Sphingoid bases are taken up by Escherichia coli and Staphylococcus aureus and induce ultrastructural damage.

Sphingoid bases found in the outer layers of the skin exhibit antimicrobial activity against gram-positive and gram-negative bacteria. We investigated the uptake of several sphingoid bases by Escherichia coli and Staphylococcus aureus, and assessed subsequent ultrastructural damage. E. coli and S. aureus were incubated with D-sphingosine, dihydrosphingosine, or phytosphingosine at ten times their MIC for 0.5 and 4 h, respectively, to kill 50% of viable bacteria. Treated bacterial cells were immediately prepared for SEM, TEM, and analyzed for lipid content by QTLC. E. coli and S. aureus treated with sphingoid bases were distorted and their surfaces were concave and rugate. Significant differences were observed in the visual surface area relative to controls for both E. coli and S. aureus when treated with dihydrosphingosine and sphingosine (p < 0.0001) but not phytosphingosine. While sphingoid base-treated S. aureus exhibited disruption and loss of cell wall and membrane, E. coli cytoplasmic membranes appeared intact and the outer envelope uncompromised. Both E. coli and S. aureus cells contained unique internal inclusion bodies, likely associated with cell death. QTLC demonstrated extensive uptake of sphingoid bases by the bacteria. Hence, sphingoid bases induce both extracellular and intracellular damage and cause intracellular inclusions that may reflect lipid uptake.

Organization, barrier function and antimicrobial lipids of the oral mucosa.

As one moves from the skin across the vermilion region of the lip and into the oral cavity, the oral mucosa is encountered. The oral mucosa consists of connective tissue known as the lamina propria covered by a stratified squamous epithelium. In the regions of the hard palate and gingiva, the epithelium is keratinized like the epidermis. In the buccal region, the floor of the mouth and the underside of the tongue, the epithelium is non-keratinized. The epithelium on the dorsum of the tongue is a specialized epithelium, but can be approximated as a mosaic of keratinized and non-keratinized epithelia. The non-keratinized epithelial regions do not produce a stratum corneum. Nuclei with intact DNA are retained in the superficial cells. In all regions, the outer portions of the epithelium provide a protective permeability barrier, which varies regionally. Antimicrobial lipids at the surfaces of the oral mucosa are an integral part of innate immunity.

CD8(+) T cell-mediated injury in vivo progresses in the absence of effector T cells.

Tissue injury is a common sequela of acute virus infection localized to a specific organ such as the lung. Tissue injury is an immediate consequence of infection with lytic viruses. It can also result from the direct destruction of infected cells by effector CD8(+) T lymphocytes and indirectly through the action of the T cell-derived proinflammatory cytokines and recruited inflammatory cells on infected and uninfected tissue. We have examined CD8(+) T cell-mediated pulmonary injury in a transgenic model in which adoptively transferred, virus-specific cytotoxic T lymphocytes (CTLs) produce lethal, progressive pulmonary injury in recipient mice expressing the viral target transgene exclusively in the lungs. We have found that over the 4-5 day course of the development of lethal pulmonary injury, the effector CTLs, while necessary for the induction of injury, are present only transiently (24-48 h) in the lung. We provide evidence that the target of the antiviral CD8(+) T cells, the transgene expressing type II alveolar cells, are not immediately destroyed by the effector T cells. Rather, after T cell-target interaction, the type II alveolar cells are stimulated to produce the chemokine monocyte chemoattractant protein 1. These results reinforce the concept that, in vivo, the cellular targets of specific CTLs may participate directly in the development of progressive tissue injury by activating in response to interaction with the T cells and producing proinflammatory mediators without sustained in vivo activation of CD8(+) T cell effectors.

Comparative whole-genome analysis of Streptococcus mutans isolates within and among individuals of different caries status.

