High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.

BACKGROUND: The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment. RESULTS: We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration. CONCLUSIONS: Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.

Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells.

OBJECTIVE: In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells. MATERIALS AND METHODS: Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC). RESULTS: In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells. CONCLUSION: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.

Family history of colorectal adenomatous polyps as a risk factor for colorectal cancer.

The aim of this study was to evaluate the risk of common colorectal cancer among first-degree relatives of patients with colorectal adenomatous polyps. In a population screening programme, 59406 subjects underwent an immunochemical faecal occult blood test. In a medical check-up-based cross-sectional study, 6139 subjects had a colonoscopic examination. They were divided into two groups, according to the results of a questionnaire on family history of colorectal adenomatous polyps, and the detection rates for colorectal cancer were compared in the groups positive or negative for a family history of colorectal adenomatous polyps. In the screening programme-based cross-sectional study, the detection rate for colorectal cancer was 0.57% (95% confidence interval (CI): 0.38-0.76) and 0.15% (95% CI: 0.12-0.18) in subjects with and without a family history of colorectal adenomatous polyps, respectively, showing a significant difference in the detection rate for colorectal cancer between the two groups (P<0.05). In the medical check-up-based cross-sectional study, the detection rate for colorectal cancer was 2.31% (95% CI: 1.15-3.47) and 0.53% (95% CI: 0. 34-0.72) in subjects with and without a family history of colorectal adenomatous polyps, respectively, indicating a significant difference between the two groups (P<0.05). These findings indicate that first-degree relatives of patients with colorectal adenomatous polyps have an elevated risk for common colorectal cancer, and that people with a family history of colorectal adenomatous polyps should be considered as a priority group for colorectal cancer screening.

Comparison of ascorbic acid and ascorbic acid 2-O-alpha-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts.

Ascorbic acid 2-O-alpha-glucoside (AA-2G) is a stable ascorbate derivative which has vitamin C activity in vivo and in vitro. We studied whether AA-2G exerts a prooxidant action in cultured fibroblasts from chick embryo and human skin, as does ascorbic acid. At concentrations of 0.1-1.0 mM, ascorbic acid markedly reduced the viable cell number of low density cultures within 24 hr, whereas AA-2G had no such effect. The ascorbate cytotoxicity was dependent on the cell density at the time of its addition and it was characteristic of low density cultures. This cytotoxicity was completely prevented by catalase and partially by an Fe3+ ion chelator, desferrioxamine. In the early culture stage at which a morphological change in the fibroblasts began to occur, intracellular ascorbate concentrations in low density cultures after addition of ascorbic acid were much higher than in high density cultures. However, at the same concentrations, AA-2G did not cause an elevation even in low density cultures and it was also effective on collagen synthesis at high and medium densities. These results suggest that the abnormally accumulated ascorbic acid in the cells cultured at low density possibly amplifies the generation of oxygen radicals through the reduction of Fe3+ ions and subsequent oxidative reactions, leading to cell death. Therefore, it is concluded that AA-2G which supplies an adequate amount of ascorbic acid during culture period is a bioavailable ascorbate source without cytotoxicity.

Rabeprazole, amoxycillin and low- or high-dose clarithromycin for cure of Helicobacter pylori infection.

BACKGROUND: Rabeprazole sodium is a proton pump inhibitor. AIM: To evaluate the efficacy and safety of 1-week triple therapy with rabeprazole, amoxycillin and clarithromycin for the eradication of Helicobacter pylori. METHODS: A total of 100 subjects with H. pylori were randomly divided into two groups of 1-week triple therapy with rabeprazole 10 mg b.d., amoxycillin 750 mg b.d. and either clarithromycin 200 mg b.d. (RAC400, n=50) or clarithromycin 400 mg b. d. (RAC800, n=50). Endoscopic examination with four biopsies (two specimens from the antrum and two from the gastric body) was performed. The status of H. pylori infection was determined using culture and histology (Giemsa stain) of the biopsy specimens. Sensitivity to clarithromycin was determined using the E-test: MIC > 8 g/mL was considered to be resistant, whereas MIC < 2 g/mL was considered to be sensitive. Cure was defined as no evidence of H. pylori infection 1 month after completion of treatment. RESULTS: There were no significant differences in the clinical characteristics of the two groups. Eradication rates (intention-to-treat and per protocol, respectively) were: RAC400: 86% (95% CI: 76-95%) and 89% (95% CI: 80-97%); RAC800: 94% (95% CI: 87-100%) and 97% (95% CI: 94-100%). There was no significant difference between the eradication rates of either regimen. Three subjects with failed eradication in the RAC400 group were all infected with a clarithromycin-resistant strain before beginning the therapy. Haemorrhagic colitis was the only severe adverse event, which was observed in one patient in the RAC800 group. CONCLUSION: One-week triple therapy with rabeprazole, amoxycillin and low-dose clarithromycin is effective for the eradication of H. pylori infection.

