Background: Squamous differentiation/squamous metaplasia is often associated with endometrial adenocarcinoma and benign lesions, such as endometrial hyperplasia and chronic endometritis. Morules have distinct histological characteristics, and are referred to as squamous metaplasia or squamoid metaplasia. Aim: To focus on the histological characteristics of morules and clarify the difference between morules and squamous differentiation. Materials/Methods: Twenty endometrioid carcinomas with morules or squamous differentiation, five adenosquamous carcinomas, and eight non-carcinomatous endometrial lesions with morules were investigated. Numerous antibodies for epithelial membrane antigen (EMA), involucrin, cytokeratins, neuropeptides, and oncofetal antigens were used for immunohistochemistry. In situ hybridisation and polymerase chain reaction were used to detect human papillomavirus (HPV). Results: The morules observed were uniform cell clusters, with no squamous differentiation. They were immunonegative for epithelial antigens including involucrin, EMA, and cytokeratins, but were positive for neurone specific enolase. A few morules were immunopositive for acetylcholine esterase, and one case was positive for somatostatin; neither oncofetal nor proliferative cell markers, including blood group A, B, and AB, or other neuropeptides were demonstrated in the morules. HPV DNA was not found in either the morules in the carcinomas or in the benign lesions. However, true squamous differentiation tissue in four endometrioid carcinomas and two adenosquamous carcinomas was HPV positive using in situ hybridisation. Conclusion: Morules are histologically distinct from squamous metaplasia/squamous differentiation tissue. Morules are thought to be neuroectodermal-like cell clusters, and are not infected with HPV. In contrast, some of the true squamous differentiation tissue was associated with HPV infection.

Aim: In Okinawa, a subtropical island located between the East China Sea and Pacific Ocean, 2000 km south of mainland Japan, the incidence of oral squamous cell carcinoma is 1.5 times higher than that seen in mainland Japan, and a large number of these patients have been reported to be infected with the Epstein-Barr virus (EBV). This study aimed to gain a better understanding of the pathogenesis of this malignancy in this area by carrying out genomic analysis of EBV. Methods: Fifty four patients with oral squamous cell carcinoma reported from 1997 to 1999 in Okinawa were compared with 21 and 20 patients from Kitakyushu and Kumamoto in Kyushu, mainland Japan, respectively. Diagnosis was confirmed by conventional histological examination of paraffin wax sections. EBV was detected by non-isotopic in situ hybridisation (NISH) and the polymerase chain reaction (PCR) (Bam HI-F, EBV nuclear antigen 2 (EBNA2), and latent membrane protein 1 (LMP-1) regions). Sequence analysis of the PCR products was also carried out. Results: In Okinawa, 25 patients were found to be infected with EBV type A by analysing the 3′ sequence divergence of the EBNA2 genes. Six patients were positive for EBV type B, and eight for both type A and B. Therefore, type A virus infection was demonstrated in 33 of 54 patients, and type B in 14 of 54. In total, 39 of 54 patients were infected with EBV. However, the "f" variant was shown in only one patient, who was also infected with type A virus. In contrast, 97.0% of EBV type A infected patients showed a 30 bp deletion of the LMP-1 gene, but those infected with EBV type B did not. Sequence analysis of the type A virus EBNA2 gene revealed slight variations of the sequence (mutations)-48991 G→T and 48998 C→A-in 18 of 33 cases compared with the B95-8 strain, and in 14 cases, in addition to these, a further mutation of 48917 T→C was demonstrated; in the single remaining case, only one mutation at 49137 A→G was detected. The mutations at 48991 (G→T), and 49137 (A→G) are associated with amino acid changes Arg→Met and Thr→Ala, respectively. In contrast, no mutation was seen in the EBNA2 DNA from the 14 cases of type B virus when compared with that of the Jijoye strain. In Kitakyushu and Kumamoto, only 10 of 41 patients (six in Kitakyushu and four in Kumamoto) were infected with EBV. Among them, nine patients were infected with type A virus, and only one patient from Kitakyushu was infected with type B virus. The 48991 G→T and 48998 C→A mutations of the EBNA2 region were demonstrated in type A virus, but the 48917 T→C and 49137 A→G mutations were not when compared with the B95-8 strain. In the case of type B virus no mutation was noted. A 30 bp deletion was found in these nine cases of type A, but not in type B. The sequence analysis of EBV type A in Okinawa, Kitakyushu, and Kumamoto showed slight variations when compared with B95-8, but EBV type B LMP-1 did not when compared with the Jijoye strains. Conclusion: In Okinawa, EBV infection was frequently demonstrated in oral squamous cell carcinoma (p < 0.001)....

