Microdissection is essential for gene expression profiling of clinically resected cancer tissues.

The gene expression array method enables us to achieve expression profiling with thousands of genes. Clinically resected bulk cancer tissues, however, contain not only cancer cells but also stromal cells, which may affect gene expression profiling and hamper accurate analysis of the cancer cells per se. Therefore, a procedure for dissecting specific cells, such as laser capture microdissection, is neededfor the clinical application of a gene expression array. There has been no study actually comparing 2 gene expression profiles, one obtained using RNA extractedfrom cancer cells by laser capture microdissection and one obtained using RNA extractedfrom bulk cancer tissues. Wefirst demonstrated the difference in expression patterns between them, without any amplification procedures. In addition, differential expression analysis between tumor and nontumor tissue yielded quite different patterns between the 2 methods. We conclude that microdissection is essential for gene expression profiling of clinical specimens.     

Suppression of eosinophilic airway inflammation by treatment with alpha-galactosylceramide

To clarify the essential role of NKT cells in allergy, we investigated the contribution of NKT cells to the pathogenesis of eosinophilic airway inflammation using alpha-galactosylceramide (alpha-GalCer), a selective ligand for NKT cells. Although continuous administration of alpha-GalCer during ovalbumin (OVA) sensitization increased OVA-specific IgE levels and worsened eosinophil inflammation, a single administration of alpha-GalCer at the time of OVA challenge completely prevented eosinophilic infiltration in wild-type mice. This inhibitory effect of alpha-GalCer was associated with a decrease in airway hyperresponsiveness, an increase in IFN-gamma, and decreases in IL-4, IL-5 and IL-13 levels in the bronchoalveolar lavage fluids. Analysis of lung lymphocytes revealed that production of IFN-gamma increased in NK cells, but not in T or NKT cells, following alpha-GalCer administration. Induction of vascular cell adhesion molecule-1 in the lungs of wild-type mice was also significantly attenuated by treatment with alpha-GalCer. These effects of alpha-GalCer were abrogated in J alpha281-/- mice, which lack NKT cells, and in wild-type mice treated with anti-IFN-gamma Ab. Hence, our data indicate that alpha-GalCer suppresses allergen-induced eosinophilic airway inflammation, possibly by inducing a Th1 bias that results in inhibition of eosinophil adhesion to the lung vessels.     

Genotype,serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus

Hepatitis B virus (HBV) genotypes have distinct geographical distribution. HBV sequences among hepatitis B carriers in Malawi have not been evaluated thus far. HBsAg serotype and genotype of HBV was determined in 20 serum samples from Malawian chronic HBV carriers, and two complete genomes and 13 entire pre-S2/S genes were sequenced directly. Genotype A HBV isolates were found in all of the samples, and serotype with adw2 and ayw2 were detected in three and 17 samples, respectively. In phylogenetic analyses, two complete genomes were classified into a subgroup A' that was described previously in South African isolates of the virus, and were separated from HBV isolates in Western countries with nucleotide differences ranging from 4.1-6.2%. The separation of subgroup A' was also evident in the tree topology of the entire pre-S1/S2, X and precore/core region, but not evident in the small-S region. The nucleotide divergences in subgroup A' were higher than those among genotype A without subgroup A' in the complete genomes as well as each of four open reading frames. All of the 13 pre-S2/S sequences were classified into the subgroup A', and clustered with known HBV isolates with ayw2 in carriers from South Africa and Zimbabwe. Three amino acids in the pre-S2/S gene were characteristic of subgroup A' with ayw2. In conclusion, unique HBV isolates of subgroup A' with ayw2 are prevalent in Malawi, and subgroup A' with a relatively higher nucleotide diversity may be a HBV isolate characteristic of the indigenous population of some African countries.     

Identification of p46 Shc expressed in the nuclei of hepatocytes with high proliferating activity: Study of regenerating rat liver

