[Quantitative determination of lipoprotein by agarose gel electrophoresis and enzymatic staining of cholesterol and triglyceride: effects of age and gender].

290 normal duplicate samples (152 males and 138 females), were subjected to lipoprotein analysis by agarose gel electrophoresis to yield HDL, VLDL and LDL fractions which were respectively stained with Cholesterol and Triglyceride reagents. HDL-Cholesterol were significantly higher in female than male, and VLDL-TG were significantly higher in male than female. Assay C.V.'s varied from 0.96 to 5.75 for cholesterol fractions and 2.00 to 4.34 for triglyceride fractions. Comparison of electrophoretic HDL-Cholesterol and LDL-Cholesterol concentrations with the results of a direct method (HDL-EX and LDL-EX, Denka Seiken) gave correlation coefficients of 0.967 and 0.952 respectively. This method is simple, rapid and can provide the simultaneous assessment of the cholesterol and triglyceride component in each Lipoprotein fraction. Additionally, the method is useful for evaluating lipoprotein assays.

Experimental myelitis in BALB/cN and C57BL/6N mice caused by herpes simplex virus type 1 compared with herpes simplex virus type 2.

Intraperitoneal and footpad inoculations of herpes simplex virus type 1 (HSV) into BALB/cN (HSV-susceptible) and C57BL/6N (HSV-resistant) mice were carried out to induce experimental myelitis. Standard laboratory strains (McIntyre, F, RK, and recently Okinawa strain R1) were inoculated in mice. As a control, the HSV 2 standard laboratory strain SAV was also inoculated. The McIntyre strain was the most virulent, while the F strain was the least. RK and R1 were both moderately virulent. Myelitis was induced in BALB/cN mice after intraperitoneal and footpad inoculations of low to high doses of the McIntyre strain, and intraperitoneal inoculation of moderate and high doses of the RK and R1 strains. Symptoms of paraplegia of the hind legs and rectal and urinary incontinence were observed, but not until 3-5 hours before death. The symptoms caused by footpad inoculation were slightly different from those following intraperitoneal inoculation; rectal incontinence, in particular, was inconspicuous in the former. In the case of footpad inoculation of RK and R1, only one mouse inoculated with R1 showed symptoms and histology of myelitis. The F strain caused no symptoms. In the case of C57BL/6N mice, high dose intraperitoneal and footpad inoculations of the McIntyre strain also caused myelitis, and the symptoms were observed about 6-7 hours before death. In only one C57BL/6N mouse intraperitoneally inoculated with a high dose of R1 did symptoms appear about 6 hours before death. The same symptoms caused by intraperitoneal and footpad inoculations of HSV 2 (SAV) were observed more clearly and for a longer period (half to one day) than those caused by HSV 1 inoculation. Spinal cord necrosis was noted with McIntyre, RK and R1 inoculations, but it was not marked with randomly located foci, when compared with that caused by SAV. Further, the foci of necrosis in C57BL/6N mice were smaller than in BALB/cN mice, even when high dose McIntyre strain was used. Nuclear pyknosis and edema of the brain in the dead mice following HSV 1 inoculation were more marked than in those killed by SAV.

Comparative study of oral squamous cell carcinoma in Okinawa, Southern Japan and Sapporo in Hokkaido, Northern Japan; with special reference to human papillomavirus and Epstein-Barr virus infection.

In Okinawa, a subtropical island in Southern Japan, the incidence of oral squamous cell carcinoma is 1.5 times higher than that in mainland Japan. Sixty cases of oral squamous cell carcinoma from 1993 to 1996 in Okinawa and 42 cases over the same period in Sapporo were examined histologically. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) were detected by polymerase chain reaction (PCR) amplification with primers specific for HPV and EBV. In situ hybridisations of the viruses were also carried out. In the case of Epstein-Barr virus, in situ PCR was also performed. Thirty-five (58.3%) Okinawan tumours were well-differentiated in type, but in Sapporo, 18 (42%) were of such type. In Okinawa, tumours of the mouth floor (10 cases, 16.7%) and oropharynx (12 cases, 20%) were frequently observed, whereas in Sapporo only five cases (12%) of each were found. HPV was demonstrated in 78% of Okinawan cases and 26.2% of Sapporon cases by PCR or non-isotopic in situ hybridisation (NISH). There were 76.6% (46 cases) of Okinawan and 38.1% (16 cases) of Sapporo cases positive for EBV by PCR. In only 12 Okinawan cases and 4 Sapporon cases, were positive signals demonstrated by in situ PCR on the cancer cells themselves. EBV was demonstrated in the large number of infiltrating lymphocytes, most of which were CD3+, and a few were CD19+. In Okinawa, HPV might be an important causative factor of oral squamous cell carcinoma and EBV a less important factor, whereas in Sapporo HPV and EBV might play only a small part in the aetiology of the tumour.

