Crest toothpaste. Animals were sedated with a 1:1 mixture of ketamine and xylazine solutions and given a mechanical prophylaxis prior to the trial period, then scored for area and extent of calculus accumulation at two, four, six, and eight weeks thereafter. The data showed that the mineral-supplemented groups accumulated calculus at a significantly faster rate than the unsupplemented or dentifrice-treated groups, but the differences were no longer significant at eight weeks. This demonstrated that the ferret is a suitable model for the study of calculus, that dietary mineral content influenced calculogenesis, and that the application of regular dentifrice initially slowed, but did not prevent, calculus accumulation.

Modification of food cariogenicity in rats by mineral-rich concentrates from milk.

Dairy products, including milk, cheese, and casein, can reduce the caries-causing potential of cariogenic substrates as measured in various animal, plaque acidity, and in vitro systems. Although the mechanisms responsible for protection are not completely identified, substances containing Ca and P may contribute to the protective potential by reducing demineralization and/or promoting remineralization of enamel. Casein may reduce demineralization by forming a protective coat on the enamel surface. By means of a rat model, this study evaluated the ability of three casein-free milk mineral concentrates with various levels of whey protein, calcium, and phosphate to modify the cariogenicity of a powdered diet containing 20% sucrose. Analysis of these data indicates that there were no significant differences among groups for weight gain, total food consumption, or feeding frequency, as monitored by a computer-based infrared activity monitor. All three mineral concentrates significantly reduced buccal caries, and two of the three reduced sulcal caries by from 10 to 30%. The analysis further shows that casein-free milk mineral fractions can modify the cariogenicity of sucrose-containing foods in a rat model.

Effect of cheese, with and without sucrose, on dental caries and recovery of Streptococcus mutans in rats.

The objective of this study was to determine the effect of aged and young cheddar cheese with and without added sucrose on dental caries and the associated recovery of implanted Streptococcus mutans. Very little caries was observed in rats consuming cheese without sucrose. There was an increase in caries in rats fed cheeses with 20% sucrose, but this increase was not significant. There was significantly greater caries activity in rats fed standard diets containing 20% or 5% sucrose (SLS or MIT 305) than in rats fed cheeses containing 20% sucrose. Rats fed cheese or powdered diets containing sucrose had significantly higher frequency of recovery and higher levels of S. mutans infection than did rats fed cheese containing no sucrose. This study confirms the low cariogenic potential and possible cariostatic activity of cheddar cheese in rats. Since cheddar cheese with sucrose did not significantly interfere with S. mutans implantation, the cariostatic mechanism is apparently unrelated to a direct antimicrobial effect on S. mutans.

Infantile agranulocytosis with survival into adolescence: periodontal manifestations and laboratory findings. A case report.

A case of infantile agranulocytosis with survival into adolescence is presented. The polymorphonuclear leukocyte is considered an important source of lysosomal enzymes in gingival crevicular fluid, and evaluation of connective tissue-degrading enzymes in the fluid was performed. The activity of beta-glucuronidase, a ground substance-degrading enzyme that may serve as a marker for polymorphonuclear leukocytes, was markedly reduced in the fluid compared to samples from systemically healthy adults with periodontitis. The activities of the ground substance-degrading enzyme arylsulfatase, and collagenase, were in the low-normal range. The plaque microbiology, as characterized by dark-field microscopy and selective culturing, was consistent with advanced periodontitis. A review of the medical history revealed a series of bacterial infections since infancy. Improvement in the systemic health of the patient occurred at about the age of 15, and the intake of antibiotics to control infections was correspondingly reduced after this time. An exacerbation of the patient's periodontal disease, as evaluated by loss of alveolar bone on radiographs, occurred 1 to 2 years later. The progression of periodontal disease observed in this patient was apparently associated with the withdrawal of antibiotics administered for control of systemic (nonoral) infections.

Differences in periodontal disease-associated microorganisms of subgingival plaque in prepubertal, pubertal and postpubertal children.

It is generally accepted that normal prepubertal children do not develop periodontitis, and that the severity of gingivitis in prepubertal children is usually less than that observed in children after puberty. One possible explanation is that the bacteria associated with periodontal diseases cannot become established in great numbers prior to puberty. Studies by Kornman and Loesche and others suggest that levels of black pigmented Bacteroides, especially B. intermedius, increase with increased levels of gonadotrophic hormones in pregnant women. Delaney and Kornman have found that there is a similar increase in levels of black pigmented Bacteroides with puberty. The present study involved cultural and microscopic characterization of the subgingival plaque flora of prepubertal, circumpubertal and postpubertal children with similar Silness and Löe plaque index scores. Puberty was confirmed through examination of wrist radiographs. Populations of black pigmented Bacteroides were very low in prepubescent children and were much higher in circumpubertal and postpubertal children. However, B. intermedius predominated only in circumpubertal plaques. Levels of total motile bacteria increased at each age level, but levels of spirochetes above 2% were observed only in the postpubertal group. These results support those of previous investigators who postulated a relationship between hormone levels and black pigmented Bacteroides levels in subgingival plaque and suggest that differences in the subgingival environment profoundly influence the proportions of suspected periodontopathic species in plaque.

Short-term microbiological and clinical effects of subgingival irrigation with an antimicrobial mouthrinse.

