Addiction to the IGF2-ID1-IGF2 circuit for maintenance of the breast cancer stem-like cells.

The transcription factor nuclear factor-κB (NF-κB) has important roles for tumorigenesis, but how it regulates cancer stem cells (CSCs) remains largely unclear. We identified insulin-like growth factor 2 (IGF2) is a key target of NF-κB activated by HER2/HER3 signaling to form tumor spheres in breast cancer cells. The IGF2 receptor, IGF1 R, was expressed at high levels in CSC-enriched populations in primary breast cancer cells. Moreover, IGF2-PI3K (IGF2-phosphatidyl inositol 3 kinase) signaling induced expression of a stemness transcription factor, inhibitor of DNA-binding 1 (ID1), and IGF2 itself. ID1 knockdown greatly reduced IGF2 expression, and tumor sphere formation. Finally, treatment with anti-IGF1/2 antibodies blocked tumorigenesis derived from the IGF1Rhigh CSC-enriched population in a patient-derived xenograft model. Thus, NF-κB may trigger IGF2-ID1-IGF2-positive feedback circuits that allow cancer stem-like cells to appear. Then, they may become addicted to the circuits. As the circuits are the Achilles' heels of CSCs, it will be critical to break them for eradication of CSCs.

Autosomal recessive cystinuria caused by genome-wide paternal uniparental isodisomy in a patient with Beckwith-Wiedemann syndrome.

Approximately 20% of Beckwith-Wiedemann syndrome (BWS) cases are caused by mosaic paternal uniparental disomy of chromosome 11 (pUPD11). Although pUPD11 is usually limited to the short arm of chromosome 11, a small minority of BWS cases show genome-wide mosaic pUPD (GWpUPD). These patients show variable clinical features depending on mosaic ratio, imprinting status of other chromosomes, and paternally inherited recessive mutations. To date, there have been no reports of a mosaic GWpUPD patient with an autosomal recessive disease caused by a paternally inherited recessive mutation. Here, we describe a patient concurrently showing the clinical features of BWS and autosomal recessive cystinuria. Genetic analyses revealed that the patient has mosaic GWpUPD and an inherited paternal homozygous mutation in SLC7A9. This is the first report indicating that a paternally inherited recessive mutation can cause an autosomal recessive disease in cases of GWpUPD mosaicism. Investigation into recessive mutations and the dysregulation of imprinting domains is critical in understanding precise clinical conditions of patients with mosaic GWpUPD.

Agonistic anti-human Fas monoclonal antibody induces fibroblast-like synoviocyte apoptosis in haemophilic arthropathy: potential therapeutic implications.

Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti-Fas monoclonal antibody (mAb) was found to induce RA fibroblast-like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti-Fas mAb on HA-FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti-Fas mAb at different concentrations, alone or in combination with tumour necrosis factor-α (TNF-α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST-1 assay. Active caspase-3 levels were measured using ELISA and Western blot. A strong Fas-immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA-FLS. Anti-Fas mAb had a significant cytotoxicity on HA-FLS in a dose-dependent manner, either alone or in combination with TNF-α and bFGF. These cytotoxic effects were due to the ability of anti-Fas to induce HA-FLS apoptosis, as shown by the increased active caspase-3 levels. Anti-Fas mAb exhibited a more pronounced pro-apoptotic effect on HA-FLS than RA-FLS. Fas antigen is highly expressed on HA-FLS and its stimulation by anti-Fas mAb may be an effective strategy to induce HA-FLS apoptosis.

Do biologics-naïve patients with rheumatoid arthritis respond better to tocilizumab than patients for whom anti-TNF agents have failed? A retrospective study.