Stromal cell-dependent ex vivo expansion of human cord blood progenitors and augmentation of transplantable stem cell activity.

In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications. Thrombopoietin (TPO) and flt3/flk2 ligand (FL) have been suggested to support the proliferation of primitive hematopoietic progenitors and the expansion of transplantable stem cells in culture. In this study, we examined the synergistic effects of the murine stromal cell line MS-5 and a combination of the two cytokines, TPO and FL, on the ex vivo expansion of human cord blood primitive progenitors and transplantable stem cells. A monolayer of MS-5 cells with TPO/FL synergistically supported a more than 600-fold expansion of human cord blood CD34+ cells and CD34+CD38- cells in 2 weeks of culture. Colony-forming unit in culture (CFU-C) and 5-week and 8-week cobblestone area-forming cells (CAFC) were also expanded approximately 300-, 4- and 13-fold, respectively. When MS-5 cells were physically separated from progenitors by a Transwell filter, the synergy was reduced to a quarter of the control, suggesting that direct cell-cell contact between MS-5 cells and progenitors is required for maximum expansion. The severe-combined immunodeficient (scid) mouse-reconstituting cell (SRC) assay demonstrated the slight augmentation of transplantable stem cell activity in culture. These results indicated that MS-5 cells provide a milieu that stimulates the proliferation of primitive progenitors including transplantable stem cells.

Comparisons of cancer detection rate and costs of one cancer detected among different age-cohorts in immunochemical occult blood screening.

PURPOSE: This study was carried out to compare the detection rate for colorectal cancer and the average costs to detect one patient with colorectal cancer among three different age-cohorts in immunochemical occult blood screening by OC-Hemodia. METHODS: In a population-screening program, 17,432 subjects received an immunochemical fecal occult blood test. In a medical checkup for colorectal cancer 7,232 subjects received colonoscopy. They were divided into three groups according to their ages: younger (4,049 years); middle (50-59); and older (60+) groups. The detection rate for colorectal cancer and the average costs to detect one patient with colorectal cancer were evaluated among the three groups. RESULTS: In the screening program-based study, the cancer detection rate and the average costs for one colorectal cancer detected were calculated as 0.09% and $13,352, 0.28% and $4,555, 0.29% and $4,461 for the younger, middle, and older groups, respectively. In addition, in the medical checkup-based study, the detection rate and the average costs were calculated as 0.3% and $6,851, 1.5% and $1,517, 1.7% and $1,391 for the younger, middle, and older groups, respectively. In these two studies, the cancer detection rates were significantly different between the younger and middle groups (P < 0.05), and between the younger and older groups (P < 0.05). CONCLUSIONS: These findings indicate that the subjects aged under 50 have some disadvantage when carrying out the immunochemical fecal occult blood test--OC-Hemodia for colorectal cancer screening--from the viewpoint of screening efficiency as well as cost-effectiveness.

Nerve growth factor applied onto the olfactory epithelium alleviates degenerative changes of the olfactory receptor neurons following axotomy.

The olfactory neuroepithelium of the mammalian nervous system manifests continuous neurogenesis throughout life. Recent studies suggest that neurotrophic factors and their receptors may play a role in the regulation of development and regeneration in the olfactory system. However, there have been very few in vivo studies investigating the effect of exogenous neurotrophic factors in the olfactory system. In the present study, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were administered into the rat olfactory mucosa for 5 days just after the transection of the olfactory nerve. We then examined the effect of exogenous neurotrophic factors on the degenerative changes in axotomized olfactory receptor neurons (ORNs). Further, we examined the location of their receptors, Trk A and Trk B. We found that both mature and immature ORNs expressed more intense signals for olfactory marker protein and beta-tubulin mRNAs, respectively, when NGF was applied to the axotomized olfactory neuroepithelium for 5 days, compared to the ORNs of saline-treated controls. BDNF at a 10 microg total dose did not show this effect. The effect of NGF applied onto the olfactory epithelium is consistent with the immunohistochemical finding that Trk A was present in the dendrites and axon bundles in normal and axotomized ORNs. These results suggest that NGF may protect the degenerative changes in mature and immature ORNs following axotomy through the binding to the Trk A receptor located on the surface of the olfactory epithelium.