Fifty chronic adult periodontitis patients completed a 6-week controlled, double-blind, split mouth clinical study to determine the effects of subgingival irrigation with an antimicrobial mouthrinse on periodontal microflora, supragingival plaque, and gingivitis when used as an adjunct to normal oral hygiene. Qualifying subjects had at least four sites, two on each side of the mouth, with probing depths between 4 and 6 mm, which bled on gentle probing. Following baseline examinations, subjects received a half mouth scaling and prophylaxis and full mouth subgingival irrigation with either the antimicrobial mouthrinse or sterile colored water control professionally delivered. Subjects continued irrigation at home once daily for 42 days with their assigned rinse delivered via a subgingival delivery system. All sites in the mouth were scored at baseline and at day 42 for supragingival plaque, bleeding on probing, and redness. For the four selected periodontitis sites, probing depth and attachment level were measured at baseline and on day 42; additionally, supragingival plaque and gingival redness were scored on days 7 and 21. Subgingival plaque samples for microbiological analysis were harvested from the selected periodontal sites at baseline and on days 7, 21, and 42. Microbiologically, irrigation with the antimicrobial mouthrinse resulted in statistically significant reductions compared to control in putative periodontopathogens, including black pigmenting species, which persisted at 42 days. Clinically, subgingival irrigation with the antimicrobial mouthrinse produced a significant reduction in supragingival plaque (P < 0.001), bleeding on probing (P = 0.019), and redness (P = 0.017) compared to the control, whether or not the area irrigated received a prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical efficacy of a dentifrice and oral rinse containing sanguinaria extract and zinc chloride during 6 months of use.

The efficacy of combined use of toothpaste and oral rinse containing sanguinaria extract and zinc chloride was compared to placebo products in a 6-month clinical trial. Sixty subjects with moderate levels of plaque and gingivitis were randomly assigned to active and placebo groups. Noninvasive measures of plaque and gingivitis were assessed at baseline and at 2, 6, 8, 14, 20, and 28 weeks. Bleeding on probing was measured at baseline and 6, 14, and 28 weeks. Active group scores were significantly lower (P less than .0001) than placebo scores at each post-baseline time point for all indices, with the exception of plaque at 2 weeks. The 28 week active group scores were 21% lower than the placebo group for plaque, 25% lower for gingivitis, and 43% lower for bleeding on probing. No dental staining or taste alteration was reported in the active group. Three of 30 active group subjects exhibited minor soft tissue irritations that resolved spontaneously without discontinuation of product use. Results indicate that the test products showed good levels of safety and efficacy when administered in a combined use regimen for 6 months.

Effect of 6 months use of a dentifrice and oral rinse containing sanguinaria extract and zinc chloride upon the microflora of the dental plaque and oral soft tissues.

This study documented the effect upon the oral flora of twice daily brushing with a dentifrice containing 0.075% sanguinaria extract and 2% zinc chloride, followed by use of a mouthrinse containing 0.03% sanguinaria extract and 0.2% zinc chloride. Sixty subjects were randomly assigned to treatment or placebo groups and monitored in a 6-month double-blind clinical trial. Bacteriological samples from the tongue, buccal mucosa, and supra- and subgingival plaque were characterized at 0, 14, and 28 weeks. Microbiological monitoring showed no increases in populations of yeast, staphylococci, coliform organisms, or Pseudomonas. Total Gram-negative counts in supragingival plaque samples decreased 83% in the active group compared to a 232% increase for the control group. Populations of B. intermedius in supragingival plaque were significantly lower in the active group at 3 months and significantly lower counts of Fusobacterium sp. were observed at 3 and 6 months. Results indicate that use of the test products did not promote opportunistic overgrowth of pathogens in the oral flora. Additionally, the alterations in organisms associated with gingivitis may account for reductions in gingivitis seen in the active group.

Lysosomal and cytoplasmic enzyme activity, crevicular fluid volume, and clinical parameters characterizing gingival sites with shallow to intermediate probing depths.

The biochemical analysis of gingival crevicular fluid (GCF) may offer a sensitive means of determining periodontal disease activity, including the transition of gingivitis to periodontitis. To continue our evaluation of the relationship between clinical and GCF parameters, 552 sites with shallow to intermediate (2.0-5.0 mm) probing depths (PD) were examined. The data were collected at baseline from 33 periodontitis patients participating in a longitudinal trial examining the relationship of changes in GCF biochemistry to attachment loss. Mesiobuccal sites were scored for dichotomous measures of bleeding on probing, gingival redness, suppuration, and plaque accumulation. In addition, GCF was collected using filter paper strips inserted into the sulcus for 30 seconds, eluted in buffer and assayed for activity of the enzymes beta-glucuronidase (BG), arylsulfatase (AS), and lactate dehydrogenase (LDH), markers for ground substance-degradation and cellular necrosis, respectively. Clinical and GCF parameters were evaluated by increasing PD. Plaque accumulation and bleeding on probing increased with increasing PD, although there was considerable overlap across groups. Suppuration was present in only a very small number of sites and the proportion of sites displaying gingival redness was not related to PD. GCF volume was grouped in 0.25-microliter increments, revealing a progressive shift with increasing PD toward a normal distribution around the median range of 0.51 to 0.75 microliter at 5.0 mm. Mean enzyme activities of BG, and to a lesser extent AS and LDH increased sharply from 2.0 to 3.0 mm, were relatively stable from 3.5 to 4.5 mm, and were significantly higher in 5.0 mm than 4.5 mm sites.(ABSTRACT TRUNCATED AT 250 WORDS)