The adaptor molecule Shc is a proto-oncogene product, and it is known to be associated with cell proliferation. However, the role of Shc in the proliferation and regeneration of hepatocytes remains unknown. In the present study, we report that p46 Shc is specifically expressed in the nuclei of proliferative (or regenerative) hepatocytes, suggesting that p46 Shc protein plays a role in hepatocellular proliferation. The expression of Shc was analyzed in liver tissue after partial hepatectomy (PH) or sham operation in Wistar rats by using immunohistochemistry and/or Western blot analysis. In addition, the expression of various cell cycle-related proteins, such as Cdk4, cyclin D1, PCNA, and Cdk1 was analyzed in the tissues of regenerating rat liver. Furthermore, the tyrosine phosphorylation of Shc was studied in liver tissue after PH or sham operation by immunoprecipitation using a monoclonal phosphotyrosine antibody. Although the protein levels of p52 Shc were unchanged in liver tissues after PH or sham operation, tyrosine phosphorylation was detected only in the regenerating rat liver after PH. The levels of p46 Shc protein were markedly increased in liver tissues during the liver regenerative process. In contrast, p66 Shc was not detected in the liver tissues after PH or sham operation. Western blotting and immunohistochemistry showed that the main location of p46 Shc was in the nuclei of proliferating hepatocytes after PH. These data suggest that p46 Shc expressed in hepatocellular nuclei may be closely related to the proliferation of hepatocytes. Therefore, it is suggested that p46 Shc expressed in hepatocellular nuclei may be a useful marker for detecting hepatocytes with high proliferative activity.     

Autosomal linkage analysis of a Japanese single multiplex schizophrenia pedigree reveals two candidate loci on chromosomes 4q and 3q.

We analyzed a large multiplex schizophrenia pedigree collected in mid-eastern Japan using 322 microsatellite markers distributed throughout the whole autosome. Under an autosomal-dominant inheritance model, the highest pairwise LOD score (LOD = 1.69) was found at 4q (D4S2431: theta = 0.0), and LOD scores at two other loci 3q (ATA34G06) and 8q (D8S1128) were 1.62 and 1.46, respectively. In multipoint analysis, LOD scores of the regions on 4q and 3q remained at a similar level; however, the LOD score of the region on 8q apparently decreased. Additional dense map analysis revealed haplotypes on 4q and 3q regions shared by affected individuals. On chromosome 4q, the haplotype spanning about 8 centiMorgans (cM) was shared by four of six genotyped individuals with schizophrenia and one affected individual whose haplotype was estimated. On 3q, the haplotype spanning about 20 cM was shared by five genotyped individuals with schizophrenia. We obtained two candidate regions of major susceptibility loci for schizophrenia on chromosomes 3q and 4q.     

Frequent occurrence of in vivo clonal expansion of CD4− CD8− T cells bearing T cell receptor αβ chains in adult humans

We have previously reported 2 cases of healthy men showing in vivo monoclonal expansion of mature CD4^? CD8^? αβ T cells. In the present study, an additional 3 adults were found to exhibit such an expansion, among a total 464 adult donors studied. These 5 individuals were otherwise physiologically normal, with no history of severe illness and autoimmune disease at the time of examination. To investigate the mechanisms of the clonal expansion, further characterization of the clonal cells was attempted. No apparent preference for usage of the Tcell receptor β chain variable region was observed in the clonal T cells. These clonal T cells showed lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities, whereas they could not lyse autologous lymphoblastoid cell lines. Failure of Fas antigen expression was not observed for any of these clones. These results suggest that clonal expansion of CD4^? CD8^? αβ T cells frequently occurs in the periphery without any T cell abnormalities.     

Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin

To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.     

Two cases of limited cutaneous nodular amyloidosis with primary Sj?gren's syndrome]

We described two female patients with primary Sj?gren's syndrome associated with localized cutaneous nodular amyloidosis (LCNA), in which amyloid protein was derived from immunoglobulin light chain. Case 1; a 70-year-old female had complained with polyarthralgia, low-grade fever and parotid gland swelling. She was diagnosed as primary Sj?gren's syndrome. Three years later she noticed brown color small tumor on the thigh and yellow to brown nodules on the bilateral calves of legs. Skin biopsy from the left thigh revealed amyloid L protein deposition, which was positive for anti-lambda light chain staining, in almost entire dermis. Infiltration of lymphocytes and plasma cells around the amyloid deposit were prominent. Case 2; a 51-year-old female had noticed increasing eruption on the hip. Skin biopsy revealed amyloid L protein deposition in the dermis, which was negative for anti-lambda nor kappa light chain staining. When she was refereed to our hospital, she complained of xerostomia and xerophthalmia. She was diagnosed as primary Sj?gren's syndrome. In both cases, histological examination of a minor salivary gland biopsy revealed infiltration of lymphocytes and plasma cells but not amyloid deposit. Serum M protein and urine Bence-Jones protein were not detected. These cases represent localized amyloidosis without systemic involvement. It is widely recognized that Sj?gren's syndrome is frequently accompanied by B cell lymphoproliferative disorders. In LCNA, infiltration of plasma cells around the amyloid deposits was frequently prominent. The relation between these two disorders is discussed.