Extensive intra-alveolar haemorrhage caused by disseminated strongyloidiasis.

We describe here four cases of disseminated strongyloidiasis. In Okinawa, it has been reported that about 10% of the residents are infected with Strongyloides stercoralis, but disseminated cases are rare. Detailed histopathological examination revealed that the present four cases could clearly be separated into two groups, two acute cases and two subacute cases. The acute cases died rapidly due to extensive diffuse intra-alveolar haemorrhage in both lungs. However, there were no inflammatory infiltrates, abscesses or granulomas in the lungs. Worms were demonstrated in the alveolar spaces. No extensive bleeding was observed in any organs except the lungs. The acute cases could be diagnosed as severe diffuse intra-alveolar haemorrhage syndrome, but deposition of immune complex (parasite antigen and immunoglobulins) and complement C3c was not demonstrated in the alveolar wall and small vessels of the lung. The subacute cases exhibited no such extensive haemorrhage, but scattered microabscesses were found with sepsis. During the migration of the worms from the colon, enteric bacteria entered the circulation in the two subacute cases. The acute cases received steroid therapy before the dissemination of the worms, but the two subacute cases did not. Steroids might have influenced the Strongyloides stercoralis dissemination and/or the course of the disease.

Human papillomavirus DNA in adenosquamous carcinoma of the lung.

AIM: To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lung--which is relatively common in Okinawa but not in mainland Japan--and examine its histological features. METHODS: Of 207 cases where primary lung cancers were surgically removed between January 1995 and June 1997 in Okinawa, 23 were adenosquamous carcinoma. HPV was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for E6 and E7 regions of the HPV genome. PCR products were analysed by Southern blotting. Immunohistochemical determination of high molecular weight cytokeratin (HMC) and involucrin was also carried out. RESULTS: 18 cases were positive for HPV DNA by PCR and NISH. HPV types 6, 11, 16, and 18 were found. Seven cases were dual positive for different types of HPV. Using NISH, HPV was also found in the squamous cell components and in neighbouring enlarged adenocarcinoma cells. The HMC and involucrin were demonstrated immunohistochemically in the same areas. CONCLUSIONS: HPV DNA was found in a high proportion (78.3%) of adenosquamous carcinomas in Okinawa, a region where HPV has previously been shown to be prevalent in squamous cell carcinoma of the lung. The adenocarcinoma cells adjacent to the squamous cell carcinoma component were enlarged and positive for HPV, HMC, and involucrin. This is thought to indicate the transition from adenocarcinoma to squamous cell carcinoma.

Carcinoma of the lung in Okinawa, Japan: with special reference to squamous cell carcinoma and squamous metaplasia.

In Okinawa, a subtropical island in southern Japan, squamous cell carcinoma (SCC), especially the well-differentiated form, is prevalent, while this form is relatively rare in both the mainland and other countries (e.g. United States of America). More patients with SCC from Okinawa, moreover, were positive for human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) (79%), and harbored HPV types 6, 16 and 18, in combination. On the other hand, less than 30% of the mainland patients were positive for HPV DNA by PCR. Those patients who were positive all harbored only one HPV type. Furthermore, in Okinawa, there were a significant number of cases with adenosquamous carcinoma, and they too were positive for HPV DNA. The SCC and the adenocarcinoma cells adjacent to the SCC component in these cases were also positive for HPV DNA, and such adenocarcinoma cells were enlarged in size with relatively wide cytoplasm. The authors postulate that HPV infects adenocarcinoma cells and changes them to enlarged cells, followed by squamous metaplasia. In this report, HPV DNA was transfected to adenocarcinoma cells (cultured cell lines) and this showed that HPV causes squamous metaplasia. In addition, aberrant expression of p53 was demonstrated in a large number of the SCC cases in Okinawa. The enlarged adenocarcinoma cells adjacent to the SCC components in adenosquamous carcinomas also showed aberrant expression of p53. The recent advances in the studies of anti-oncogenes, p53, etc. and oncogenes are outlined. It is to be noted that the molecular mechanisms of carcinogenesis in the lung have been studied in general, classifying lung tumors into two groups, namely, small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). However, because human lung cancer is represented by a wide variety of histologic types, molecular genetic studies according to a more detailed histological subclassification is needed.

[Pathological examination of the lung in leprosy patients].

