Safety and efficacy of endoscopic spray cryotherapy for esophageal cancer.

Although surgery is traditionally the standard of care for esophageal cancer, esophagectomy carries significant morbidity. Alternative endoscopic therapies are needed for patients who are not candidates for conventional treatment. The objective of this study is to assess the safety, efficacy, and tolerability of spray cryotherapy of esophageal adenocarcinoma. This study includes patients with esophageal adenocarcinoma who had failed or were not candidates for conventional therapy enrolled retrospectively and prospectively in an open-label registry and patients in a retrospective cohort from 11 academic and community practices. Endoscopic spray cryotherapy was performed until biopsy proven local tumor eradication or until treatment was halted due to progression of disease, patient withdrawal or comorbidities. Eighty-eight patients with esophageal adenocarcinoma (median age 76, 80.7% male, mean length 5.1 cm) underwent 359 treatments (mean 4.4 per patient). Tumor stages included 39 with T1a, 25 with T1b, 9 with unspecified T1, and 15 with T2. Eighty-six patients completed treatment with complete response of intraluminal disease in 55.8%, including complete response in 76.3% for T1a, 45.8% for T1b, 66.2% for all T1, and 6.7% for T2. Mean follow-up was 18.4 months. There were no deaths or perforations related to spray cryotherapy. Strictures developed in 12 of 88 patients (13.6%) but were present before spray cryotherapy in 3 of 12. This study suggests that endoscopic spray cryotherapy is a safe, well-tolerated, and effective treatment option for early esophageal adenocarcinoma.

PI3K/Akt signalling is required for the attachment and spreading, and growth in vivo of metastatic scirrhous gastric carcinoma.

BACKGROUND: PI3K/Akt (PKB) pathway has been shown in several cell types to be activated by ligands to cell surface integrins, leading to the metastasis of tumour cells. The signalling pathways involved in the metastatic spread of human scirrhous gastric carcinoma cells have not been defined. METHODS: The role of the PI3K/Akt pathway in an extensive peritoneal-seeding cell line, OCUM-2MD3 and a parental cell line, OCUM-2M, was investigated by assessing in vitro adhesion and spreading assay, and in vivo peritoneal metastatic model. We also examined the correlation of PI3K/Akt pathway with integrin signals by immunoprecipitations, using cells by transfection with mutant p85 (Δp85). RESULTS: Adhesiveness and spreading of OCUM-2MD3 cells on collagen type IV was significantly decreased by PI3K inhibitors and expression of mutant p85, but not by inhibitors of protein kinase C (PKC) or extracellular signal-regulated kinase (ERK). Immunoprecipitation studies indicated that the PI3K/Akt pathway was associated with integrin signalling through Src and vinculin. In an in vivo experimental metastasis model, p85 inhibition reduced peritoneal metastasis of OCUM-2MD3 cells. CONCLUSION: PI3K/Akt signalling may be required for integrin-dependent attachment and spreading of scirrhous gastric carcinoma cells, and would be translated into generating better strategies to optimise their use in cancer clinical trials.

Linoleic acid enhances angiogenesis through suppression of angiostatin induced by plasminogen activator inhibitor 1.

BACKGROUND: The intake of dietary fatty acids is highly correlated with the risk of various cancers. Linoleic acid (LA) is the most abundant polyunsaturated fat in the western diet, but the mechanism(s) by fatty acids such as LA modulate cancer cells is unclear. In this study, we examined the role of LA in various steps in gastric cancer progression. METHODS: The difference in gene expression between LA-treated and untreated OCUM-2MD3 gastric carcinoma cells was examined by mRNA differential display. The involvement of candidate genes was examined by oligo- and plasmid-mediated RNA interference. Biological functions of several of these genes were examined using in vitro assays for invasion, angiogenesis, apoptosis, cell viability, and matrix digestion. Angiogenesis in vivo was measured by CD-31 immunohistochemistry and microvessel density scoring. RESULTS: LA enhanced the plasminogen activator inhibitor 1 (PAI-1) mRNA and protein expression, which are controlled by PAI-1 mRNA-binding protein. LA-stimulated invasion depended on PAI-1. LA also enhanced angiogenesis by suppression of angiostatin, also through PAI-1. LA did not alter cell growth in culture, but increased dietary LA-enhanced tumour growth in an animal model. CONCLUSION: Our findings suggest that dietary LA impacts multiple steps in cancer invasion and angiogenesis, and that reducing LA in the diet may help slow cancer progression.

Genotoxicity and subchronic toxicity evaluation of Active Hexose Correlated Compound (AHCC).