OBJECTIVES: To determine responses to tocilizumab between patients with rheumatoid arthritis (RA) who switched to anti-TNF agents and those who are biologics-naïve. METHODS: This retrospective study investigated 107 patients with RA who were treated with tocilizumab. At baseline, 61 of them had already been treated with anti-TNF agents (switched group; 46 for inefficacy and 15 for adverse events), and 46 were biologics-naïve (naïve group). Treatment responses to tocilizumab at week 12 and 24 were compared between the switched and naïve groups using the disease activity score 28 (DAS28). RESULTS: Forty-two (91.3%) and 50 (82.0%) patients in the naïve and switched groups, respectively, completed 24 weeks of tocilizumab treatment. The DAS28-ESR and DAS28-CRP values (means±SD) at weeks 12 and 24 compared to baseline decreased significantly for the naïve and switched groups. The DAS28-ESR and DAS28-CRP values at weeks 12 and 24 were significantly decreased in the naïve group, compared to the switched group. Disease activity was improved in the naïve patients compared to the switched patients. CONCLUSIONS: Tocilizumab was safe, tolerable, and clinically effective for patients with inadequate responses to anti-TNF therapy and for those who were biologics-naïve, and it was more effective among the latter.

A margin model to account for respiration-induced tumour motion and its variability.

In order to reduce the sensitivity of radiotherapy treatments to organ motion, compensation methods are being investigated such as gating of treatment delivery, tracking of tumour position, 4D scanning and planning of the treatment, etc. An outstanding problem that would occur with all these methods is the assumption that breathing motion is reproducible throughout the planning and delivery process of treatment. This is obviously not a realistic assumption and is one that will introduce errors. A dynamic internal margin model (DIM) is presented that is designed to follow the tumour trajectory and account for the variability in respiratory motion. The model statistically describes the variation of the breathing cycle over time, i.e. the uncertainty in motion amplitude and phase reproducibility, in a polar coordinate system from which margins can be derived. This allows accounting for an additional gating window parameter for gated treatment delivery as well as minimizing the area of normal tissue irradiated. The model was illustrated with abdominal motion for a patient with liver cancer and tested with internal 3D lung tumour trajectories. The results confirm that the respiratory phases around exhale are most reproducible and have the smallest variation in motion amplitude and phase (approximately 2 mm). More importantly, the margin area covering normal tissue is significantly reduced by using trajectory-specific margins (as opposed to conventional margins) as the angular component is by far the largest contributor to the margin area. The statistical approach to margin calculation, in addition, offers the possibility for advanced online verification and updating of breathing variation as more data become available.

Surgical resection of solitary adrenal metastasis from esophageal carcinoma following esophagectomy.

The prognosis of esophageal carcinoma following esophagectomy is poor due to a high frequency of metastasis to periesophageal lymph nodes and distant organs. However, we experienced a case with good prognosis following resection of a solitary adrenal metastatic tumor. The patient was a 70-year-old man diagnosed with type 2 esophageal cancer (Lt-Ae, T2N1M0, Stage IIB) who was treated with esophagectomy. Eight months following surgery, solitary adrenal metastasis was detected by CT, and was resected. At 42 months follow-up he has had a good quality of life in the community without evidence of recurrence. To the best of our knowledge, only five cases with resected solitary adrenal metastases including our case, have been reported, and show a greater than 1-year survival. Consequently, we suggest that resection of solitary organ metastases is a good alternative, even following esophagectomy.

Tryptase enhances release of vascular endothelial growth factor from human osteoarthritic chondrocytes.

OBJECTIVE: A contribution of mast cells and its mediators in the pathogenesis of arthritis has been postulated. We aimed to clarify the role of mast cell-derived serine protease tryptase and proteinase activated receptor (PAR)-2-mediated signaling in chondrocytes. METHODS: Human articular cartilage specimens were obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA) and with traumatic fracture without arthritis (PT; as controls) who underwent joint surgery. Isolated chondrocytes were cultured in vitro by monolayer, and confluent cells were incubated with recombinant human lung Beta tryptase or with a PAR-2 agonist peptide. The secreted level of vascular endothelial growth factor (VEGF) in culture supernatant was measured using commercially available ELISA kits, and expression of VEGF mRNA was analyzed using real-time PCR. RESULTS: The tryptase-stimulated chondrocytes from OA or RA, but not from PT patients, produced significantly higher amount of VEGF in their supernatants. The response was blocked by a G-protein receptor inhibitor pertussis toxin, however, was not reproduced by incubation of cells with the PAR-2 agonist, suggesting a presence of non-PAR-2 dependent signals for the VEGF induction. In addition, actinomycin D and cycloheximide did not exert significant inhibition, indicating a regulation of VEGF release by tryptase. CONCLUSION: The inflammatory mediator, mast cell-derived protease tryptase may modulate chondrocyte metabolism through induction of VEGF release.

Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1beta, TNF-alpha and TGF-beta.

OBJECTIVE: To investigate the modulation of expression of proteinase-activated receptor-2 (PAR-2) in articular chondrocytes by inflammatory cytokines. DESIGN: Articular synovium and cartilage tissues were collected from eight patients with osteoarthritis (OA), and three patients without arthropathy ("normal"). Chondrocytes were stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta1. The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (PCR), Western blotting and immunofluorescence. Quantitative PCR was performed to assess the expression levels of PAR-2 messenger RNA (mRNA). RESULTS: The expression of PAR-2 mRNA was demonstrated in both OA and normal chondrocytes as well as in synovial fibroblasts. However, the level of PAR-2 in OA chondrocytes was much higher than in normal chondrocytes. Long-term culture revealed that PAR-2 mRNA expression was maintained up to three passages in OA but not in normal chondrocytes. IL-1beta and TNF-alpha both upregulated PAR-2 expression in normal and OA chondrocytes. In contrast, TGF-beta1 significantly decreased expression of PAR-2 in OA chondrocytes but increased PAR-2 in normal chondrocytes. CONCLUSIONS: Overexpression of PAR-2 in OA chondrocytes is upregulated by proinflammatory cytokines IL-1beta and TNF-alpha, and down-regulated by regulatory cytokine TGF-beta1. PAR-2 may be involved in the pathogenesis of OA.

The role of subchondral bone resorption pits in osteoarthritis: MMP production by cells derived from bone marrow.

OBJECTIVE: The vascular invasion of bone marrow tissue into the subchondral plate is often observed in articular cartilage and we named it the subchondral bone absorption pit; however, its implication in the pathogenesis of osteoarthritis (OA) has been poorly understood. The purpose of this study was to evaluate its characteristics and roles in osteoarthritic conditions. METHODS: Articular cartilage specimens from 11 patients with medial type knee OA and 7 non-arthritic cadavers were analyzed with HE staining. OA sections were stained with safranin-O, TRAP (tartrate resistant acid phosphatase) and immunostained with anti-MMP-1, MMP-3, MMP-13, vitronectin receptor (VNR)-alpha chain, vimentin and bone morphogenic protein (BMP) 2/4 antibodies. RESULTS: Subchondral bone resorption pits were classified according to the extent of invasion: pits with bone marrow tissue were located within uncalcified cartilage below the tidemark in grade I and invaded beyond the tidemark in grade II, while no invasion was seen in grade 0. Grade II pits were dominant in OA compared to non-arthritic joints, especially medial condyles. Proteoglycan detected with safranin-O staining was lost around the tip of grade II pits and the density of pits was related to the modified Mankin Score. Cells in pits expressed vimentin, MMP-1, MMP-3 and MMP-13. Some polynuclear cells co-expressed VNR-alpha chain and MMP-13, whereas pits showed reparative features expressing BMP. CONCLUSION: These results suggest that subchondral bone resorption pits contribute to cartilage degradation by expressing matrix metalloproteinases in OA.

Implication of interleukin 18 in production of matrix metalloproteinases in articular chondrocytes in arthritis: direct effect on chondrocytes may not be pivotal.

OBJECTIVE: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes. METHODS: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Ralpha, IL18Rbeta, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Ralpha were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein levels of signalling molecules were assayed by western blotting. RESULTS: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1beta stimulation. Flow cytometric analysis showed that IL1beta, tumour necrosis factor alpha, and IL18 up regulated IL18Ralpha expression levels. The level of IL18Rbeta mRNA was much lower than that of IL18Ralpha, and was slightly up regulated by IL1beta. In chondrocytes responding to IL18, IL18 (1-100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-kappaB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation. CONCLUSION: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.

Plasminogen activator inhibitor-1 is elevated, but not essential, in the development of bleomycin-induced murine scleroderma.

