MERKEL CELL TUMOR COEXPRESSING CYTOKERATIN AND NEUROFILAMENT PROTEINS

Whorled filaments 10 nm in width were identified by anti-intermediate filaments antibodies in a Merkel cell tumor from a 52-year-old man. Immunohistochemlcal tests revealed that the tumor was stained with anti-keratin antibody and antibodies against the 68-kd and 200-kd subunits of neurofilament proteins but not antibody against the 150-kd subunlt. This is the first reported case of Merkel cell tumor expressing a 200-kd subunit of neurofilament proteins.     

Interaction of poly(styrene sulfonic acid) with the alternative pathway of the serum complement system

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.     

Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication

The Rad17-replication factor C (Rad17-RFC) and Rad9-Rad1-Hus1 complexes are thought to function in the early phase of cell-cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17-/- and Rad9-/- DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17-/- and Rad9-/- but not ATM-/- cells are defective in S-phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.     

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.     

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

A der(13)t(7;13)(p13;q14) with monoallelic loss of RB1 and D13S319 in myelodysplastic syndrome

Deletions or translocations of chromosome band 13q14, the locus of the retinoblastoma gene (RB1), have been observed in a variety of hematological malignancies including myelodysplastic syndrome (MDS). We describe here a novel unbalanced translocation der(13)t(7;13)(p13;q14) involving 13q14 in a patient with MDS. A 66-year-old woman was diagnosed as having MDS, refractory anemia with excess of blasts (RAEB-1) because of 7.4% blasts and trilineage dysplasia in the bone marrow cells. G-banding and spectral karyotyping analyses showed complex karyotypes as follows: 46,XX,der(6)t(6;7)(q11;?),der(7)del(7)(?p13)t(6;7)(q?;q11)t(6;13)(q?;q?),der(13)t(7;13)(p13;q14). Fluorescence in situ hybridization (FISH) analyses demonstrated that one allele of the RB1 gene and the microsatellite locus D13S319, located at 13q14 and telomeric to the RB1 gene, was deleted. Considering other reported cases, our results indicate that submicroscopic deletions accompanying 13q14 translocations are recurrent cytogenetic aberrations in MDS. The RB1 gene or another tumor suppressor gene in the vicinity of D13S319, or both, may be involved in the pathogenesis of MDS with 13q14 translocations by monoallelic deletion.     

Adult Still's disease reflects a Th2 rather than a Th1 cytokine profile

Adult Still's disease (ASD) is a chronic multisystemic disease. Extraordinarily high serum levels of IL-18 in ASD patients have been described, whereas the mechanism remains to be clarified. This study aimed to evaluate proinflammatory cytokines and to consider their pathological roles. In patients with rheumatic diseases (n = 151), blood samples were taken at the active phase and the serum levels of IL-18 and other proinflammatory cytokines were measured by ELISA. The extra-high levels of IL-18 were confirmed selectively in ASD patients (n = 10). In the active phase of ASD patients, the levels of IL-6 were elevated accordingly, but IL-1beta and TNF-alpha were undetectable. As to Th1-Th2 cytokines, the levels of IL-4 and IL-13, but not INF-gamma, IL-12, or IL-2, were elevated in all ASD patients examined. Moreover, the serum levels of IL-18 showed a good correlation with those of IL-4, suggesting that ASD reflects a Th2 rather than a Th1 cytokine profile.