Prognostic significance of CD44 variant 2 upregulation in colorectal cancer.

BACKGROUND: CD133 and CD44 are putative cancer stem cell (CSC) markers in colorectal cancer (CRC). However, their clinical significance is currently unclear. Here, we evaluated primary CRC cell isolates to determine the significance of several CSC markers, including CD133 and CD44, as predictors of tumourigenesis and prognosis. METHODS: CD133- and CD44-positive cells from fresh clinical samples of 77 CRCs were selected by flow cytometric sorting and evaluated for tumourigenicity following subcutaneous transplantation into NOD/SCID mice. Cancer stem cell marker expression was examined in both xenografts and a complementary DNA library compiled from 167 CRC patient samples. RESULTS: CD44(+), CD133(+) and CD133(+)CD44(+) sub-populations were significantly more tumourigenic than the total cell population. The clinical samples expressed several transcript variants of CD44. Variant 2 was specifically overexpressed in both primary tumours and xenografts in comparison with the normal mucosa. A prognostic assay using qRT-PCR showed that the CD44v2(high) group (n=84, 5-year survival rate (5-OS): 0.74) had a significantly worse prognosis (P=0.041) than the CD44v2(low) group (n=83, 5-OS: 0.88). CONCLUSIONS: CD44 is an important CSC marker in CRC patients. Furthermore, CRC patients with high expression of CD44v2 have a poorer prognosis than patients with other CD44 variants.

Adhalin gene mutations in patients with autosomal recessive childhood onset muscular dystrophy with adhalin deficiency.

Homozygous adhalin gene mutations were found in three patients from two consanguineous families with autosomal recessive childhood onset muscular dystrophy. Muscle biopsies from patients in each family showed complete absence of adhalin. Sequencing of adhalin cDNA prepared from skeletal muscle by reverse transcription PCR demonstrated a cytosine to thymidine substitution at nt 229 in the patient in family 1 and an adenine to guanine substitution at nt 410 and a 15-base insertion between nt 408 and 409 in the two patients in family 2. Sequencing of genomic DNA prepared from peripheral blood leukocytes by PCR confirmed these mutations. The parents in each family were found to be heterozygous for the respective mutations. These adhalin gene mutations are presumed to be responsible for the absence of adhalin in the skeletal muscle. Adhalin deficiency likely causes disruption of the muscle cell membrane, resulting in dystrophic changes in the skeletal muscle similar to dystrophin deficiency in Duchenne muscular dystrophy.

Perinatal bisphenol A affects the behavior and SRC-1 expression of male pups but does not influence on the thyroid hormone receptors and its responsive gene.

Bisphenol A (BPA) has been shown to interfere with thyroid hormone receptors (THRs) and to influence the expression of THR-responsive elements in vivo and in vitro, while some studies reported hyperactivity induced by BPA treatment. In the present study, our purpose was to investigate the effect of BPA exposure on behavioral alteration and its mechanism of action, especially focusing on the thyroid hormone pathway. Significant sexual difference on behaviors was observed in perinatal BPA exposure, as manifested by hyperactivity and impaired spatial learning/memory in male pups after matured. Dams treated with 0.1mg/l BPA showed transient hypothyroidism, while male pups were found to exhibit a transient hyperthyroidism followed by hypothyroidism. Furthermore, significant up-regulated expression levels of mRNA and protein of SRC-1 in the hippocampus were observed in male pups by 0.1mg/l BPA treatment. However the expression of THRalpha/beta and RC3/neurogranin were not affected by BPA treatment. These results indicate that perinatal BPA exposure at a very low level may influence thyroid function and then consequently affects brain development, but at the same time, suggest that thyroid hormone receptor may not be a direct target of BPA action, but instead, another factor may be involved in this action.

High frequencies of human T-lymphotropic virus type I (HTLV-I) infection and presence of HTLV-II proviral DNA in blood donors with anti-thyroid antibodies.