INTRODUCTION: Genotypic analyses of Streptococcus mutans using fingerprinting methods depend on a few genetic loci being different but do not reveal the underlying genome-wide differences between strains. METHODS: We used comparative genomic hybridization (CGH) with 70-mer oligonucleotide microarrays containing open reading frames (ORFs) from S. mutans strain UA159 to examine the genetic diversity of 44 isolates from nine children selected from a local study population in Eastern Iowa. RESULTS: Unique strains (clones) within each child initially identified by arbitrary-priming polymerase chain reaction were confirmed by CGH. There was a wide range of variation in the hybridization patterns of the 1948 ORFs among the test isolates examined. Between 87 and 237 ORFs failed to give a positive signal among individual isolates. A total of 323 of the UA159 ORFs were absent from one or more of the test strains. These 323 variable genes seemed to be distributed across the entire UA159 genome and across all the predicted functional categories. CONCLUSION: This set of very close geographically and temporally collected S. mutans isolates had a degree of gene content variation as high as a previously examined global set of strains. Comparing the frequency of these variable genes, the majority of which have unknown function, among strains of different origins (i.e. different caries status) could help to determine their relevance in S. mutans cariogenicity.

Evidence of Early Emergence of the Primary Dentition in a Northern Plains American Indian Population.

The purposes of this study were to describe primary tooth emergence in an American Indian (AI) population during the first 36 mo of life to compare 1) patterns of emergence between male and female children and 2) tooth emergence between these AI children and other U.S. ethnic groups. Data were derived from a birth cohort of 239 AI children from a Northern Plains tribe participating in a longitudinal study of early childhood caries, with examination data at target ages of 8, 12, 16, 22, 28, and 36 mo of age (±1 mo). Patterns of emergence in AI children were characterized and sex comparisons accomplished with interval-censored survival methodology. Numbers of erupted teeth in AI children at each age were compared via Kruskal-Wallis tests against those in children of the same age, as drawn from a cross-sectional study of dental caries patterns in Arizona; these comparisons were based on the dental examinations of 547 White non-Hispanic and 677 Hispanic children. Characterization of time to achievement of various milestones-including emergence of the anterior teeth, the first molars, and the complete primary dentition-provided no evidence of sex differences among AI children. AI children had significantly more teeth present at 8 mo (median, 3) than either White non-Hispanic (P < 0.0063) or Hispanic (P < 0.0001) children (median, 2 each). This was also true at 12 mo (P < 0.001; medians, 8 vs. 6 and 7, respectively) and 16 mo (P < 0.001; medians, 12 vs. 11 each). Less pronounced differences were seen at 22 mo (P < 0.0001). White non-Hispanic and Hispanic children did not differ at any time considered (P > 0.05). These results provide evidence of earlier tooth emergence in AI children than in the other 2 ethnicities. Although the underlying etiology of the severity of early childhood caries in AI children is likely to be multifactorial, earlier tooth emergence may be a contributing factor. Knowledge Transfer Statement: The findings of this study have practical implications for practitioners providing childhood oral health care to ethnic groups with early tooth emergence. It may be important to provide parents with information on toothbrushing, dentist visits, and other practices supportive of good oral health as early as possible to protect their children's primary dentition.

Effect of an alcohol-free, 1% chlorhexidine gel as an adjunct to a fluoridated dentifrice using an intraoral crown model.

