Motoo Kitano

Bacterial invasion into dentinal tubules of human vital and nonvital teeth

The difference in resistance to bacterial invasion into the dentinal tubules between vital and nonvital teeth has not been determined. This study was conducted to clarify the effect of vital pulp on bacterial invasion into the dentinal tubules. The specimens were 19 intact pairs of bilateral upper third molars of 19 healthy, young adult male volunteers. In each case, 30 or 150 days before extraction, pulpectomies and root canal fillings were carried out unilaterally and a class V cavity involving the dentin was made on the palatal surface of both the pulpectomized tooth and the nonpulpectomized opposite tooth. The cavities were left unprotected to expose them to oral flora until the extractions were done, and the extracted teeth were examined histologically. When extraction followed 150-day exposure to the oral flora, there was a statistically significant difference in the bacterial invasion rate between the vital and nonvital teeth. It was postulated that vital teeth were much more resistant to bacterial invasion into the dentinal tubules than were nonvital teeth, thereby suggesting that the vital pulp plays some important role in this process.

Biological and Genetic Characterization of a Human Immunodeficiency Virus Strain Resistant to CXCR4 Antagonist T134

The chemokine receptors CXCR4 and CCR5 are considered to be potential targets for the inhibition of HIV-1 replication. We have reported that T134 and T140 inhibited X4 HIV-1 infection specifically because they acted as CXCR4 antagonists. In the present study, we have generated a T134-resistant virus (trHIV-1(NL4-3)) in a cell culture with gradually increasing concentrations of the compound. The EC(50) of T134 against trHIV-1(NL4-3) recovered after 145 passages was 15 times greater than that against wild-type HIV-1(NL4-3). This adapted virus was resistant to other CXCR4 antagonists, T140, AMD3100, and ALX40-4C, and SDF-1; from 10 to 145 times greater than that against wild-type HIV-1(NL4-3). On the other hand, T134, T140, and ALX40-4C were still active against AMD3100-resistant viruses (arHIV-1(018A)). The trHIV-1(NL4-3) contained the following mutations in the V3 loop of gp120: N269K, Q278T, R279K, A284V, F285L, V286Y, I288T, K290E, N293D, M294I, and Q296K; an insertion of T at 290; and Delta274-275 (SI). In addition, many other mutations were recognized in the V1, V2, and V4 domains. Thus, resistance to T134 may be the consequence of amino acid substitutions in the envelope glycoprotein of X4 HIV-1. The trHIV-1(NL4-3) could not utilize CCR5 as an HIV infection coreceptor, although many amino acid substitutions were recognized. The trHIV-1(NL4-3) acquired resistance to vMIP II, which could inhibit both X4 and R5 HIV-1 infection. However, neither the ligands of CCR5, RANTES, and MIP-1alpha, nor a CCR5 low molecular antagonist, TAK-779, were able to influence the infection of trHIV-1(NL4-3). Those results indicated that alternation of coreceptor usage of trHIV-1(NL4-3) was not induced.

Higher osteoclastic demineralization and highly mineralized cement lines with osteocalcin deposition in a mandibular cortical bone of autosomal dominant osteopetrosis type II: ultrastructural and undecalcified histological investigations.

In this study we report on histological and ultrastructural investigations of the mandibular cortical bone in a case of autosomal dominant osteopetrosis type II complicated by mandibular osteomyelitis. Histologically, there was a marked increase in the number and size of osteoclasts on the inner bone surface. An undecalcified preparation showed a pair of deeply stained (highly demineralized) and stain-phobic (highly mineralized) layers on the bone surface just beneath the osteoclasts. The layers were incorporated into the bone matrix during the remodeling process as thickened cement lines. A contact microradiogram of the cortical bone revealed highly mineralized layers at the cement lines, which were closely correlated with immunohistochemical evidence of deposition of osteocalcin at the thickened cement lines. Ultrastructural examination showed that the osteoclasts had a typical clear zone, but they were deficient in ruffled border formation and had numerous lysosomal vacuoles containing dense substances. An electron-dense amorphous material layer was present on the bone surface just beneath the osteoclasts as well as at the cement lines. The layer was partly composed of a short fibrillar material, and it partially revealed the lamellar structure. Consequently, an osteoclastic malfunction might be primarily involved in the process of bone matrix resorption rather than demineralization, resulting in higher demineralization and abnormal material deposition on the bone surface and at the cement lines. Furthermore, evidence of active osteoclastic bone resorption with a brush border formation at the bone involved in the inflammatory lesion in this case suggests that the osteoclastic malfunction is influenced and recovered by a microenvironment such as inflammatory cytokines.

