Characterization of liver-cirrhosis nodules by analysis of gene-expression profiles and patterns of allelic loss

To disclose genetic mechanisms involved in development or progression of hepatocellular carcinoma (HCC), we used a genome-wide cDNA microarray consisting of 8,448 genes to compare gene-expression profiles among 12 liver-cirrhosis nodules (LCNs) and five specimens of HCC excised from a single patient and carefully prepared by laser-capture microdissection (LCM). The expression patterns enabled us to identify 72 genes that were frequently upregulated and 57 that were downregulated specifically in the LCN specimens as compared to the HCCs. We also documented upregulation of 31 genes and downregulation of seven others in both HCC and LCN tissues. Several types of intracellular kinase, including receptor-type kinase, were upregulated in LCNs. Expression patterns of HCCs and LCNs generally represented two genetically distinct groups when subjected to a hierarchical clustering analysis, although expression profiles of two of the LCNs resembled the HCC pattern. Analysis of allelic losses at microsatellite loci revealed that LCNs showed frequent loss of heterozygosity (LOH) (33%) in chromosomal regions 6q and 22q; over half of the LCNs had lost an allele for at least one of the 28 loci examined. The presence of early genetic changes among LCNs, with additional genetic changes occurring during formation of HCCs, suggests that hepatocellular carcinogenesis follows the multistep model established for colon cancers and that some LCNs may be precancerous lesions.     

Kinetic analysis of pepsin digestion of chicken egg white ovomucoid and allergenic potential of pepsin fragments

BACKGROUND: The allergenic potential of chicken egg white ovomucoid (OVM) is thought to depend on its stability to heat treatment and digestion. Pepsin-digested fragments have been speculated to continue to exert an allergenic potential. OVM was digested in simulated gastric fluid (SGF) to examine the reactivity of the resulting fragments to IgE in sera from allergic patients. METHODS: OVM was digested in SGF and subjected to SDS-PAGE. The detected fragments were then subjected to N-terminal sequencing and liquid chromatography/mass spectrometry/mass spectrometry analysis to confirm the cleavage sites and partial amino acid sequences. The reactivity of the fragments to IgE antibodies in serum samples from patients allergic to egg white was then determined using Western blotting (n=24). RESULTS: The rate of OVM digestion depended on the pepsin/OVM ratio in the SGF. OVM was first cleaved near the end of the first domain, and the resulting fragments were then further digested into smaller fragments. In the Western blot analysis, 93% of the OVM-reactive sera also bound to the 23.5- to 28.5-kDa fragments, and 21% reacted with the smaller 7- and 4.5-kDa fragments. CONCLUSION: When the digestion of OVM in SGF was kinetically analyzed, 21% of the examined patients retained their IgE-binding capacity to the small 4.5-kDa fragment. Patients with a positive reaction to this small peptide fragment were thought to be unlikely to outgrow their egg white allergy. The combination of SGF-digestibility studies and human IgE-binding experiments seems to be useful for the elucidation and diagnosis of the allergenic potential of OVM.     

A perforin/granzyme-positive MDS-derived T cell line, K2-MDS, induces apoptosis in CD34+ cells through the fractalkine-CX3CR1 system

Fractalkine (CX3CL1) and its receptor CX3CR1 play an important role in natural killer (NK) cell- and cytotoxic T cell-mediated endothelium damage. Here we describe the cytotoxicity of myelodysplastic syndrome (MDS)-derived T cell line, K2-MDS, through the fractalkine-CX3CR1 system. K2-MDS cells induced apoptosis against CD34(+) cells from normal bone marrow (BM) in a direct cell contact manner. K2-MDS cells expressed perforin and granzyme B, but they lacked Fas ligand expression. A specific inhibitor for perforin, concanamycin A, blocked K2-MDS-dependent cytotoxicity. Furthermore, a CX3C-chemokine, fractalkine, was expressed in CD34(+) cells, and its receptor, CX3CR1, was expressed on K2-MDS cells. The neutralizing monoclonal antibody (MoAb) for fractalkine and soluble fractalkine significantly inhibited K2-MDS-dependent cytotoxicity. K2-MDS cells also induced the cytotoxicity against human umbilical cord endothelial cells (HUVECs) expressing fractalkine. These data indicate that K2-MDS may be a perforin-granzyme-positive T cell line that exerts a cytotoxic effect on CD34(+) cells mediated through the fractalkine-CX3CR1 system.     

Characterization of chromosome structures of Falconinae (Falconidae, Falconiformes, Aves) by chromosome painting and delineation of chromosome rearrangements during their differentiation

Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7–10 pairs of large- and medium-sized macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with chicken chromosomes 1–9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S–28S rRNA genes and telomeric (TTAGGG) _n sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed, and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently in the lineages of Accipitridae and Falconinae.     