BACKGROUND/AIMS: The use of chlorhexidine as a topically applied oral antiseptic is well documented; however, clinical studies examining the effects of chlorhexidine gel on in situ dental caries are limited. This study utilized an in situ caries model and a modified crossover design to examine whether the addition of a biweekly topical, alcohol-free, 1% chlorhexidine digluconate gel to a daily fluoridated dentifrice inhibited artificial caries in dental tissues better than the fluoridated dentifrice alone when compared to a nonfluoridated placebo dentifrice. METHODS: Thirty patients were recruited based on their need for a mandibular, full crown. Artificial caries lesions were created in extracted human teeth and enamel and root tissue sections 100 mum in thickness were characterized using polarized light microscopy. The sections were fixed in the crown and placed on the prepared tooth. The participants were assigned a placebo toothpaste, a toothpaste with 1,100 ppm F or a 1,100 ppm F toothpaste followed by 1 ml of 1% chlorhexidine gel at day 1 and day 14 (chlorhexidine+). Patients were instructed to brush twice daily for 4 weeks. Following each round, the sections in the crown were replaced with new sections. The sections were recharacterized and the mean changes were compared using ANOVA at alpha = 0.05. RESULTS: The chlorhexidine + Fdentifrice and the F dentifrice alone significantly reduced lesion area in enamel tissue when compared to the placebo dentifrice. Both treatments also inhibited lesion progression and initiation in root tissue better than control in this model system. Although the chlorhexidine+ group enhanced remineralization and inhibited lesion progression better than the F(-) dentifrice alone for all outcomes measured, the differences were not significant. CONCLUSIONS: The chlorhexidine, in conjunction with a fluoride dentifrice, was no more effective than the fluoride dentifrice alone. Further study is needed before this 1% alcohol-free chlorhexidine gel should be recommended as an adjunct to a fluoride dentifrice in the treatment of dental caries.

The effects of contamination on biological monitoring.

We investigated the frequency and patterns of biological-monitoring-test contamination and the effect of contamination on the growth of test organisms. Overall, the contamination rate was 0.81%, but the rate of contamination varied significantly by sterilization method. Contamination did not appear to inhibit growth of test organisms.

Patterns of expression of viral and cytokine gene transcripts during mouse polyoma virus infection.

CD8(+) cytotoxic T lymphocytes are critical for clearance of infection and prevention of tumors caused by mouse polyoma virus. High susceptibility to polyoma-induced tumors is manifested by neonatal inoculation of mice belonging to particular H-2(k) haplotype inbred strains. We previously reported that tumor-susceptible mice generate polyoma-specific CD8(+) T cells, but at a frequency approximately 20-fold lower than tumor-resistant H-2(k) mice. To determine whether susceptibility or resistance may also be associated with a cytokine microenvironment conducive for promoting cell-mediated (i.e., type 1 cytokines) or humoral (i.e., type 2 cytokines) immune responses, we used quantitative bioluminescence RT-PCR to measure in vivo message levels for viral proteins and cytokines during infection of neonatal mice. We found that the level of polyoma viral transcripts peaked higher and fell with significantly slower kinetics in tumor-susceptible mice than in tumor-resistant mice. Interestingly, message for VP1, the major viral capsid protein, persisted in multiple organs of mice of both susceptible and resistant strains, indicating chronic productive infection regardless of tumor susceptibility. IL-1beta, IL-12, IL-2, IFN-&gama; and IL-4 message levels were all higher in infected susceptible than resistant mice. Although both susceptible and resistant mice expressed transcripts for IFN-&gama; and IL-4, the signature type 1 and type 2 cytokines, respectively, a dominance of IL-4 message, with concomitant drop in IFN-&gama; message, was seen only in the susceptible mice. These results suggest that a type 2 pattern of cytokine expression may contribute to susceptibility to polyoma virus tumorigenesis.

Synergistic, growth-inhibitory effects of chlorhexidine and copper combinations on Streptococcus mutans, Actinomyces viscosus, and Actinomyces naeslundii.

The purpose of this investigation was to determine whether chlorhexidine combined with copper would exert a synergistic, growth-inhibitory effect against selected oral bacteria. Actinomyces viscosus, Actinomyces naeslundii, and Streptococcus mutans were all susceptible to chlorhexidine individually, with S. mutans displaying the highest sensitivity. Much higher concentrations of copper were needed to achieve growth inhibition of the micro-organisms tested. Determination of minimum inhibitory concentrations (MICs) of chlorhexidine and Cu2+ combinations suggested synergistic activity. Bactericidal kinetics assays confirmed synergism of chlorhexidine and Cu2+ combinations with 1 to 2 log greater decreases in viable cell numbers compared with chlorhexidine alone. Under the constraints of the conditions employed, these data demonstrate the efficacy of chlorhexidine and Cu2+ combinations against the oral bacteria tested. These studies suggest that a chlorhexidine and copper formulation could be useful as a mouthrinse in helping control cariogenic micro-organisms and/or in the treatment of gingivitis.