Relationships between a sign of rectal bleeding and the results of an immunochemical occult blood test, and colorectal cancer.

A cross-sectional study based on medical check-up was carried out to investigate the association between signs of rectal bleeding and colorectal cancer, and the results of an immunochemical faecal occult blood test. The 9625 patients received both an immunochemical faecal occult blood test using a two-consecutive-day method and colonoscopy. They were then divided into two groups, according to the results of a self-completed questionnaire on the signs of rectal bleeding. The positivity rate of the immunochemical faecal occult blood test as well as the positive predictive value for colorectal cancer were determined in these two groups. The faecal occult blood test was positive in 9.3% of patients with rectal bleeding and in 4.4% of patients without rectal bleeding, and the positive predictive value for colorectal cancer was 0.79 and 0.27 in patients with and without rectal bleeding, respectively. This indicates a significant difference in the positivity rate (P < 0.001) as well as the positive predictive value (P < 0.05) between these two groups. The results suggest that there are positive associations between the signs of rectal bleeding and the results of immunochemical faecal occult blood test, and between the patients presenting with rectal bleeding and colorectal cancer.

In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression.

To determine the limits of the duration of in vivo transferred foreign gene expression, we conducted electroporation (EP), a powerful non-viral means of gene transfer for living animals, into skeletal muscle of rats and mice with a luciferase, GFP or erythropoietin (EPO)-encoding reporter plasmid. The luciferase reporter plasmid was used for optimization of EP conditions, while GFP and EPO plasmids were used for monitoring the duration of gene expression. In the rat, increased hematocrit levels were maintained for at least 9 weeks with approximately a 3-fold increase in plasma EPO protein concentration at 4 weeks post-transfection. In the mouse, the GFP plasmid transfer confirmed that the reporter gene expression lasted as long as 3 months post-transfection. By introducing the EPO gene in vivo in the mouse, increased hematocrit levels revealed that duration of reporter gene expression was at least 14.5 months after in vivo gene EP into skeletal muscle. These results implicate an excellent potential of in vivo gene EP, applicable to both experimental and therapeutic purposes.

Elevated gastrin secretion by in vivo gene electroporation in skeletal muscle.

Whether or not in vivo gene transfer of gastrin gene into skeletal muscle by electroporation could modify gastrin secretion was examined. The expression plasmid vector, either pMEPrGaspA encoding the rat gastrin gene or pEGFP-N1 encoding the GFP reporter gene was injected into M. rectus abdominis of rats or M. biceps formis of mice. Subsequently, square electric pulses of direct current were applied six times at 25 V with a loading period of 100 msec per pulse. Clear foreign gene expression in the skeletal muscle was demonstrated by both GFP fluorescence and immunostaining of rat gastrin. Time course changes in plasma gastrin levels after transfection revealed that in rats, gastrin gene transfer significantly increased the plasma gastrin level for 4 weeks post-transfection (P<0.05), but the difference diminished at the end of the 10-week period. In mice, plasma gastrin level elevated similarly for 3 weeks, and pH of gastric contents decreased in the gastrin gene transfected group compared with the control counterpart (P<0.05). These findings suggest that localized in vivo gene transfer by electroporation allows skeletal muscle to become an artificial endocrine tissue for hormonal manipulation of animals.

Endocytosis and transepithelial transport of endolymph in the endolymphatic sac.

The fate of cationized ferritin (CF) injected into the endolymphatic space of the endolymphatic sac was observed by transmission electron microscopy. At 10 min after the injection, CF particles bound to the apical plasma membrane of epithelial cells of the sac and were then endocytosed with coated pits. However, they never passed through the junctional complexes between the epithelial cells. At 30 min after the injection, the CF particles were transferred to endosomes and lysosomes by small vesicles of 100-150 nm in diameter. CF particles were also found in small vesicles close to Golgi cisternae and in multivesicular bodies. Acid phosphatase positive lysosomes were found close to endosomes containing CF particles. In addition, a small fraction of the small vesicles containing CF particles became inserted into the basolateral plasma membrane. At 60 min after the injection, many CF particles were found in acid phosphatase positive secondary lysosomes. These observations suggest that endocytosis of endolymph is actively performed by the epithelial cells of the sac, and transepithelial vesicular transport across the epithelial cells occurs.