Active Hexose Correlated Compound (AHCC), a mushroom extract rich in α-1,4 linked glucans, is associated with immunostimulatory effects. AHCC is used in Japan as a dietary supplement to boost immune function and it also is purported to improve the symptoms of cancer and liver disease patients. A series of toxicological studies were conducted on a freeze dried preparation of AHCC (AHCC-FD) to further develop the body of evidence supporting the safety of this ingredient. AHCC-FD was not mutagenic to Salmonella typhimurium and did not exhibit clastogenicity in a mouse micronucleus assay. In a 90-day study, Sprague-Dawley rats were administered 1000, 3000, or 6000 mg/kg body weight/day by gavage. No changes attributable to AHCC-FD treatment were observed in overall condition, body weight, food consumption, ophthalmology findings, hematology and clinical chemistry parameters, and absolute and relative organ weights. Changes in urinary pH values observed in high-dose animals and mid-dose females were considered physiological rather than adverse effects given the acidic nature of AHCC-FD. Urinary protein also was increased in the same dose groups. As this finding was associated with decreased urinary pH and no evidence of kidney dysfunction was observed, it was considered of no toxicological significance. Histopathological changes related to AHCC-FD administration were observed in the limiting ridge of the stomach and in the liver of the high-dose group. The NOAEL was considered to be 3000 mg/kg body weight/day.

Focal segmental glomerular sclerosis, a type of intractable chronic glomerulonephritis, is a stem cell disorder.

The etiopathogenesis of focal and segmental glomerular sclerosis (FGS) remains unknown. Using a new animal model for FGS (FGS mouse), we demonstrate here that bone marrow transplantation from normal mice to FGS mice with a high grade of proteinuria (+ + +) ameliorates FGS, and that the transplantation of bone marrow cells or purified hemopoietic stem cells (HSCs) from FGS mice induces FGS in normal mice. These findings strongly suggest that FGS is a stem cell disorder; the abnormalities may be genetically programmed at the level of HSCs.

Co-registered spectrally encoded confocal microscopy and optical frequency domain imaging system.

Spectrally encoded confocal microscopy and optical frequency domain imaging are two non-contact optical imaging technologies that provide images of tissue cellular and architectural morphology, which are both used for histopathological diagnosis. Although spectrally encoded confocal microscopy has better transverse resolution than optical frequency domain imaging, optical frequency domain imaging can penetrate deeper into tissues, which potentially enables the visualization of different morphologic features. We have developed a co-registered spectrally encoded confocal microscopy and optical frequency domain imaging system and have obtained preliminary images from human oesophageal biopsy samples to compare the capabilities of these imaging techniques for diagnosing oesophageal pathology.

Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide.

NO-mediated inhibition of base excision DNA repair may potentiate oxidativeDNA damage in cells and could be relevant to carcinogenesis associated with chronic inflammation. Because 8-oxoguanine, a ubiquitous oxidative DNA lesion, is repaired predominantly by human 8-oxoguanine glycosylase (hOgg1), our aim was to determine whether NO directly inhibits its repair activity. Neither induction of NO-generating enzyme inducible NO synthase nor treatment with S-nitroso-N-acetyl-D-L-pencillamine altered expression of hOgg1 in a human cholangiocarcinoma cell line (KMBC). In contrast, both treatments completely inhibited activity of hOgg1 immunoprecipitated from KMBC cells overexpressing hOgg1 and in a cell-free system. Both NO and peroxynitrite were capable of inhibiting hOgg1 activity. Inhibition of hOgg1 protein was characterized by formation of S-nitrosothiol adducts and loss/ejection of zinc ions. Our data indicate that NO, an inflammatory mediator, directly inhibits a key base excision repair enzyme (hOgg1) responsible for base excision repair of 8-oxoguanine. These data support the concept that NO-mediated inhibition of DNA contributes to the mutagenic environment of chronic inflammation.

Two novel missense mutations in calcium-sensing receptor gene associated with neonatal severe hyperparathyroidism.

Familial hypocalciuric hypercalcemia (FHH) is characterized by lifelong asymptomatic hypercalcemia without PTH hypersecretion and is inherited as an autosomal dominant trait with near 100% penetrance. In contrast, neonatal severe hyperparathyroidism (NSHPT) is a life-threatening disorder characterized by marked hypercalcemia and PTH hypersecretion. FHH/NSHPT results from inactivating mutations of the human calcium-sensing receptor (Casr) gene on chromosome 3q13.3-24. Nearly 30 different mutations of the Casr gene associated with FHH/NSHPT have been reported previously. In this report, genetic analysis of 1 Japanese NSHPT family revealed 2 novel mutations at codon 185 (CGA-->TGA/Arg-->Ter) in exon 4 of the Casr gene and at codon 670 (GGG-->GAG/Gly-->Glu) in exon 7. The Arg185Ter change was shown to occur in the proband's unaffected father and paternal grandmother as well as in the proband. The other mutation in exon 7 was shown in the proband's unaffected mother of Philippine origin as well as in the proband. This family is the first case of manifestation of more than 1 mutation in a proband's chromosomes; 1 mutation was obtained from the unaffected father, and the other was from the unaffected mother. Our observations have given us important keys to help elucidate the structure-function relationships of the Casr.