Effects of aged fluoride-containing restorative materials on recurrent root caries.

Accumulated evidence has illustrated that secondary caries is the major reason for the failure of amalgam and resin composite restorations. The purpose of this study was to assess the cariostatic effects of aged fluoride-containing restorative materials on the formation of secondary root caries. Fifty sound human molars were selected and randomly assigned to five material groups: non-fluoride-containing amalgam (NA), fluoride-containing amalgam (FA), non-fluoride-containing composite (NC), fluoride-containing composite (FC), and glass-ionomer cement (GIC). After standardized class V cavity preparations and placement of restorations, teeth within each group were randomly divided into two subgroups, "non-aged" and "aged". The aged subgroup was immersed in an inorganic buffer solution for 2 wks before being thermocycled. After being thermocycled and subjected to four cycles of caries formation in a bacterial model system, the teeth were sectioned. Depths of outer lesions and areas of lesions on the cavity walls were measured by polarized light microscopy. The results showed that the FA and GIC groups, whether aged or not, had significantly smaller outer lesion depth than the non-fluoride-containing NA and NC groups. After aging, the FA group demonstrated significantly greater lesion depth (p = 0.0002), while the GIC group exhibited no significant changes in lesion depths. The NA group had a greater wall lesion area than the NC group, while both demonstrated no significant changes following 2 weeks of aging. The FA and GIC groups had similar inhibition areas along the cavity walls, whereas both inhibition areas increased significantly after the aging process. It is concluded that the fluoride-containing amalgam and the glass-ionomer cement, even after a two-week aging process, can still elicit a significant preventive effect on recurrent root caries in an in vitro bacterial model system.

Photosynthetic gas exchange and temperature-induced damage in seedlings of the tropical alpine species Argyroxiphium sandwicense.

The capacity of Argyroxiphium sandwicense (silverword) seedlings to acclimate photosynthetic processes to different growing temperatures, as well as the tolerance of A. sandwicense to temperatures ranging from -15 to 60° C, were analyzed in a combination of field and laboratory studies. Altitudinal changes in temperature were also analyzed in order to explain the observed spatial distribution of A. sandwicense. A. sandwicense (Asteraceae) is a giant rosette plant that grows at high elevation on two Hawaiian volcanoes, where nocturnal subzero temperatures frequently occur. In addition, the soil temperatures at midday in the open alpine vegetation can exceed 60° C. In marked contrast to this large diurnal temperature variation, the seasonal variation in temperature is very small due to the tropical maritime location of the Hawaiian archipelago. Diurnal changes of soil and air temperature as well as photosynthetic photon flux density were measured on Haleakala volcano during four months. Seedlings were grown in the laboratory, from seeds collected in ten different A. sandwicense populations on Haleakala volcano, and maintained in growth chambers at 15/5, 25/15, and 30/25° C day/night temperatures. Irreversible tissue damage was determined by measuring electrolyte leakage of leaf samples. For seedlings maintained at each of the three different day/night temperatures, tissue damage occurred at -10° C due to freezing and at about 50° C due to high temperatures. Tissue damage occurred immediately after ice nucleation suggesting that A. sandwicense seedlings tend to avoid ice formation by permanent supercooling. Seedlings maintained at different day/night temperatures had similar maximum photosynthetic rates (5 µmol m-2 s-1) and similar optimum temperatures for photosynthesis (about 16° C). Leaf dark respiration rates compared at identical temperatures, however, were substantially higher for seedlings maintained at low temperatures, but almost perfect homeostasis is observed when compared at their respective growing conditions. The lack of acclimation in terms of frost resistance and tolerance to high temperatures, as well as in terms of the optimum temperature for photosynthesis, may contribute to the restricted altitudinal range of A. sandwicense. The small seasonal temperature variations in the tropical environment where this species grows may have prevented the development of mechanisms for acclimation to longterm temperature changes.