To investigate the relationship between human T-lymphotropic virus (HTLV) types I and II and the pathogenesis of autoimmune thyroid diseases, we examined serum anti-thyroid antibodies in 1019 blood donors with or without serum anti-HTLV-I antibody as well as proviral DNA for HTLV-II in leukocyte DNA by the polymerase chain reaction in 395 blood donors with or without anti-thyroid antibodies. The frequency of donors with anti-HTLV-I antibody who also showed anti-thyroid antibodies (7.9%) tended to be higher than that (6.3%) among donors who did not have the anti-HTLV-I antibody. The frequency of anti-thyroid antibodies in 125 young male donors aged 16-39 years with anti-HTLV-I antibody (4.8%) was significantly higher (P < 0.05) than that (0.6%) in 164 control donors without the antibody. In blood donors with anti-thyroid antibody, 25.0% of those with anti-HTLV-I antibody and 14.3% of those without the antibody had HTLV-II proviral DNA. In contrast, in donors without anti-thyroid antibody HTLV-II proviral DNA was detected in 2.3% of those with anti-HTLV-I antibody and in 0.6% of those without the antibody. Thus the detection rates in donors with anti-thyroid antibody were significantly higher (P < 0.001) than those in donors without the antibody, regardless of HTLV-I infection. These results suggest that HTLV-I infection and the presence of HTLV-II proviral DNA may be independently related to the pathogenesis of autoimmune thyroid diseases.

Evidence of HTLV-I in thyroid tissue in an HTLV-I carrier with Hashimoto's thyroiditis.

Human T-lymphotropic virus type l (HTLV-I) protein and messenger RNA (mRNA) for HTLV-I were examined in thyroid tissues from two patients with Hashimoto's thyroiditis and serum anti-thyroid antibody. The virus envelope protein and signals for the mRNA were detected in many of the follicular epithelial cells of the thyroid tissue from one of the patients, respectively, by immunohistochemistry and in situ hybridization. PCR-Southern blotting revealed the presence of HTLV-I DNA in the thyroid tissue, in which the viral protein and mRNA were detected, although no virus particles were found in the epithelial cells by electron microscopy. HTLV-I virus was not present in the thyroid tissue from the second patient. The present findings suggest that infection of thyroid tissue with HTLV-I is associated with the pathogenesis of Hashimoto's thyroiditis in some patients.

Graves' disease in HTLV-I carriers.

Three carriers of human T-lymphotropic virus type I (HTLV-I) with Graves' disease are reported. All three cases were complicated with uveitis, and one also showed chronic arthropathy. Anti-HLTV-I antibody was found in the serum by the particle agglutination method and western blotting, and HTLV-I proviral DNA was detected in peripheral lymphocytes by the polymerase chain reaction and Southern blotting. HTLV-I is a causal agent of adult T-cell leukemia and HTLV-I associated myelopathy/tropical spastic paraparesis, and is believed to be related to the pathogenesis of diseases such as chronic arthropathy, uveitis, chronic bronchoalveolitis, and Sjögren's syndrome. On the other hand, retrovirus infection is considered to cause autoimmune diseases. Thus, the pathogenesis of Graves' disease in the present patients might be associated with HTLV-I infection.

Trimethyltin syndrome as a hippocampal degeneration model: temporal changes and neurochemical features of seizure susceptibility and learning impairment.

The effects of trimethyltin on the hippocampus were investigated in terms of changes in histology, depth electroencephalography, learning acquisition and memory retention, choline acetyltransferase and neuropeptides, and seizure-induced c-fos messenger RNA expression. The results were as follows. (1) Morphologically, trimethyltin produced a progressive loss of hippocampal CA3 and CA4 pyramidal cells, starting from four days after peroral treatment with trimethyltin hydroxide (9 mg/kg), as described previously. (2) Neurophysiologically, the increased seizure susceptibility to pentylenetetrazol treatment reached a maximum at four days post-trimethyltin and then declined after five days post-trimethyltin. The maximal seizure susceptibility at four days post-trimethyltin was confirmed by the immediate and long-lasting appearance of spike discharge in the hippocampus. However, this was not verified by the expression of c-fos messenger RNA in the hippocampus, which was comparable between trimethyltin-treated and control rats. (3) Behaviorally, the time-courses of aggression and learning impairment were similar to that of the seizure susceptibility. (4) Neurochemically, trimethyltin treatment caused changes of neurochemical markers, which were manifested by the elevation of neuropeptide Y content in the entorhinal cortex, and of choline acetyltransferase in the hippocampal CA3 subfield. Trimethyltin may offer potential as a tool for investigations on the relationship between neuronal death in the hippocampus and the development of seizure susceptibility and learning impairment. Alterations in glucocorticoids, glutamate and neuropeptides may all contribute to the manifestation of the trimethyltin syndrome.

Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HC II Tokushima.

We found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bhv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC I1 in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC I1 mRNA was transcribed at a similar level as that of wild-type. Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.

Effects of percutaneous transvenous mitral commissurotomy on levels of plasma atrial natriuretic peptide during exercise.

To clarify the factors that influenced the secretion of human atrial natriuretic peptide (ANP) during exercise, we studied the relations between the changes in ANP, transmitral pressure gradient, heart rate and blood pressure at exercise in 16 patients with mitral stenosis before and after percutaneous transvenous mitral commissurotomy (PTMC). Before PTMC, ANP levels increased from 107 +/- 70 to 183 +/- 96 pg/ml during exercise testing (p less than 0.01), concomitant with the increment in mean transmitral pressure gradient, heart rate and systolic blood pressure. After PTMC, ANP levels also increased from 78 +/- 43 to 117 +/- 64 pg/ml, concomitant with the increment of those parameters. However, increments of ANP, mean transmitral pressure gradient and heart rate after PTMC were lower than those before PTMC. Because the most important factor influencing the secretion of ANP was unclear, the differences between these parameters were calculated at submaximal exercise before and after PTMC. There was a significant relation only between the change in ANP and mean transmitral pressure gradient (r = 0.70, p less than 0.01). These results suggest that the most important factor influencing the secretion of ANP during exercise is the change in transmitral pressure gradient in patients with mitral stenosis.

Successful combination therapy--flunarizine, pentoxifylline, and cholestyramine--for spur cell anemia.

Spur cell anemia, a hemolytic anemia observed in patients with alcoholic cirrhosis, is characterized by unusual erythrocyte morphology and an increased ratio of free cholesterol to phospholipid in the erythrocyte membrane. The prognosis of spur cell anemia is usually extremely poor, however, we describe here a patient with spur cell anemia who was successfully treated with combination therapy consisting of flunarizine, pentoxifylline, and cholestyramine. Initial therapy with flunarizine alone for 6 weeks did not significantly decrease the number of spur cells on peripheral blood smears. So pentoxifylline was added to the regimen. The patient recovered from the anemia, showed remarkable improvement with regard to the hyperbilirubinemia, and the changes were accompanied by a significant decrease in the number of spur cells in peripheral blood smears. To correct the hypercholesterolemia, cholestyramine was added to the regimen, which resulted in a reduction in the serum level of free cholesterol and an increase in the molar ratio of free cholesterol to phospholipid in erythrocyte membrane. However, 6 months later a skin eruption developed that was considered an adverse reaction to the drugs, so the flunarizine and pentoxifylline were discontinued. With cholestyramine therapy alone, the remission of spur cell anemia was maintained for more than 11 months. These observations suggest that non-invasive combination therapy with flunarizine, pentoxifylline, and cholestyramine is effective and valuable in the treatment of patients with spur cell anemia.

Morphologic alterations and cytokinetic studies of tracheal autograft epithelium in rabbits.