Accumulative data have demonstrated that plasminogen activator inhibitor-1 (PAI-1) plays an important role in the extracellular matrix metabolism; however, the involvement of PAI-1 in scleroderma has not been fully elucidated. In this study, we investigated the role of PAI-1 in bleomycin-induced murine scleroderma. 100 microg of bleomycin was injected subcutaneously to the back skin of C3H/HeJ mice on alternate day for 4 weeks. Histopathological findings revealed that PAI-1 was positive in macrophage-like cells and fibroblastic cells in the dermis, in parallel with the induction of dermal sclerosis. PAI-1 mRNA expression in the whole skin was up-regulated at 1 and 4 weeks. The production of active PAI-1 protein in the lesional skin was significantly increased 3 and 4 weeks after bleomycin treatment. Next, we examined whether dermal sclerosis is induced by bleomycin in PAI-1-deficient (PAI-1-/-) mice. 10 microg of bleomycin was subcutaneously injected to PAI-1-/- and wild type (WT) mice 5 days per week for 4 weeks. Histological examination revealed that dermal sclerosis was similarly induced even in PAI-1-/- as well as WT mice. Dermal thickness and collagen contents in the skin were significantly increased by bleomycin injection in both PAI-1-/- and WT mice, and the rate of increase was similar. These data suggest that PAI-1 plays an important role, possibly via TGF-beta pathway activation. However, the fact that PAI-1 deficiency did not ameliorate skin sclerosis suggest that PAI-1 is not the essential factor in the development of bleomycin-induced scleroderma, and more complex biochemical effects other than PA/plasmin system are greatly suspected.

Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1 beta and tumour necrosis factor alpha.

OBJECTIVE: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1 beta and TNF alpha. METHODS: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 microg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-kappa B ligand (RANKL), osteoprotegerin (OPG), IFN gamma, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay. RESULTS: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1 beta, or TNFalpha increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1 beta, and TNF alpha did not induce M-CSF, GM-CSF, IFN gamma, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1 beta, but less potent than that of TNF alpha. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1 beta, and greatly inhibited induction by TNF alpha. CONCLUSION: IL18, IL1 beta, or TNF alpha can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1 beta, but less potent than TNF alpha.

Telomerized presenescent osteoblasts prevent bone mass loss in vivo.

Previously, we showed that human osteoblasts expressing the human telomerase reverse transcriptase (hTERT) gene exhibited specific survival advantages--the result of breaching the replicative senescence barrier and maintaining the phenotypic and functional properties of primary osteoblasts in vitro over the total replicative capacity of primary osteoblasts. We postulated that rejuvenated osteoblasts may have a potential to correct bone loss or osteopenia in age-related osteoporotic diseases. In the present study, we studied whether telomerized presenescent osteoblasts prevent bone mass loss in vivo. After obtaining the informed consent from a patient with osteoarthritis who underwent the arthroplastic knee surgery, osteoblastic cells were isolated from donor bone sample. We transfected the gene encoding hTERT into human osteoblastic cells. Human bone fragments from a donor were incubated with human hTERT-transfected presenescent (in vitro aged) osteoblasts or mock-transfected presenescent osteoblasts in culture medium containing Matrigel. We subcutaneously implanted human bone fragments with telomerized presenescent osteoblasts or primary presenescent osteoblasts as three-dimensional Matrigel xenografts in severe combined immunodeficiency (SCID) mice (each group: six mice) and analyzed the grafts at 6 weeks after implantation. We also determined whether telomerized osteoblasts affect the bone-forming capacity in vivo, using a well-established mouse transplantation model in which ceramic hydroxyapatite/tricalcium phosphate particles are used as carrier vehicle. Telomerized presenescent osteoblasts were rejuvenated, and maintained the functional properties of young osteoblasts in vitro. Bone mineral content (BMC) and bone mineral density (BMD) were measured by ash weight and dual-energy X-ray absorptiometry, respectively. Whereas BMC and BMD of human bone fragments, which were inoculated with aged osteoblasts in SCID mice, decreased with time, telomerized presenescent osteoblasts maintained the BMC and BMD of human bone fragments, indicating that telomerized and rejuvenated osteoblasts may be functional to prevent bone mass loss in vivo. In xenogenic transplants, telomerized osteoblasts generated more bone tissue with lamellar bone structure and cellular components, than did control osteoblasts. These findings suggest that telomerized/rejuvenated presenescent osteoblasts may be used in the development of tissue engineering or cell-based therapy for bone regeneration and repair.

Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts.

OBJECTIVE: Fas-mediated apoptosis is preferentially observed in synoviocytes of patients with rheumatoid arthritis (RA) and is associated with the pathophysiological process of RA. To clarify the molecular mechanisms of Fas-mediated apoptosis of RA synoviocytes, we investigated the role of the mitochondrial pathway and tumour suppressor p53 in this process. METHODS: Cultured synovial fibroblasts were prepared from RA patients. After treatment of RA synovial fibroblasts with anti-Fas monoclonal antibody, the expression levels of activated caspase-9 and -3, Bid cleavage, cytochrome c release and phosphorylation of p53 at Ser15 were assessed using immunoblot analysis. The mitochondrial membrane potential (DeltaPsim) was evaluated with a fluorescence-based detection assay. Apoptotic cells were determined by a DNA fragmentation assay in the presence or absence of caspase inhibitors. Expression of p53-regulated apoptosis-inducing protein 1 (p53AIP1) was measured by real-time PCR. RA synovial fibroblasts stably transfected with a dominant-negative (DN) p53 were prepared in order to investigate the role of p53 during Fas-induced apoptosis. RESULTS: Fas ligation induced Bid cleavage, loss of DeltaPsim, cytochrome c release to the cytosol and activation of caspase-9 and -3 in RA synovial fibroblasts. Treatment with a caspase-9-specific inhibitor almost completely inhibited Fas-mediated apoptosis. Moreover, p53 activation after Fas ligation was evidenced by its phosphorylation at Ser15 and up-regulation of the p53 target gene p53AIP1. Fas-mediated apoptosis was significantly suppressed by anti-sense p53 oligonucleotides and by p53DN. CONCLUSION: Our findings strongly suggest the involvement of mitochondria and p53 in Fas-mediated apoptosis of RA synovial fibroblasts.

Measurement of Dermatophagoides mite allergens on bedding and human skin surfaces.

BACKGROUND: The level of the Dermatophagoides mite group 1 (Der 1) allergens in reservoir dust has been used as an index of exposure in most studies. However, the mite allergen level in reservoir dust cannot directly reflect the personal exposure level. OBJECTIVE: We sought to develop a new method for quantifying the Der 1 allergens on bedding and human skin surfaces as an index of exposure to mite allergens. METHODS: Samples were obtained with a small adhesive tape from the forearm skin of 30 healthy volunteers and from their regularly used mattresses. The level of Der 1 allergens collected onto the adhesive tape was measured by a newly developed sensitive fluorometric ELISA for Der p 1 and Der f 1. RESULTS: The Der 1 allergens could be detected in all the samples from bedding surfaces and in 28 of the 30 samples from skin surfaces. The Der 1 levels by adhesive tape sampling from the mattresses correlated with those by reservoir dust sampling. The sampling of the skin and bedding surface with adhesive tape correlated, but skin sampling did not correlate with reservoir sampling. CONCLUSION: The Der 1 allergens on bedding surfaces and on human skin surfaces could be quantified with a very simple sampling technique. The system developed in this study will provide a new tool for the assessment of mite allergen exposure in daily life.

Enhanced expression of p16 in seborrhoeic keratosis; a lesion of accumulated senescent epidermal cells in G1 arrest.