To study pulmonary lesions in leprosy, five autopsy cases were histopathologically examined. Case 1 (BL type) died of renal failure, case 2 (LL type) of myocardial infarction, case 3 (TT type) and 4 (BL type) of cerebral hemorrhage and case 5 (BL type) of pancreas cancer. The mean age was 78 years old. Uremic lung (case 1), old tuberculous lesion (case 3) and metastatic adenocarcinoma (case 5) were demonstrated. Pulmonary congestion and mild emphysema were seen in all cases. Aspiration pneumonia was found in all but case 4. Small foci of adenomatous hyperplasia were found in cases 1 and 5. In addition, pulmonary findings of 13 outpatients with leprosy were roentgenologically examined. No patient except one with mild emphysema showed any abnormality in the lung. In our cases, no characteristic pulmonary lesion to leprosy was found.

Experimental myelitis caused by herpes simplex virus type 2 in C57BL/6N and BALB/cN mice.

Intraperitoneal and intracranial inoculation of herpes simplex virus type 2 (HSV 2) into BALB/cN and C57BL/6N mice was carried out to induce experimental myelitis. The myelitis was clearly observed in C57BL/6N mice following intraperitoneal inoculation. Within 24 hours before death, the mice showed urinary and rectal incontinence and paraplegia of the hind legs. Randomly distributed, severe necrosis was demonstrated in the spinal cord, mainly at the lower cord. In BALB/cN mice the clinical symptoms were not clearly observed, as the mice died shortly after their onset. Although spinal cord necrosis was more prominent in C57BL/6N mice than BALB/cN mice, brain necrosis was only found in the latter, and not in the former. Both strains of mouse showed marked nuclear pyknosis of the nerve cells and slight nuclear pyknosis of the astrocytes in the brain where HSV 2 antigen was demonstrated immunohistochemically. The antigen was also detected in the necrotic spinal cord. In contrast, intracranial inoculation of the virus into both strains did not cause myelitis. Spinal cord necrosis was not demonstrated and virus DNA was not detected, by PCR, in spinal cord samples. In the brain, however, the virus was demonstrated by both PCR and immunohistochemistry.

Pathologic studies and comparison of the virulence of herpes simplex virus type 2 from Okinawa, Japan and Chiang Mai, Thailand.

The virulence of four herpes simplex virus type 2 (HSV 2) strains (K1-K4) isolated in Okinawa, Japan was investigated, and compared with four strains (C1-C4) from Chiang Mai, Thailand and a standard laboratory strain SAV. Virulence was tested on BALB/c and C57/black mice. After viral inoculation intraperitoneally, the distribution with the passage of time of the virus in the brain and other organs was also studied using the polymerase chain reaction (PCR) method and immunohistochemistry. Generally, Okinawan HSV 2 (K1, K3 and K4) were less virulent than Chiang Mai strains (C1-C4). Among the Okinawan strains, K2 was the most virulent, but slightly less so than C3 and C4. All four Chiang Mai strains (C1-C4) and one Okinawan strain (K2) were more virulent than SAV strain. The virulence of K3 was very weak and no animals died from the intraperitoneal inoculation. In the brain, viral DNA from each strain was demonstrated at 1-9 days after inoculation by the PCR method. However, K3 strain was detected in the brain only between one day and 3 days after virus inoculation, and not after day 5. Immunohistochemically, the virus antigen was first demonstrated around the 3rd ventricle at one day after viral inoculation, then strongly at the ventral hypothalamus and the temporal lobe at 3 days after viral inoculation, and slightly in the frontal lobe, hippocampus, pons and cerebellum (on day 5 after inoculation). Furthermore, in Kupffer cells in the liver and macrophages in the spleen, numerous viral antigens were demonstrated from one to 9 days after viral inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Massive spinal cord necrosis associated with adult T-cell leukaemia caused by Aspergillus.

A man with adult T-cell leukaemia (ATL) underwent massive spinal cord necrosis caused by Aspergillus infection. Leukopenia due to anti-cancer chemotherapy resulted in pulmonary Aspergillus infection. The aspergilloma was reduced in size by anti-fungal chemotherapy, but paraplegia occurred. At autopsy, the pulmonary aspergilloma was encapsulated and showed no contiguous extension of the infection to the epidural or subdural spaces or spinal cord. However, at the T5 level of the spinal cord, there was marked necrosis with haemorrhage caused by Aspergillus infection, but without leukaemic cell infiltration of the spinal cord. Dichotomously branching Aspergillus hyphae filled the blood vessels of the T5 level of the spinal cord.