APC and p53 mutations in de novo colorectal adenocarcinomas

To investigate genetic features in small and flat colorectal carcinomas that arise de novo, we searched for genetic alterations in six sporadic tumors by examining their APC, K-ras, and p53 genes. Two of the six tumors carried detectable mutations within the mutation cluster region (MCR) of the APC gene; both mutations were predicted to cause truncation of the gene product. Four tumors carried mutations of the p53 gene; three were missense mutations in exon 5, and the other was a 3-bp deletion in exon 6. However, neither codon 12 nor codon 13 of K-ras contained detectable mutation in any tumors. Hence, as “adenoma-carcinoma sequence” model of development of colorectal carcinoma, inactivation of the APC and p53 genes appear to be Involved in development of the de novo type of colorectal carcinoma even though the adenoma stage is not observed. © 1994 Wiley-Liss, Inc.     

A light and electron microscopic study of the mouse visceral yolk sac endodermal cells in the middle and late embryonic periods, showing the possibility of definitive erythropoiesis

Hematological studies have revealed the importance of the visceral yolk sac (VYS) in the primitive erythropoiesis of mouse embryos at an early stage before day 12. We examined the possibility of the occurrence of extra-embryonic erythropoiesis at a stage later than embryonic day 12 by light and electron microscopic analyses. Surprisingly, a novel structure in the form of erythrocyte-like globules was observed in the VYS endodermal cells. They were consistently present in the VYS endodermal cells from embryonic day 12 until day 18 (birth is day 19), by immunocytochemical and enzyme histochemical analyses. They were immuno-positive for mouse erythrocyte antibody and also positive for the benzidine reaction showing the presence of hemoglobin. The erythrocyte-like globules were shown to be the erythrocytes present in the cytoplasm. These results indicated that erythropoiesis in the VYS endodermal cells continues from the early embryonic stage, as primitive erythropoiesis, until the late stage.     

Expression and function of Fas antigen on activated murine B cells

We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up-regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti-IgM and interleukin-4 (IL-4) alone did not induce significant Fas expression but enhanced CD40L-mediated up-regulation of Fas expression. The T cell-derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas-mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas-mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas-mediated death signal was observed in 2-day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas-mediated apoptosis than were cells stimulated with CD40L plus anti-IgM or IL-4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co-stimulation of B cells in the presence of CD40L and anti-Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.     

Osteopetrotic (op/op) mice have reduced microglia, no Aβ deposition, and no changes in dopaminergic neurons

Background  

Activation of microglia is a part of the inflammatory response in neurodegenerative diseases but its role in the pathophysiology of these diseases is still unclear. The osteopetrotic (op/op) mouse lacks colony-stimulating factor-1 (CSF-1) and thus has a deficiency in microglia and macrophages. Prior reports have demonstrated that op/op mice deposit amyloid β (Aβ) plaques, similar to those found in Alzheimer's disease. The purpose of these studies was to confirm this and to determine if the lack of CSF-1 affects the development of dopaminergic neurons and the expression of CD200, a known microglial inhibitory protein.     

Case of interstitial cystitis accompanied by food allergy]

A case is a 62 years old woman who consulted her family physicion with pollakiuria and sharp pain at the time of urinary bladder distention. Since anti-H1-antagonist but not antibiotics was partially effective against the symptoms and some specific food ingestion appeared to increase the bladder pain, she was referred to our hospital. Treatment with spulatast tosilate and elimination of food products that revealed the presence of specific IgE antibodies and positive skin reactiont resulted favorable clinical response. Cystoscopic examination showed reduction in bladder capacity, mucosal injection and vascularization, besides pinpoint submucosal hemorrage and linear ulcer by hydrodistension. The diagnosis of interstitial cystitis was established by those evidences and histological findings. The patient got apparent remission after the bladder hydrodistension and is now in steady state with an addition of suplatast tosilate, a Th2 cytokine inhibitor, on the treatment mentioned above. Interstitial cystitis is a very rare disease which is characterized by cystitis-symptoms with normal urinary finding and non-effectiveness of antibiotics. The pathogenesis of the disease is unclear but considered as allergic inflammation. We report a case of interstitial cystitis accompanied by food allergy.     

Optimizing anti-CD3 affinity for effective T cell targeting against tumor cells

Bispecific antibodies binding to the TCR/CD3 complex and to a tumor-associated surface molecule can be used to target cytotoxic T lymphocytes against tumor cells. We reasoned that high-affinity binding to CD3 may reduce the efficiency of T cell stimulation and target the bispecific reagent to T cells rather than to tumor cells in vivo. We therefore mutated a bispecific single-chain antibody (BscAb) directed to human CD3 and EpCAM to generate variants that bind to CD3 with higher or lower affinity. When compared to the wild-type molecule, a mutant with increased binding to CD3 showed lower capacity to target T cells against an EpCAM+ tumour. In contrast, mutants with decreased binding to CD3, in spite of rapid dissociation, efficiently triggered T cell activation and cytotoxicity, especially when present on tumor cells at low copy number. These results are consistent with the TCR serial triggering model and suggest that BscAb with extremely low affinity for the TCR-CD3 complex could be exploited therapeutically because of their preferential localization to tumor cells.