Microbial contamination of laboratory constructed removable orthodontic appliances.

OBJECTIVES: This study aims to determine whether laboratory constructed removable orthodontic appliances are free from microbial contamination prior to clinical use and to evaluate the dental hospital cross-infection procedures to ensure that patient-derived contamination does not enter the construction process, thereby propagating a cycle of cross-contamination. MATERIALS AND METHODS: The construction process of removable orthodontic appliances from three individuals was evaluated at every stage, from impression to final delivery of the appliance using molecular microbiological techniques. The bacterial profiles at each stage of appliance construction were obtained using denaturing gradient gel electrophoresis, along with the bacterial profiles of the three participants' saliva. This enabled the bacterial profiles found at each stage of construction to be compared directly with the saliva of the person for whom the appliance was being constructed. Bacteria were identified at each stage using 16S rDNA PCR amplification and sequence phylogeny. RESULTS: There was no evidence of bacterial cross-contamination from patients to the laboratory. The current process of disinfection of impression appears to be adequate. Contamination was found on the final removable appliances (0.97 × 10(2)-1.52 × 10(3) cfu ml(-1)), and this contamination occurred from within the laboratory itself. CONCLUSIONS: Every effort is made to reduce potential cross-infection to patients and dental professionals. Newly constructed removable appliances were shown not to be free from contamination with bacteria prior to clinical use, but this contamination is environmental. Further studies would be required to determine the level of risk this poses to patients. CLINICAL SIGNIFICANCE: Dental professionals have a duty of care to minimise or eradicate potential risks of cross-infection to patients and other members of the team. To date, much less attention has been paid to contamination from the orthodontic laboratory, so contamination and infection risks are unknown.

The effects of different orthodontic appliances upon microbial communities.

OBJECTIVES: Orthodontic appliances can promote accumulation of dental plaque, with associated enamel decalcification or gingival inflammation. The aim of this study was to examine longer-term microbiological changes during orthodontic treatment with fixed appliances. MATERIALS AND METHODS: Twenty-four orthodontic patients aged 11-14 years undergoing fixed appliance therapy were recruited into the study. Each was randomized for cross-mouth assignment of molar bands and bonded molar tubes to contralateral quadrants of the mouth. All patients received self-ligating brackets, but again using randomization, one upper lateral incisor bracket (left or right) also received an elastomeric ligature. Plaque samples from the molars and upper lateral incisors were obtained at intervals during treatment and up to 1 year after appliance removal. Denaturing gradient gel electrophoresis and 16S rDNA microarray were used to compare plaque microbial fingerprints. RESULTS: Plaque populations changed within 3 months of commencing treatment at all sites. The greatest differences in plaque composition were seen with self-ligating brackets with an elastomeric ligature. Post-treatment plaque associated with both types of molar attachment contained increased levels of periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Eubacterium nodatum, while Campylobacter rectus, Parvimonas micra, and Actinomyces odontolyticus were also elevated with bonds. CONCLUSIONS: The results suggest that orthodontic treatment may cause sustained changes in plaque microbiotas and that molar bond-associated plaque may have raised disease potential.

Molecular insights into the effects of sodium hyaluronate preparations in keratinocytes.

BACKGROUND: Hyaluronic acid (HA) preparations are widely used in clinical practice to accelerate wound healing, but it is not clear whether HA can exert direct effects on epidermal keratinocytes. AIM: To investigate the molecular and functional changes induced by HA preparations in keratinocytes by measuring global gene expression and wound healing. METHODS: Human skin keratinocytes were used for this study. They were treated with either sodium hyaluronate (SH) alone or a commercial adjuvant gel (Aminogam(®)) containing SH in combination with a pool of synthetic amino acids (L-proline, L-leucine, L-lysine and glycine). Global gene expression of nearly 55,000 transcripts was investigated with a chip array (Affymetrix Human Genome U133 2.0 Plus). RESULTS: We found that keratinocytes expressed all major HA receptors at the transcriptional level. In a fibroblast-free system, both SH and the adjuvant gel could effectively promote wound healing of keratinocytes. Major gene expression changes induced by HA preparations involves proteolysis, proteinase inhibitors, cellular metabolism and cytoskeleton. In total, 21 genes were differentially transcribed by SH and the adjuvant gel. CONCLUSIONS: Keratinocytes represent a previously underestimated target for HA action in wound healing. HA preparations induce transcriptional changes in keratinocytes and stimulate wound closure. Furthermore, the addition of synthetic amino acids to SH induces a distinct transcriptional profile.

Time-dependent recontamination rates of sterilised dental instruments.