Synergistic effect of pyrophosphate and sodium dodecyl sulfate on periodontal pathogens.

Our previous studies have shown that pyrophosphate (PPi), the anticalculus component of tartar-control dentifrices, inhibits the growth of organisms associated with coronal and root surface caries. The purposes of this investigation were to: 1) determine if periodontal pathogens are similarly susceptible to the growth-inhibitory properties of PPi; and 2) determine if combinations of pyrophosphate-sodium dodecyl sulfate (PPi-SDS) inhibit growth synergistically. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Eikenella corrodens, and Campylobacter rectus (formerly Wolinella recta) were cultured in appropriate enriched media under anaerobic conditions. Inhibition assays were performed in tubes containing media supplemented with PPi and/or SDS. A range of concentrations of PPi and SDS in 2-fold increments was employed, with each concentration assayed in triplicate. Minimal inhibitory concentration (MIC) analyses revealed all of the bacteria were susceptible to PPi and SDS, with MICs of 0.67% (25 mM) and 0.01% w/v respectively. Combination studies with PPi-SDS showed much greater growth inhibition against P. gingivalis and A. actinomycetemcomitans than achieved with the agents individually. Determination of fractional inhibitory concentration indices indicated a synergistic growth-inhibitory effect. Under the constraints of the conditions employed, these studies demonstrate the efficacy of PPi-SDS combinations in inhibiting the growth of periodontal pathogens. It is conceivable that these compounds may have clinical benefit as a subgingival irrigant.

Bacterial retention in canal walls in vitro: effect of smear layer.

When dentin is planed by endodontic instruments, a smear layer forms. Whether this layer should be removed is unknown and controversial. This study was conducted to assess the effect of the smear layer on retention of bacteria using an in vitro root canal bacterial colonization model. Canals of 26 extracted human canines were step-back prepared using 2.5% NaOCl. Teeth were then randomly divided into two groups based on the type of high volume final flush: 1-20 ml of sterile saline (0.85% wt/vol) or (2-10 ml of 17% EDTA followed by 10 ml of 2.5% NaOCl which removes smear layer. Streptococcus anginosus (milleri) was cultured in trypticase soy broth supplemented with 0.5% yeast extract at 37 degrees C in 5% CO2. Cells were harvested by centrifugation and resuspended in fresh media. Serial dilutions were performed to achieve inocula of 10(6) colony-forming units in a 30-microliters volume. Teeth were inoculated and incubated for 2 h in 5% CO2 at 37 degrees C. Following incubation, teeth were split and processed for microbiological analysis. Numbers of colonizing bacteria were determined by a spiral-plating system. Enumeration of the numbers of bacteria revealed a reproducible, order of magnitude difference (p = 0.0002) between teeth with smear layer (10(4) colony-forming units) versus teeth without smear layer (10(5) colony-forming units). This suggests that smear layer produced during root canal therapy may inhibit bacterial colonization of root canals. One suggested mechanism is that smear layer may block bacterial entry into dentinal tubules.

Primary bacterial colonization of implant surfaces.

The purpose of this study was to assess the effects of modifying titanium surfaces, in terms of wettability, roughness, and mode of sterilization, on the ability of the oral bacterium Streptococcus sanguis to colonize. An in vitro model system was developed. All surfaces were colonized by the bacteria, but to significantly different levels. Titanium samples that exhibited rough or hydrophobic (low wettability) surfaces, along with all autoclaved surfaces, were preferentially colonized (P < .01). Titanium surfaces that had been repeatedly autoclaved were colonized with the levels of bacteria 3 to 4 orders of magnitude higher than other modes of sterilization. This may have implications relative to the commonly used method of autoclaving titanium implants, which may ultimately enhance bacterial biofilm formation on these surfaces.