Solitary fibrous tumor in the mental region

Solitary fibrous tumor (SFT) is a rare, benign, soft tissue tumor that most commonly occurs in the pleura; however, it has recently been described in other sites of the body. To date, eight examples of oral SFT have been reported. This paper is a description of the first case of an SFT occurring as a soft tissue tumor in the mental region. Histologically, the tumor was composed predominantly of rather uniform spindle‐shaped fibroblastic cells arranged in vague fascicles or in a haphazard fashion, intermingled with abundant collagen fibers. Immunohistochemically, the tumor cells were positive for CD34 and vimentin, and weakly positive for muscle actin and α‐smooth muscle actin. The diagnosis of SFT may be difficult as this tumor shares a number of histological features with other soft tissue tumors. Awareness of its occurrence in the oral cavity is important so that confusion with other spindle cell neoplasms can be avoided.

Histomorphometric analysis of age changes in the human inferior alveolar artery

Angiography is often used to investigate age-related changes in the inferior alveolar artery, the major nutrient artery of the mandible. Although histological examinations have been made from several viewpoints, e.g. age change, pathogenesis of osteoradionecrosis, and relation to tooth extraction, these studies have used a limited number of samples and simple histometric methods. The purpose here was to describe histopathological and histomorphometric age-related changes, and to investigate the relation between dentate status and the histomorphometry of the artery. Inferior alveolar arteries from 162 autopsy cases (age range 3-86 years) were examined histometrically with a mathematically standardized method. Histologically, there was diffuse fibrous intimal thickening, but no atheroma formation. Histometric analyses revealed a very gradual increase in both the radius of the artery and the thickness of the media with age, but the luminal radius did not correlate with age. Intimal thickness increased exponentially with age with very different features from those of the increase in the media. The relative radius of the lumen decreased with age after the sixth decade; this is thought to be an index for senile changes in the artery. Among the variables of arterial architecture examined, no particular difference was found between the dentate and non-dentate cases in the molar region.

Five Quantitative Trait Loci Affecting 4‐Nitroquinoline 1‐Oxide‐induced Tongue Cancer in the Rat

In our previous study, Dark‐Agouti (DA) rats were found to be highly susceptible to 4‐nitroquino‐line 1‐oxide (4NQO)‐induced tongue carcinoma (TC), whereas Wistar/Furth (WF) rats were barely susceptible. Interval mapping analysis of reciprocal backcross rats showed two quantitative trait loci (QTL) on rat chromosomes (RNO) 1 and 19. In the present study, a composite interval mapping analysis was applied to 4NQO‐induced TC in 130 (DAxWF) F2 rats, demonstrating five independent QTL, Tongue squamous cell carcinoma 1‐5 (Tscc1‐5), responsible for phenotypic differences in the size and number of TCs in the two strains. Two of these QTL were mapped on RNO1, and the others were mapped on RNO4, 14, and 19. The DA allele at these loci consistently yielded semidominant susceptibility to TC. Out of the five loci detected in this F2 generation, Tscc1 and 2 were identical to Stcl and Rtc1 described in our previous study, but the other three were novel. We propose a new nomenclature consistent with their function. Genome‐wide screening of the F2 progeny also suggested the presence of three additional QTL on RNO5, 6, and 10. The possible roles of these loci in tongue carcinogenesis are discussed.

Increased Fractal Complexity of the Epithelial-connective Tissue Interface in the Tongue of 4NQO-treated Rats

The effect of the carcinogen 4-nitroquinoline 1-oxide (0.001% in drinking water) on the irregularity of the epithelial connective tissue interface (ECTI) of the ventral surface of the tongue was quantified in Dark Agouti and Wistar Furth rats. Histological tongue sections stained with the Azan-Mallory method were digitised (111 images, resolution 1 pixel = 3.1 microns), and the limit between epithelium and stroma of the ventral surface was extracted and analysed using a fractal geometry technique (local connected dimension). The results showed that although none of the images included carcinomas on the ventral surface of the tongue (all cases had other oral carcinomas), the epithelial profiles of the treated cases showed a statistically significant increase in irregularity when compared to controls. Canonical discriminant analysis of the parameters describing the irregularity of the ECTI classified 81.1% of the images in the original groups (treated or control). Fractal analysis is capable of detecting subtle architectural changes in the oral epithelium of the rat occurring after exposure to the carcinogen, even when full malignant transformation has not yet taken place. Fractal analysis, which may prove useful for monitoring the progression of carcinogenesis in this animal model, is a morphometrical parameter in the diagnosis of oral epithelial dysplasia.