OBJECTIVES: To determine time-related recontamination rates of sterilised instruments, following guidance from the UK Department of Health (HTM 01-05) that such instruments within primary dental care may only be stored for 60 days following sterilisation using a vacuum autoclave. MATERIALS AND METHODS: A total of 25 used examination mirrors underwent a washer-disinfector cycle, individual packaging and finally vacuum autoclaving. Immediately after autoclaving, time zero, five mirrors were tested for microbial contamination by aerobic and anaerobic culture. At 31, 60, 90 and 124 days a further five mirrors were removed from their packaging and were similarly tested for microbial contamination. RESULTS: There was no bacterial growth on blood-enriched media under both aerobic and anaerobic conditions after 5 days of incubation at 37°C at any time period from 0 to 124 days post-sterilisation. CONCLUSIONS: There was no recontamination of sterilised instruments in this investigation over the test period of 124 days. This exceeds the recommended limit of 60 days stated by the UK Department of Health. The new guidance, HTM 01-05, appears to place an extra burden on primary care dentists. This burden is not without associated costs, and at present does not appear to be based on published evidence.

Deregulation of PERK in the autoimmune disease pemphigus vulgaris occurs via IgG-independent mechanisms.

BACKGROUND: Serum and IgG isolated from patients with the autoimmune blistering disease pemphigus vulgaris (PV) trigger complex intracellular pathways in keratinocytes, including alterations of the cell cycle and metabolism, which ultimately lead to cell-cell detachment (acantholysis). We have shown previously that one of the earliest pathogenic events in PV is the activation of protein kinases, including the PKR-like endoplasmic reticulum (ER) kinase PERK. OBJECTIVES: In the present study we investigated in more detail the role of PERK in the pathogenesis of PV. METHODS: PERK levels were assessed by Western blotting and in-cell enzyme-linked immunosorbent assay, and PERK expression was silenced by siRNA technology. The effects of PV sera/IgG on keratinocyte cultures were investigated by flow cytometry, MTT and adhesion assays. RESULTS: We show that PERK is activated in keratinocytes exposed to PV serum, as demonstrated by an increase in phosphorylated PERK levels and phosphorylation of eIF2α. Decreased expression of PERK by siRNA reduced the effects of PV serum on the cell cycle and keratinocyte viability, two key events in PV pathophysiology. As impairment of metabolic activity in PV is partially due to non-IgG serum factors, we then investigated the activation of PERK in keratinocytes incubated with whole PV serum, purified PV IgG and IgG-depleted PV serum. The data demonstrated that PV sera depleted of IgG, but not PV IgG, triggered PERK phosphorylation and this correlated with a marked reduction of metabolic activity in keratinocytes exposed to IgG-free serum. Knockdown of PERK by siRNA abrogated the changes in the cell cycle and apoptosis induced by IgG-depleted PV serum. Finally, the reduction of metabolic activity observed in keratinocytes exposed to IgG-depleted PV serum was almost absent in PERK-deficient cells. CONCLUSIONS: Taken together, the results demonstrate that activation of PERK participates in the reduction of metabolic activity and cell viability seen in PV and that this phenomenon depends on non-IgG factors. PERK activation may represent a novel signalling mechanism linking ER stress and acantholysis in PV.

A new method for placental/cord blood processing in the collection bag. I. Analysis of factors involved in red blood cell removal.

We describe a new procedure for large-scale CB processing in the collection bag, thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C, the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal, the cell pellet was resuspended and the percent recovery of total WBC, CD34+ progenitor cells, CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300, 600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC, CFU, CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy, effective and particularly suitable for large-scale banking under GMP conditions.

HLA compatibility and human reproduction.

Studies carried out on inbred strains of mice have shown that conceptuses which differ at the MHC antigens from their mothers appear to enjoy a selective advantage when compared with conceptuses which are more compatible. In humans a highly significant degree of MHC compatibility can be found in couples with a history of repetitive spontaneous abortions with unknown aetiology. We HLA - typed 28 selected couples with a history of three or more consecutive spontaneous abortions of unknown aetiology and 28 normal couples as control. We found that 22/23 (79%) aborter couples shared common HLA antigens, while normally fertile couples only 7/28 (25%) (p less than 0.001). The finding of a significant HLA compatibility in couples having abortions might be consistent with the hypothesis that blocking antibodies, formed in early pregnancy as response to HLA antigens, are perhaps necessary for a successful gestation. The factor causing abortion in couples sharing HLA antigens might also refer to the homozygosity for fetal genes in linkage with HLA alleles. The sharing of HLA alleles could be a marker for other genes of the same region which are lethal for the embryo in the homozygous state.

HLA typing in couples with repetitive abortion.

Highly inbred strains of mice have a reduced reproductive capacity. In contrast, an increased capacity is associated with an immune response to placental antigens. In humans, repeated miscarriages seem to be associated to a higher frequency of HLA allele BW35. Twenty couples with repetitive spontaneous abortions of unknown etiology and 20 control couples with normal fertility were HLA typed. The sera of wives of infertile couples were tested against the husbands lymphocytes in complement dependent lympholysis test in order to detect anti-paternal HLA antibodies. The data show an HLA significant compatibility between the husbands and the wives with a history of repetitive spontaneous abortions. The best probability of evaluating different loci is that of HLA-A (p less than 0.01). The frequency of HLA-BW35 in women with repeated miscarriages is higher than in control women or than in the general Italian population. In infertile couples, all the sera collected from the wife were negative against the husband lymphocytes.