Coronal leakage of mixed anaerobic bacteria after obturation and post space preparation.

OBJECTIVES: The purpose of this study was to assess bacterial leakage of a mixed anaerobic community of organisms in obturated canals after post space preparation. STUDY DESIGN: A mixed microbial community of strict anaerobic organisms (F. nucleatum, P. micros and C. rectus) was developed. With the use of an in vitro model system, coronal leakage was assessed in 40 anterior teeth after obturation and post space preparation. The specific leakage time in days for each organism to penetrate through the obturating material was determined. In addition, colonization of the apical canal space was assessed by scanning electron microscope after longitudinal splitting of randomly selected specimens. RESULTS: Eighty percent of the teeth demonstrated coronal leakage of F. nucleatum and C. rectus by the 90 day interval. Bacterial penetration occurred from 48 days to 84 days. Scanning electron microscope examination showed a heterogeneous biofilm of coccal and bacillary species colonizing the apical portion of the canal wall. CONCLUSIONS: This study demonstrated that coronal leakage phenomena do occur after loss of coronal seals. The model system developed using mixed, anaerobic bacterial cultures is more clinically relevant and may be used to assess bacterial penetration through gutta percha obturation.

Coronal leakage: bacterial penetration through obturated canals following post preparation.

Coronal leakage of bacteria from saliva into root canal filling materials is a potential cause of failure. This problem may be more pronounced when only a small volume of obturating material remains in the canal, such as after post preparation. In this study, coronal leakage of bacteria through unsealed, apically obturated canals was investigated using a new in vitro model system.

The antimicrobial activity of Prevention mouthrinse.

Prevention mouthrinse was designed to serve as a vital supplement to normal oral hygiene procedures. To determine the antimicrobial potency of this mouthrinse, minimal inhibitory concentrations (MICs), bactericidal kinetics, and short-term exposure studies were conducted. A spectrum of oral microorganisms was employed in this investigation: Streptococcus sanguis, Streptococcus anginosus, Streptococcus mutans, Actinomyces viscosus, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Eikenella corrodens, Porphyromonas gingivalis, Serratia marcescens and Candida albicans. Microorganisms were cultured in standard enriched media and under appropriate atmospheric conditions. Inhibition assays were conducted in tubes, with each mouthrinse dilution assayed in triplicate. MIC determinations revealed that all of the microorganisms studied were highly susceptible to Prevention mouthrinse, with MICs ranging from 16-fold to 128-fold dilutions. Bactericidal kinetics assays showed rapid killing of the test organisms in the presence of the mouthrinse. Brief (5-minute) exposure of S. mutans to 8-fold diluted mouthrinse resulted in a substantial delay in growth. Under the constraints of this type of study, Prevention mouthrinse exhibited potent antimicrobial activity against all of the microorganisms studied. We support the notion that Prevention mouthrinse may be a valuable supplement to normal oral hygiene procedures. A 6-month clinical trial assessing the in vivo efficacy of Prevention mouthrinse is currently being conducted.

Enhanced bactericidal activity of Arm and Hammer Dental Care.