Cellular localization of rat Isk protein in the stria vascularis by immunohistochemical observation.

A novel rat membrane protein, termed Isk protein, that exhibits a voltage-dependent potassium channel activity was first reported through molecular cloning combined with an electrophysiological assay (Takumi et al., 1988). In the present study, we made an attempt to identify the cellular localization of the rat Isk protein in the stria vascularis using two types of antibodies that specifically react with the distinct parts of the rat Isk protein. Immunohistochemical analysis showed that the rat Isk protein was present only on the endolymphatic surface of the marginal cell. The possibility that the Isk protein is involved in potassium permeation in the luminal membrane of the marginal cell will be also discussed.

Failure of forskolin to elevate the endocochlear potential in kanamycin-poisoned animals.

The effect of forskolin (FSK) on the endocochlear potential (EP), K+ activity (AK), Na+ activity (ANa) and Cl- activity (ACl) in scala media (SM) was compared between normal and kanamycin (KM)-poisoned guinea pigs by means of double-barrelled ion-selective microelectrodes. The perfusion of the scala vestibuli (SV) with FSK (200 microM) produced EP elevation in normal animals whereas FSK failed to do it in KM-poisoned animals. FSK increased ACl of SM with no significant change in AK and ANa of SM in both groups of animals. Histological examination of KM-poisoned animals showed damaged outer and inner hair cells with an intact appearance of the stria vascularis. The mechanism underlying the failure of FSK to elevate the EP in KM-poisoned animals is discussed.

Argon plasma surgery for the inferior turbinate of patients with perennial nasal allergy.

OBJECTIVES: Argon plasma coagulation (APC) is a new electrosurgical modality. The advantages of APC are coagulating of the target tissue without contact and the creation of uniformly deep devitalized and coagulated zones. The objectives of the present study were to determine the clinical effects of APC for the inferior turbinate of patients with nasal allergy and to clarify the histological changes in the mucosa after APC. STUDY DESIGN: In a prospective study, 95 patients with perennial nasal allergy were treated with APC. Nasal symptoms and intranasal findings were evaluated preoperatively and 1, 3, and 6 months, and 1 year after the APC. Mucosal specimens from the turbinates were examined under light and electron microscopy. RESULTS: Nasal stuffiness was improved in 77 of 79 (97.5%) patients after 1 month, in 50 of 51 (98.0%) patients after 3 months, in 20 of 23 (87.0%) patients after 6 months, and in 9 of 12 (75.0%) patients at 1 year after the APC. Rhinorrhea was improved in 46 of 75 (61.3%) patients after 1 month, in 40 of 51 (78.4%) patients after 3 months, in 16 of 21 (76.2%) patients after 6 months, and in 6 of 10 (60.0%) patients at 1 year after the APC. The sneezing was improved in 32 of 54 (59.3%) patients after 1 month, in 21 of 35 (60.0%) patients after 3 months, in 10 of 14 (71.4%) patients after 6 months, and in 6 of 8 (75.0%) patients at 1 year after the APC. In the intranasal findings, congestion of the inferior turbinate improved in 75 of 76 (98.7%) patients after 1 month, in 49 of 52 (94.2%) patients after 3 months, in 20 of 23 (87.0%) patients after 6 months, and in 7 of 11 (63.6%) patients at 1 year after the APC. The nasal discharge was reduced in 40 of 75 (53.3%) patients after 1 month, in 32 of 52 (61.5%) patients after 3 months, in 15 of 22 (68.2%) patients after 6 months, and in 5 of 11 (45.5%) patients at 1 year after the APC. No patients needed nasal packing after the APC. CONCLUSIONS: This is the first report on the clinical effects of turbinate surgery for nasal allergy using APC. APC was useful fer turbinate surgery of nasal allergy, especially for nasal stuffiness and congestion of the turbinate.

Vascular permeability of the stria vascularis and morphology of the endolymphatic sac in two types of experimental endolymphatic hydrops.