Background/Aim: It has been reported previously in cases of adenosquamous carcinoma of the lung in Okinawa, a subtropical island 2000 km south of mainland Japan, that the squamous cell carcinoma components were positive for human papillomavirus (HPV) by non-isotopic in situ hybridisation (NISH). The adenocarcinoma cells adjacent to the squamous cell carcinoma components were enlarged and also positive for HPV. This is thought to indicate that after adenocarcinoma cells are infected with HPV, they undergo morphological changes, and that "squamous metaplasia" follows. In this present study, the effects of HPV transfection into adenocarcinoma cells were examined. The relation between the region expressing the HPV gene and squamous metaplasia was also studied. Methods: Plasmid pBR322 containing HPV type 16 (HPV-16) was transfected into cultured colonic adenocarcinoma (DLD-1) and lung adenocarcinoma (PC-14) cells using the calcium phosphate method. Neomycin was used as a selection marker. The presence of HPV E1, E2, E4, E5, E6, E7, L1, and L2 mRNAs and also transglutaminase 1, involucrin, cyclin dependent kinases (CDKs), cyclins, caspases, apoptosis inducing factor, DNase γ, Fas, and Fas ligand mRNAs in HPV transfected cells was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The G0-G1 cell population was analysed by flow cytometry. Morphological examination under light and electron microscopes was also carried out. Results: The virus transfected cells showed squamous metaplasia when they were injected into severe combined immunodeficient mice, expressing the high molecular weight keratin (Moll's number 1 keratin) and involucrin molecules immunohistochemically, and involucrin and transglutaminase I mRNAs by RT-PCR. The squamous metaplasia was most conspicuous in the HPV transfected DLD-1 cell when compared with HPV transfected PC-14 cells. Squamous metaplasia was most clearly demonstrated in one HPV transfected DLD-1 cell clone, which expressed not only E2 but also E6-E7 fusion gene mRNA. Viral L1 mRNA expression was absent in HPV transfected cell clones, and was not related to squamous metaplasia. The growth rate of HPV transfected cells was reduced. Transfection of the virus into the cultured adenocarcinoma cells increased the G0-G1 cell population greatly, as assessed by flow cytometer analysis. Furthermore, in the virus transfected cells, apoptosis was also observed by means of the terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling method. Conclusion: HPV transfection into adenocarcinoma cells induced clear squamous metaplasia. One of the HPV transfected cell clones that expressed E2 and E6-E7 fusion gene mRNA showed the squamous metaplasia particularly clearly, and apoptosis was also demonstrated.

Background: Morules have been reported in pulmonary blastoma (PB), well differentiated fetal adenocarcinoma of the lung (WDFA), and uterine endometrioid carcinoma (EC), and rarely in other carcinomas. b Catenin gene mutation has been associated with morule formation. Aims: To compare and clarify the cellular characteristics of morules in carcinomas in various organs and show that morules are distinct from epithelial cellular nodules. Methods: Twenty tumours were studied: two PBs, three WDFAs, three papillary lung adenocarcinomas, 11 ECs, and one papillary thyroid carcinoma. Numerous epithelial cell, oncofetal, and neuropeptide antibodies were used for immunohistochemistry. b Catenin gene mutation was investigated. Results: Morules in PBs and ECs were uniform cell clusters distinct from squamous differentiation. All were immunonegative for epithelial cell and oncofetal antigens, but those in ECs were positive for neurone specific enolase c (NSEc). Synaptophysin, encephalin, and somatostatin were sporadically immunopositive in PB morules. Morules were not seen in the other carcinomas and WDFAs, although morule-like features closely resembling morules histopathologically were seen. These were positive for epithelial cell and oncofetal antigens, and showed squamous differentiation. Their nuclei were more atypical and slightly larger than those in morules. Morule-like features were seen in WDFAs. b Catenin gene mutation was demonstrated in one EC and PB, and in two WDFAs. Conclusion: Morules were non-epithelial cell clusters showing neuronal differentiation. There were two types: endometrioid type, expressing NSEc, and blastoma type, expressing neuropeptides. In contrast, similar morule-like features were epithelial nodules. Although the number of cases was small, the presence of morules showed no clear prognostic correlations.

SUMMARY Twenty-eight cases of mirror writing were seen during a period of three and a half years. These consisted of 12 patients with essential tremor, nine with Parkinson's disease, three with spino-cerebellar degeneration and four other cases. There were no cases of hemiparesis, aphasia, apraxia, agnosia or confusion. Fragmentary reversals were excluded from this study. Since essential tremor, Parkinsonian tremor and cerebellar tremor can be abolished by a stereotaxic produce applied to the thalamus, a common neural pathway via the thalamic nuclei may exist in these disorders. The existence is therefore proposed of some neural mechanism that controls the higher cerebral function of writing via the thalamus. were unskilfully written and drawn because of his action tremor. When he was instructed to write with the left hand, however, he wrote kanji from right to left in mirror fashion without any hesitation. He did not even recognise his own mirror writing. In the case of the clock face, not only the numerals, but also the position of the clock digits were reversed. Prospective Evaluation of the Clinical Cases Following our discovery of this initial case, we decided to evaluate the incidence of mirror writing in cases of neurological disorders which included not only essential tremor, but also Parkinson's disease, spinocerebellar degeneration, cerebrovascular disease, and various other neurological complaints, on the basis of work in an outpatients clinic. In addition, 258 medical and nursing students were asked to cooperate by writing their names with both right and left hands without any further suggestions. Although these students were much younger than the patients and were therefore unsuitable as perfectly exact age and sex matched controls, they can nevertheless be used as examples of how normal subjects write with their left hands.The mirror writing we report on here was, of course, induced mirror writing, which is somewhat different from the many types of case with spontaneous mirror writing classified by Critchley. Yet in every case, mirror writing was demonstrated when our patients were instructed to write letters with their left hands.