Host genes controlling the susceptibility and resistance to squamous cell carcinoma of the tongue in a rat model.

Development of tongue carcinoma (TC) in rats by 4‐nitroquinoline 1‐oxide (4NQO), a potent carcinogen, is under host genetic control. The inbred Dark‐Agouti (DA) strain rats showed a much higher susceptibility to TC than the Wistar–Furth (WF) strain. The author’s previous study on crosses between two strains postulated a susceptibility gene in DA and a resistance gene in WF rats. This hypothesis was confirmed by the genetic analysis of the backcrosses to either parent and F2 with a simple sequence repeat polymorphism analysis. In the crosses between the DA and WF strains of rats, two major independently segregating host loci that influenced the cancer development by application of 4NQO positively or negatively were identified and mapped. DA rats had a semidominant susceptibility gene, Stc1, closely linked with D19Mit9 on chromosome 19, which was on the segment syntenic to human chromosome 16. In contrast, WF rats had a semidominant resistance gene, Rtc1, closely linked with D1Rat320 on chromosome 1, which is syntenic to human chromosome 11. The presence of other susceptibility and resistance genes on some chromosomes of both DA and WF rats was suspected, and they will be clarified in the near future. These findings provide powerful evidence that chemically induced tongue carcinogenesis is a multigenetic event.

CARCINOGENESIS MODIFIER LOCI IN RAT TONGUE ARE SUBJECT TO FREQUENT LOSS OF HETEROZYGOSITY

Rats of the DA strain are highly susceptible to 4NQO‐induced TCs, whereas WF rats are barely susceptible. In (DA × WF)F2 rats, 5 QTL, Tscc1–5, are responsible for most of the phenotypic variations, though they do not account for all of the phenotypic differences between WF and DA rats. Analysis of 40 tongue tumors >5 mm in diameter from (DA × WF)F1 rats for LOH at the Tscc loci revealed a high frequency of LOH in chromosomal regions where the Tscc2, ‐3 and ‐4 loci map. In most cases of LOH, the allele of the barely susceptible WF strain was lost, suggesting that these loci in the WF strain encode tumor‐suppressor genes. Analysis of the same tumors for somatic mutations in oncogenes indicated frequent alteration of Ha‐ras, which maps in the Tscc3 region, but rare mutation of the p15INK4B and p16INK4A genes or the p53 and Msh2 genes. Frequent LOH was also found on rat chromosomes 5 (RNO5) and 6 (RNO6). Tumors of large size accumulated LOH at multiple loci, suggesting the involvement of Tscc loci in tumor progression.

Polygenetic susceptibility and resistance to 4-nitroquinoline 1-oxide-induced tongue carcinomas in the rat

Summary Oral administration of 4-nitroquinoline 1-oxide (4NQO) to rats induced a high incidence of tongue carcinomas (TCs). The inbred Dark-Agouti (DA) strain of rats showed much higher susceptibility to 4NQO-induced TCs than the Wistar-Furth (WF) strain. Our previous study on crosses between the two strains postulated a semidominant susceptibility gene in DA and a semidominant resistance gene in WF rats. This hypothesis was confirmed by the genetic analysis of the back-crosses to either parent with PCR-based microsatellite assay. Using the number of TCS with >5 mm diameter as a quantitative parameter, we mapped a quantitative trait locus Stc1 (Susceptibility to TC) favouring TC development near the locus D19Mit9 on Chr. 19 with a peak LOD score of 6.08. Two other regions in Chr. 3 and Chr. 14 showed weak linkage for susceptibility, but were not statistically significant. On the other hand, another quantitative trait locus Rtc1 (Resistance to TC) providing resistance to TCs was mapped on Chr. 1 between the loci of D1Mit1 and D1Mit3 with a peak LOD score of 3.30. Quantitative parameters such as the number of tumours in the tongue or upper alimentary tract, the frequency of larger tumours and their maximum size were closely correlated and principally determined by Stc1 and Rtc1 . Therefore the susceptibility to 4NQO-induced TCs in crosses between DA and WF is explained by the combinations of genotypes at these two loci. Possible candidate genes for Stc1 and Rtc1 are discussed.