We produced two kinds of experimental endolymphatic hydrops. One was induced by obliteration of the endolymphatic sac (ES), and the other was induced by both systemic keyhole limpet hemocyanin immunization and secondary keyhole limpet hemocyanin challenge into the ES. The vascular permeability of the stria vascularis in acute and chronic phases was compared between both models by light and electron microscopy using the tracer method of horseradish peroxidase. Both models showed that the number of strial capillaries showing horseradish peroxidase leakage was significantly higher in the acute phase than in the chronic phase. In the acute phase, the ES showed acute inflammation in both models. In the chronic phase, extensive fibrosis occurred in the ES obliteration model, whereas the ES appeared to have a normal shape in the immunologically induced model. The mechanism of hydrops formation will be compared between the two models.

CYFRA 21-1 as a tumor marker used in measuring the serum fragment of cytokeratin subunit 19 by immunoradiometric assay.

Serum levels of cytokeratin subunit 19 (CYFRA 21-1) were measured in 42 healthy volunteers, 104 cases of malignant diseases, 30 patients with chronic renal failure and 13 patients with nonmalignant and infectious diseases. The reliability of the method was demonstrated after dilution of serum samples and intra- and inter-assay reproducibility. Serum CYFRA-21-1 concentrations were less than 2.00 ng/ml in all healthy controls and 86% of the malignant cases had high serum CYFRA 21-1 levels. However slightly elevated values of CYFRA 21-1 were observed in most chronic renal failure patients. High correlation was observed between serum CYFRA 21-1 and Tissue Polypeptide Antigen (TPA) values (r = 0.90, n = 10) but not with serum alpha-feto protein (AFP) concentrations. Furthermore, cross binding tests with the CYFRA 21-1 tracer/CYFRA 21-1 antibody-coated beads and CYFRA 21-1 tracer/TPA antibody-coated beads also gave an almost linear graph. These results indicate that CYFRA 21-1 and TPA share similar type of antigens.

Cytochemical effects of in vitro dopamine treatment on the Na-KATPase activity in strial marginal cells.

Dopamine is often used clinically for the treatment of patients with shock. In a previous study, the Na-KATPase (K-NPPase) activity of strial marginal cells was inhibited after the repeated in vivo administration of dopamine hydrochloride. In the present study, the K-NPPase activity of strial marginal cells was determined using a cerium-based cytochemistry after an in vitro incubation with dopamine. The enzyme reaction product was found in untreated normal strial marginal cells, but it was almost completely undetectable after an in vitro treatment with 10 mM dopamine. This finding suggested that dopamine directly inhibited the Na-KATPase activity of strial marginal cells, and that these cells might have a dopamine receptor.

Transport of HRP through Reissner's membrane in experimental endolymphatic hydrops.

Unilateral endolymphatic hydrops was produced in guinea pigs by cauterization of the endolymphatic sac. Measurements of compound action potential (CAP), cochlear microphonics (CM) and negative summating potential (-SP) confirmed endolymphatic hydrops three months after surgery. In both control and hydropic ears, reaction product of HRP was observed only on the perilymphatic surface of the epithelial cells of Reissner's membrane after 10 min perfusion, while it was observed on both the endolymphatic and perilymphatic surfaces after 30 min perfusion. Epithelial tight junctions were not stained and labelled pinocytotic vesicles were observed in the epithelial cells. These findings suggest that the transport of HRP through Reissner's membrane is unchanged in endolymphatic hydrops and that the epithelial junctions are tight regardless of the distension of Reissner's membrane.

Cholesteatoma otitis media with intact ossicular chain.

Thirty-one cases of cholesteatoma with intact ossicular chain were reviewed to examine the extension of cholesteatoma, operation procedures, pre- and postoperative hearing levels, and postoperative condition of the ear drum. They were operated on at Departments of Hyogo College of Medicine and Osaka University Medical School from 1989-1996, which were 20.4% of all of the primary cholesteatoma cases (n = 152). Twelve cases located at attic were operated by atticotomy or canal wall up and scutumplasty, 17 cases located at attic/antrum were operated by canal wall down and reconstruction, and two cases located around stapes were operated through the ear canal. Preoperative hearing level was mild (34.2 +/- 18.4 dB) and postoperative one was slightly improved (28.7 +/- 11.2 dB). In ossicular reconstruction, modified Wullstein type III method showed better hearing gain than Wullstein type I method. Postoperative attic retraction and/or pocket occurred in 15 cases (48.4%), but no recurrent cholesteatoma was found during the present follow-up period. These findings suggested that cholesteatoma with intact ossicular chain showed good postoperative hearing results, but postoperative retraction of the ear drum should be followed up for a long time.