Adriamycin-Lipiodol suspension for i. a. chemotherapy of hepatocellular carcinoma

Summary Physicochemical properties of two types of adriamycin preparation, suspensions and emulsions prepared for i.a. chemotherapy of hepatocellular cacinoma, were investigated. A suspension was prepared by dispersing adriamycin directly into the lipid contrast medium, Lipiodol, whereas an emulsion was obtained by emulsifying an aqueous solution of adriamycin into Lipiodol. The dispersibility of the drug in each preparation was examined microscopically. The chemical stability of and drug release from the preparation were determined by high-performance liquid chromatography and spectrophotometry, respectively. The suspension was then given to ten patients with primary hepatocellular carcinoma. The suspension maintained good dispersibility without coagulation of drug particles, whereas coalescence of aqueous droplets and the resultant phase separation occurred 4 h after preparation of the emulsion. Both preparations maintained the initial drug content for at least 1 week at room temperature. The release of adriamycin was more prolonged in the suspension than in the emulsion. After i.a. administration of the suspension, a selective accumulation of Lipiodol in the tumor and decrease in serum -fetoprotein (AFP) levels were found in most patients. A significant amount of adriamycin was still detected in hepatic speciments resected from two patients 1 and 2 months after treatment. These findings suggest that the adriamycin-Lipiodol suspension may be a useful preparation for targeting chemotherapy to hepatocellular carcinoma.     

Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.     

Disease associations and altered immune function in CD45 138G variant carriers

The CD45 antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes. Expression of different patterns of alternatively spliced CD45 isoforms is associated with distinct functions. We recently identified a polymorphism in exon 6 (A138G) of the gene encoding CD45 (PTPRC) that results in altered CD45 splicing. The 138G allele is present at a high frequency among Japanese (23.7%), with 5.1% individuals homozygous for the G allele. In this study we show that the A138G polymorphism is the cause of altered CD45 isoform expression, promoting splicing towards low molecular weight CD45 isoforms. We further report that the frequency of A138G heterozygotes is significantly reduced in number in cohorts of patients with autoimmune Graves' disease or hepatitis B infection, whereas G138G homozygotes are absent from a cohort of Hashimoto's thyroiditis patients. We also show that 138G individuals exhibit altered cytokine production in vitro and an increased proportion of memory T cells. These data suggest that the 138G variant allele strongly influences these diseases by modulation of immune mechanisms and may have achieved its high frequency as a result of a natural selection probably related to pathogen resistance.     

Patterns of C-heterochromatin and telomeric DNA in two representative groups of small apes,the genera <Emphasis Type="Italic">Hylobates</Emphasis> and <Emphasis Type="Italic">Symphalangus</Emphasis>

The course of chromosome evolution in small apes is still not clear, though painting analyses have opened the way for elucidating the puzzle. Even the C-banding pattern of the lar-group of gibbons (the genus Hylobates) is not clarified yet, although our previous studies suggested that lar-group gibbons have a unique C-banding pattern. We therefore made observations to establish C-banded karyotypes of the agile gibbons included in the lar-group. The data were compared with those of siamangs (the genus Symphalangus), which carry distinctive C-bands, to determine the chromosomal patterns in each group. C-banded chromosomes of agile gibbons showed several terminal, interstitial and paracentric bands, whose patterns are specific for each chromosome, whereas the C-bands of siamangs were located only at the terminal and centromeric regions in most chromosomes. Moreover, the C-bands of agile gibbons and siamangs were shown to be G+C-rich and A+T-rich DNA, respectively, by DAPI/C-band sequential staining. Additionally, PRINS labelling with a telomere primer revealed that agile gibbons have telomeric DNA only at chromosome ends where there is no C-band (non-telomeric heterochromatin), whereas the telomeric DNA of siamangs is located in the terminal C-banded regions (telomeric heterochromatin). Although the evolutionary mechanisms in small apes are still unknown, C-banding patterns and distribution of telomeric DNA sequences should provide valuable data to deduce the evolutionary pathways of small apes.     