Wortmannin inhibits the activation of MAP kinase following vasopressin V1 receptor stimulation.

Treatment of rat 3Y1 fibroblasts with vasopressin (AVP) results in a transient activation of MAP kinase as potent as with EGF and serum. An antagonist of vasopressin receptor V1, but not an antagonist of V2, inhibited the AVP-induced activation of MAP kinases, indicating that AVP activates MAP kinases through V1 receptor. Prolonged TPA treatment of cells resulted in partial MAP kinase activation, indicating the presence of PKC-independent pathway. The pathway was inhibited by wortmannin, an inhibitor of PI3-kinase. The results suggest that wortmannin-sensitive molecules such as PI3-kinase, are involved in the V1 receptor-mediated activation of the MAP kinase pathway independent of TPA-sensitive PKC.

Gene of rat Ca2+/calmodulin-dependent protein kinase II alpha isoform -- its cloning and whole structure.

The gene encoding the alpha isoform of rat Ca2+/calmodulin-dependent protein kinase II was cloned, and its exon-intron organization was analyzed. The coding region of cDNA consists of 18 exons spanning more than 50 kilobase pairs. Each of the discrete functional units, such as the ATP-binding site, the autophosphorylation site responsible for Ca2+-independent activity, the calmodulin-binding site, and link structure is encoded by a single exon. The largest and smallest exons consist of 229 and 41 base pairs, respectively. All splice junction sequences flanking the introns conform to the consensus splice junction sequence and the GT-AG splice rule.

A candidate tumor suppressor locus for scirrhous gastric cancer at chromosome 18q 12.2.

We investigated LOH on 5q, 16q, 17p, 17q, and 18q in scirrhous gastric cancer and identified the chromosomal locus that is frequently deleted in scirrhous gastric cancer. LOH frequency on 18q of scirrhous gastric cancer was found to be significantly higher than that of the other types of advanced gastric cancer, while no significant difference between LOH at 5q, 16q, 17p and 17q was found. Eight microsatellite markers on chromosome 18q were additionally examined in scirrhous gastric cancer. The highest rates of LOH were observed at the D18S34 locus which is located at 18q12.2. These findings suggest that the D18S34 locus might be a novel candidate tumor suppressor locus in scirrhous gastric carcinoma.

Repair mechanism of lupus nephritis in (NZB x NZW)F1 mice by allogeneic bone marrow transplantation.

We have recently found that allogeneic bone marrow transplantation (BMT) can be used to treat lupus nephritis in (NZB x NZW)F1(B/WF1), BXSB, MRL/lpr and (NZW x BXSB)F1 mice. To elucidate why and how glomerular damage is repaired by BMT, serial renal biopsies were carried out using B/WF1 mice before and after BMT. Donor-derived B cells and macrophages with normal functions developed two weeks (wks) after BMT. At this stage, the macrophages did not show immune complex (IC) clearance activity. Donor-derived T cells with normal functions were generated six wks after BMT. At this stage, visceral epithelial cells macrophages and mesangial cells in the glomeruli were activated by T cells and showed marked phagocytic activity; macrophages and mesangial cells were found to be responsible for the clearance of ICs, whereas, to our surprise, epithelial cells were found to be responsible for the repair of injured basement membranes. These findings suggest that T cells with normal functions, which have the capacity to activate macrophages, mesangial cells and epithelial cells, play a crucial role in repairing IC-mediated glomerular damage.

Flagellin-containing membrane vesicles excreted from Vibrio alginolyticus mutants lacking a polar-flagellar filament.

Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-). Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella.

Pancreatic microcirculatory changes in experimental pancreatitis of graded severity in the rat.

BACKGROUND: A technique with two complementary methods, intravital microscopy (IVM) and diffuse reflectance spectroscopy (DRS), was developed to analyze pancreatic tissue perfusion. METHODS: After initial in vivo and in vitro validation of the techniques, we studied pancreatic microcirculation in models of mild, moderate, and severe pancreatitis. Anesthetized Sprague-Dawley rats were randomly allocated to the three models or to serve as controls. Stable systemic hemodynamic parameters were maintained with normal saline solution infusion. Exocrine capillary perfusion was assessed by IVM; hemoglobin oxygenation and hemoglobin content were measured by DRS. RESULTS: Capillary perfusion in mild pancreatitis initially increased significantly at 30 minutes to 155% +/- 38% of baseline values but returned to baseline within 3 hours. Hemoglobin content and oxygen saturation remained stable. In moderate and severe pancreatitis capillary perfusion significantly decreased versus the control group to 12% +/- 6% and 6% (range, 0% to 14%) of baseline values, respectively, at 6 hours. Oxygen saturation decreased significantly in moderate pancreatitis from 48.5% +/- 2.3% to 41.6% +/- 3.5% (p < 0.05) and in severe pancreatitis from 47.2% +/- 1.5% to 38.9% +/- 0.5% (p < 0.05), whereas hemoglobin content did not change. CONCLUSIONS: We conclude that (1) IVM and DRS provide both unique and complementary data on tissue perfusion of the pancreas, (2) that moderate and severe experimental pancreatitis are accompanied by progressive tissue ischemia, and (3) that significant stasis (decreased perfusion) and decreased oxygen saturation occur whereas generalized vasoconstriction (decreased hemoglobin levels) was not found. In contrast, mild experimental pancreatitis was accompanied by initial hyperperfusion and normal oxygen delivery was maintained.