BACKGROUND: Although tracheobronchoplasty has been used widely in the field of thoracic surgery, few details of the morphologic changes in and cytokinetics of the graft epithelium have been reported. The aim of this study was to focus on these aspects in autografted rabbit tracheas. METHODS: Resected cervical tracheas were anastomosed immediately after removal, retrieved on postoperative days 1 through 28, and examined morphologically. Mitotic and bromodeoxyuridine-labeling indices of the graft epithelium were analyzed. RESULTS: On postoperative days 1 to 4, the graft epithelium showed focal desquamation at the anastomoses. Ciliated cells disappeared during postoperative days 4 to 7 and then increased gradually. Nonciliated cells retained a somewhat columnar shape on postoperative days 4 to 7, except at denuded foci. Thereafter, the grafts were covered completely with pseudostratified mucociliary epithelium. On postoperative day 4, both indices were maximal and appeared higher at the anastomotic than midgraft sites. CONCLUSIONS: Most of the graft epithelium was preserved during acute ischemia and then started to regenerate. The increased regenerative activity near the anastomoses may be attributable to mechanical damage or different nutritional conditions.

Nitric oxide synthase-containing neurons in the hippocampus are preserved in trimethyltin intoxication.

We studied the effects of trimethyltin (TMT) (9 mg/kg, p.o.) on the nitric oxide synthase (NOS)-containing neurons in the rat hippocampus by NADPH-diaphorase histochemistry and a biochemical assay of NOS activity. TMT exposure caused the typical behavioral changes and a loss of the CA3/4 pyramidal cells, which were NADPH diaphorase-negative. The scattered interneurons and the CA1 pyramidal cells, which were NADPH diaphorase-positive, were spared. Hippocampal NOS activity showed no reduction in the TMT-treated rats compared with the controls. These results provide evidence of the preservation of the NOS-containing neurons in TMT intoxication.

Oxidative damage to skeletal muscle DNA from patients with mitochondrial encephalomyopathies.

To estimate the oxidative damage to skeletal muscle DNA in mitochondrial encephalomyopathies, we studied the amount of 8-hydroxy-deoxyguanosine (8-OH-dG) and the localization of superoxide dismutase (SOD) in the skeletal muscles of patients with progressive external ophthalmoplegia (PEO) or Kearns-Sayre syndrome (KSS). The molar ratio of 8-OH-dG/deoxyguanosine in skeletal muscle from PEO or KSS patients was significantly higher than the control value. The ratio from patients with polymyositis or Duchenne's muscular dystrophy was not significantly elevated. Immunohistochemical staining for both Mn-SOD and Cu,Zn-SOD showed pronounced staining in the subsarcolemmal and intermyofibrillar regions of cytochrome-oxidase-negative ragged red fibers of KSS or PEO muscles. Our findings suggest that overproduction of 8-OH-dG and mitochondrial dysfunction with gene deletions are associated with each other in muscle cells of patients with PEO or KSS, and that free radicals may play an important role in the pathophysiology of mitochondrial encephalomyopathies.

Enhanced secretion and impaired natriuretic action of atrial natriuretic peptide in response to hypertonic saline infusion in patients with essential hypertension.

To clarify the natriuretic action of endogenous atrial natriuretic peptide (ANP) in patients with essential hypertension (EHT), we examined the relationship between ANP release and urinary sodium excretion in response to hypertonic saline infusion. Plasma ANP levels increased from 13.3 +/- 2.0 pg/ml to 37.0 +/- 3.0 pg/ml in patients with EHT and from 9.2 +/- 1.5 pg/ml to 21.1 +/- 4.0 pg/ml in normal subjects after the infusion. The area under the curve (AUC) of plasma ANP response was significantly higher in patients with EHT than in normal subjects (P < 0.05). There was a significant positive correlation between AUC and urinary sodium excretion in both groups (P < 0.01). However, the ratio of urinary sodium excretion to AUC was significantly lower in patients with EHT than in normal subjects (P < 0.01). These results suggest that impaired natriuretic response to endogenous ANP is one of the factors responsible for the development of EHT and that enhanced secretion of ANP in response to hypertonic saline infusion is a compensatory mechanism to the impaired natriuretic action of ANP in patients with EHT.

Impaired Biel and radial arm maze learning in rats with methylnitrosourea-induced microcephaly.