BACKGROUND: Seborrhoeic keratosis (SK) is a common skin disease associated with skin ageing and photoageing, but only limited studies have been performed on SK and the senescence of keratinocytes. OBJECTIVES: We sought to clarify the genetic basis of SK and the senescence of keratinocytes. METHODS: Expression of p16, cyclins A, D and E, p21, p53, retinoblastoma (Rb) gene product and telomerase-associated protein 1 (TP1) in SK was examined by immunohistochemistry. DNA fragmentation in SK was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method. We cultured keratinocytes from SK lesions and non-lesional epidermis and examined expression of p16, observed morphology of the cultured cells by light and electron microscopy and measured survival time. RESULTS: p16, a cyclin-dependent kinase inhibitor, was expressed in all cells from SK lesions, whereas normal keratinocytes expressed p16 only in the granular cells. Other factors such as cyclins A, D and E, p21, p53, Rb gene product, and TP1, were not expressed in SK cells. These results suggest that p16 expression is a marker of SK and that p16 has a role in the pathogenesis of SK. DNA fragmentation was not detected in four of five SK tissue samples; one of the SK tissue samples showed DNA fragmentation only in the superficial upper layer of an SK lesion, suggesting that apoptosis was inhibited in SK cells. In contrast, normal epidermis showed DNA fragmentation in the granular and squamous layers. Immunohistochemical examination of cultured SK cells also revealed the presence of p16. A greater number of SK cells survived after 3 weeks of culture in comparison with normal keratinocytes. Features of senescence, such as a balloon-like appearance after lengthy culture and increased amounts of tonofilaments in cytoplasm, were observed in SK cells in culture. CONCLUSIONS: These results suggest that SK is a benign neoplasm where keratinocytes in a senescent condition and G1 arrest are accumulated.

Superiority of minipool nucleic acid amplification technology for hepatitis B virus over chemiluminescence immunoassay for hepatitis B surface antigen screening.

BACKGROUND AND OBJECTIVES: The Japanese Red Cross (JRC) have developed a fully automated multiplex (MPX) nucleic acid amplification technology (NAT) system for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1). This is used to test serologically negative blood units from volunteer, non-remunerated donors. The system utilizes a 50-sample pool for NAT screening with an input volume of each pool. This results in a significantly higher sensitivity for hepatitis B than that seen with highly sensitive hepatitis B surface antigen (HBsAg) testing. MATERIALS AND METHODS: From 1 February 2000 to 15 October 2001, over 11 million donations, which were serologically negative, were tested using the MPX NAT system. Donations found to be HBV DNA positive were further tested by using the chemiluminescence immunoassay (CLIA). RESULTS: Out of 181 HBV DNA-positive donations, 96 (53%) and 76 (42%) were negative by individual enzyme immunoassay (EIA) and CLIA testing, respectively. CONCLUSIONS: The sensitivity of the 50-sample pool MPX NAT system was higher than that of individual HBsAg screening by CLIA. By adopting this NAT-screening system, the JRC has improved the safety of the blood supply and maintained supply across Japan.

Alteration of the expression of Bcl-2, Bcl-x, Bax, Fas, and Fas ligand in the involved skin of psoriasis vulgaris following topical anthralin therapy.

Anthralin (dithranol) is frequently used for the treatment of psoriasis. However, the mode of action of anthralin has not been completely elucidated as yet. Recent findings suggest that psoriatic keratinocytes are resistant to the apoptotic process. In this study, we examined the immunohistochemical expression of apoptosis-regulated protein in the involved psoriatic skin following topical anthralin therapy. Biopsy specimens were obtained from back skins treated with topical anthralin or white petrolatum (control) in 4 patients with psoriasis vulgaris. Immunohistochemical examination revealed that psoriatic keratinocytes expressed high levels of Bcl-x, which was significantly reduced after anthralin treatment. Bax was not detected in the epidermal keratinocytes in the petrolatum-treated skin, while it was present in the upper keratinocytes after anthralin therapy. Bcl-2 was detected only in basal layers of psoriatic epidermis following both petrolatum and anthralin application. Psoriatic keratinocytes expressed higher levels of Fas in the lower epidermis, while only weak expression was detected in anthralin-treated plaques. On the other hand, hyperproliferative keratinocytes strongly expressed Fas ligand (FasL) on their plasma membranes as well as infiltrating lymphocytes in the upper dermis. Furthermore, anthralin-treated psoriatic epidermis did not express FasL. In normal skin, keratinocytes expressed low to absent levels of Bcl-x and Bax, while Bcl-2 was detected only in melanocytes in basal layers. Neither Fas nor FasL were detected in the epidermis of normal skin. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed positive labeling on the majority of psoriatic keratinocytes through the epidermis in petrolatum-treated skin, whereas anthralin treatment markedly reduced TUNEL-positive keratinocytes. These in vivo results may reflect improvement of the psoriatic skin following effective anthralin therapy.