Reflectance spectroscopy of pancreatic microcirculation.

A technique employing diffuse reflectance spectroscopy (DRS) is described to assess and mirror dynamic changes of pancreatic tissue perfusion. An especially designed reflectance spectrophotometer was initially used to derive the quantitative relation between hemoglobin concentration ([Hb]) and reflectance measurements in vitro. Over a wide range of scattering related to the medium in which the measurements were made (scattering coefficient: 6.5-13 cm-1), a close, direct correlation existed with a slope of 0.376 +/- 0.012. In Sprague-Dawley rats under general anesthesia, the pancreas was isolated in situ and perfused with graded infusions of hemoglobin solutions. A correlation, comparable to the in vitro setting, was found between a [Hb] of 0 and 14 g/dl in the perfusate with slopes of 0.0037 and 0.0035. Changes in perfusion induced by adrenergic drugs produced changes in hemoglobin oxygen saturation and [Hb] that correspond with measured alterations of systemic arterial pressure and aortic blood flow. We conclude that diffuse reflectance spectroscopy reliably provides data on intrapancreatic hemoglobin oxygen saturation and [Hb] that can be a valuable tool for minimally invasive on-line evaluation of these aspects of pancreatic perfusion in the rat. This newly designed device is superior to previously used ones in that it analyzes the entire spectrum and therefore can account for changes in scattering that are very likely to occur with pathophysiological alterations such as edema formation.

Burn depth estimation using indocyanine green fluorescence.

Expedient primary excision of deep dermal and full-thickness burn wounds with subsequent skin grafting is the standard of care in most burn institutions, but differentiating full-thickness from partial-thickness burns is often difficult. Because accurate early assessment of burn depth may improve care, a variety of technical methods have attempted to measure burn depth but these methods have had limited success. We describe a new technique to determine burn depth that uses infrared (840- to 850-nm) fluorescence emission from intravenously administered indocyanine green following excitation with infrared (780 nm) and UV light (369 nm). Full-thickness and partial-thickness burns in hairless rat skin were distinguished based on the infrared-induced and UV-induced fluorescence intensity ratios relative to normal, unburned skin immediately after the burn and on post-burn days 1 through 3 and 7. Dual-wavelength excitation of indocyanine green infrared fluorescence can delineate full-thickness from partial-thickness burns at an early date, allowing prognosis, surgical planning, and early primary excision and grafting.

Skin graft take and healing following 193-nm excimer, continuous-wave carbon dioxide (CO2), pulsed CO2, or pulsed holmium: YAG laser ablation of the graft bed.

BACKGROUND: Ablative lasers have been used for cutaneous surgery for greater than two decades since they can remove skin and skin lesions bloodlessly and efficiently. Because full-thickness skin wounds created after thermal laser ablation may require skin grafting in order to heal, we have examined the effect of the residual laser-induced thermal damage in the wound bed on subsequent skin graft take and healing. In a pig model, four different pulsed and continuous-wave lasers with varying wavelengths and radiant energy exposures were used to create uniform fascial graft bed thermal damage of approximately 25, 160, 470, and 1100 microns. Meshed split-thickness skin graft take and healing on the thermally damaged fascial graft beds were examined on a gross and microscopic level on days 3 and 7, and then weekly up to 42 days. RESULTS: Laser-induced thermal damage on the graft bed measuring greater than 160 +/- 60 microns in depth significantly decreased skin graft take. Other deleterious effects included delayed graft revascularization, increased inflammatory cell infiltrate at the graft-wound bed interface, and accelerated formation of hypertrophied fibrous tissue within the graft bed and underlying muscle. CONCLUSIONS: Ablative lasers developed for cutaneous surgery should create less than 160 +/- 60 microns of residual thermal damage to permit optimal skin graft take and healing. Pulsed carbon dioxide and 193-nm excimer lasers may be valuable instruments for the removal of full-thickness skin, skin lesions, and necrotic tissue, since they create wound beds with minimal thermal damage permitting graft take comparable to that achieved with standard surgical techniques.