PURPOSE: To compare and contrast antibacterial activities of a baking soda-containing dentifrice, Arm and Hammer Dental Care (AHDC) with two fluoride dentifrices without baking soda (Crest and Colgate). MATERIALS AND METHODS: A biphasic approach was taken, utilizing newly-developed laboratory model systems to: (1) assess the activity of brief exposure to dentifrices on single and mixed cultures; and (2) determine the effect of multiple, short-term exposure of sucrose-colonized Streptococcus mutans to simulate cumulative activity against cariogenic plaque. RESULTS: The short-term exposure assays revealed that S. mutans was significantly more susceptible to AHDC than either Crest of Colgate (P<0.05). Moreover, exposure of mixed suspensions of bacteria by AHDC resulted in complete killing of Actinomyces viscosus and significantly greater decreases in S. mutans (P<0.05). This enhanced bactericidal effect was not due to an alkaline pH as pH-adjusted AHDC solutions exhibited similar activity. The comprehensive in vitro plaque studies showed that exposure of colonized S. mutans to AHDC resulted in significantly greater decreases in numbers of viable cells than Crest (P<0.05). Under the conditions employed, the baking soda-containing AHDC exhibited greater antibacterial efficacy overall than the standard Crest or Colgate pastes. These studies suggest that the use of AHDC may provide additional clinical benefit as a result of the enhanced bactericidal activity.

Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host antimicrobial peptides.

Antimicrobial peptides (AMPs) are among the repertoire of host innate immune defenses. In the oral cavity, several AMPs are present in saliva and have antimicrobial activities against oral bacteria, including Streptococcus mutans, a primary etiological agent of dental caries. In this study, we hypothesized that unique S. mutans strains, as determined by DNA fingerprinting from sixty 13-year-old subjects with or without experience of caries, would have different susceptibilities to α-defensins-1-3 (HNP-1-3), ß-defensins-2-3 (HBD-2-3) and LL-37. The salivary levels of these peptides in subjects were also measured by enzyme-linked immunosorbent assays. We found that S. mutans strains from children with active caries showed greater resistance to salivary HNP-1-2, HBD-2-3 and LL-37 at varying concentrations than those from caries-free subjects. In addition, combinations of these peptides increased their antimicrobial activity against S. mutans either additively or synergistically. The salivary levels of these peptides were highly variable among subjects with no correlation to host caries experience. However, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. Our findings suggest that the relative ability of S. mutans to resist host salivary AMPs may be considered a potential virulence factor for this species such that S. mutans strains that are more resistant to these peptides may have an ecological advantage to preferentially colonize within dental plaque and increase the risk of dental caries.

Beta 2-microglobulin knockout mice are highly susceptible to polyoma virus tumorigenesis.

Polyoma virus is highly oncogenic when inoculated into immunocompromised adult mice and neonatal mice of specific inbred strains. Although T lymphocytes are known to be essential in controlling polyoma virus tumorigenesis, the importance of class I MHC-restricted CD8+ T cells in mediating tumor resistance remains unclear. Here, we investigated the tumorigenicity of polyoma virus in adult mice rendered CD8+ T cell-deficient by homozygous (-/-) disruption of the beta 2-microglobulin (beta 2m) or CD8 alpha (CD8) genes. Nearly all (94%) of the virus-infected adult C57BL/6 beta 2m-/- mice developed tumors, and 20% of the virus-inoculated adult C57BL/6CD8-/- mice developed hindlimb paralysis, which is indicative of vertebral tumors. Only 2 of 20 virus-inoculated adult normal C57BL/6 mice developed tumors. Despite these different tumor susceptibilities, persistent viral DNA was detected in multiple organs of mice of all three strains. Multifocal lymphoplasmacytic interstitial infiltrates were present in the kidneys and lungs of virus-infected C57BL/6 beta 2m-/- and in the lungs of virus-inoculated C57BL/6CD8-/- mice. These infiltrates were composed primarily of B cells and colocalized with staining for the major viral capsid protein, VP1. No infiltrates or VP1 staining was detected in the kidneys of infected C57BL/6 mice. Using a highly sensitive RT-PCR bioluminescence immunoassay, we investigated and detected persistent polyoma T protein and VP1 messages in both C57BL/6 beta 2m-/- and C57BL/6 mice. C57BL/6 beta 2m-/- and C57BL/6 mice had equivalent serum virus-neutralizing antibody titers. These results provide in vivo evidence that class I MHC-restricted CD8+ T cells are involved in mediating protection against polyoma virus tumor development.