Metal ions induce bone-resorbing cytokine production through the redox pathway in synoviocytes and bone marrow macrophages

To evaluate the biological reactions to metal ions potentially released from prosthetic implants, we examined the ability of metal ions to produce bone-resorbing cytokines and the underlying mechanism using synoviocytes and bone marrow (BM) macrophages. The cells were incubated with NiCl(2), CoCl(2), CrCl(3) or Fe(2)(SO(4))(3) at optimal concentrations, which are detectable in joint fluid following total joint arthroplasty. The production of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha were enhanced by all metal ions tested as determined by enzyme-linked immunosorbent assay. From the results of electrophoresis mobility shift assay, all metal ions enhanced the DNA-binding activity of nuclear factor kappaB (NF-kappaB), and p50-p65 heterodimers and p50 homodimers were the major subunits. These effects of the metal ions were considerably blocked by pyrrolidine dithiocarbamate (PDTC) known as a radical scavenger. An electron spin resonance study clearly demonstrated the ability of metal ions to generate activated oxygen species (AOS), especially hydroxyl radicals (*OH), which accounts for PDTC-blockade of metal ion-induced NF-kappaB activation and subsequent cytokine production. Taken together, our data raised the possibility that small amounts of metal ions released from prosthetic implants activate synoviocytes and BM macrophages through the AOS-mediated process (i.e. the redox pathway), and contribute to the initiation of osteolysis at the bone-implant interface.     

Effect of inhaled corticosteroid on an immunoreactive thymus and activation-regulated chemokine expression in the bronchial biopsies from asthmatics

BACKGROUND: Bronchial asthma is characterized by airway inflammation, notably because of eosinophils and T cells. Thymus and activation-regulated chemokine (TARC) is known to selectively attract Th2 cells, and is increased in response to interleukin (IL)-4 and IL-13, which share a common receptor, IL-4 receptor alpha (IL-4Ralpha). While corticosteroids have proven, very effective in modifying airway inflammation, the effect of corticosteroids on TARC in asthmatics has been little studied. OBJECTIVE: We examined the effects of inhaled budesonide (BUD) on the expression of TARC and the number of inflammatory cells in bronchial biopsy specimens taken from asthma patients. METHODS: Inhaled BUD 800 mug daily, or placebo was administered for 3 months in a double-blind, parallel-group study, and bronchial biopsies were performed before and after treatment. Biopsy specimens were examined by immunocytochemistry. RESULTS: We observed a significant decrease in the epithelial expression of TARC (P < 0.01) in the BUD group compared with the placebo group. This was accompanied by decreases in the number of eosinophils (P < 0.01), CD3(+) T cells (P < 0.05), and CD4(+) T cells (P < 0.01). A significant correlation was found between changes in epithelial TARC and in IL-4Ralpha immunoreactivity (r(s) = 0.66, P < 0.01). CONCLUSIONS: These findings suggest that corticosteroid asthma treatment can reduce infiltration of the airway by inflammatory cells, an effect modulated by down-regulation of bronchial epithelial TARC expression.     

Antiphospholipid antibodies

The concept of antiphospholipid syndrome(APS) has been widely accepted. Antiphospholipid antibodies originally included anticardiolipin antibodies and lupus anticoagulants as serological marker of APS. However, recent advances have shown that most pathogenic antiphospholipid antibodies are directed to phospholipid binding proteins such as beta 2-glycoprotein I and prothrombin as well as phospholipids. The preliminary classification criteria for definite APS have been advocated as the "Sapporo criteria". Further prospective investigations are required to re-evaluate the clinical significance of so-called antiphospholipid antibodies.     

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.     

Role of p27Kip1 and Cyclin-Dependent Kinase 2 in the Proliferation of Non-Small Cell Lung Cancer

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27^Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.