Jc1:SD rats were given methylnitrosourea (MNU; 5 mg/kg, IP) on day 13 of gestation. Male offspring with MNU-induced microcephaly were examined on the Biel water maze and its mirror-image maze at 6 weeks of age and on a radial eight-arm maze at 14 weeks of age. The MNU rats showed postnatal depression in body weight. Their brain weight was about 60% of the control value, and they were thus microcephalic. The MNU animals made significantly more errors on the Biel maze and its mirror-image maze than the controls. In the radial arm maze test, they required more trials to acquire the learning criterion than the controls, and the animals with the acquired learning criterion were fewer. In the retest, however, no significant difference appeared in number of trials required for reacquisition of the criterion between the MNU and control animals. The autopsy of the MNU animals revealed the thinned cerebral cortex and hypoplastic hippocampus. The present results with the MNU rats confirmed the learning impairment and suggested no effect of microcephaly on retention of the acquired memory.

Learning/memory impairments in rat offspring prenatally exposed to phenytoin.

Phenytoin (PHT) was orally administered in dosages of 50 and 100 mg/kg/day to pregnant rats on days 7-18 of gestation. Offspring were tested on the negative geotaxis test, a figure-eight maze (F8), the Biel water maze (BM), the Morris maze (MM), and the radial maze (RM). In addition, a delayed nonmatching-to-sample (DNMTS) test was employed. The levels of neuropeptides in brain and brain weights were determined. The maturation of negative geotaxis was delayed in both PHT groups. PHT groups showed no differences in F8, BM, and MM. In the RM, the total number of choices was high, whereas the number of correct choices was low. In the DNMTS, PHT groups showed low for correct choices with a long interval. The concentrations of neuropeptides were changed in the mesolimbic cortex, hippocampus, and amygdala. Brain weights were lower at 6 weeks of age in the 100 mg/kg/day PHT group, but were comparable at 16 weeks of age. This study suggests that the RM is a detectable task for the learning/memory impairments induced by PHT. In addition, it is surmised that the learning deficit is due to a working memory impairment arising from abnormal changes in neuropeptides and an injury in the fetal hippocampus.

Hyperactivity and spatial maze learning impairment of adult rats with temporary neonatal hypothyroidism.

Temporary hypothyroidism was induced in neonatal rats by 0.02% propylthiouracil (PTU) administration to lactating dams during days 0-19 after delivery, and its effects on the behavior and learning of their male offspring were examined. The serum T4 (thyroxine) level was returned to normal around 1 week after the last PTU administration, but the body weight gain was still depressed. The open field and Biel water maze tests at the age of 6 weeks showed an increased number of ambulations and an increase in errors with prolonged swimming time in the PTU rats. The radial arm maze test started at 13 weeks revealed that the PTU animals required more trials until they showed the first well-performed trial. The total number of choices was also larger, with less correct choices, and treatment effects on the response distribution and pattern were significant. Thus, the rats, which had suffered from temporary hypothyroidism in the neonatal period, showed hyperactivity and irreversible impairment in maze learning. These results suggest an involvement of temporary neonatal hypothyroidism in hippocampal dysfunction.

Cardiac dysfunction in patients with chronic progressive external ophthalmoplegia.

BACKGROUND: Chronic progressive external ophthalmoplegia (CPEO), which includes Kearns-Sayre syndrome, is a mitochondrial disorder with large deletions of mitochondrial DNA. Recently, mtDNA deletions in cardiac muscle cells were thought to be a cause of dilated cardiomyopathy. However, the cardiac involvement in patients with CPEO is generally considered to be limited to the cardiac conduction system. HYPOTHESIS: The purpose of this study was to evaluate left ventricular function in patients with CPEO. METHODS: We evaluated the cardiac function of five patients with CPEO by means of carotid pulse recording and Doppler echocardiography. RESULTS: The ratio of the pre-ejection period to ejection time was increased to 0.67 in one patient and to 0.50 in another. Echocardiography showed left ventricular dilatation and diffuse hypokinetic wall motion in both cases. Left ventricular fractional shortening was decreased to 5 and 19%, respectively, and the mean rate of circumferential shortening was decreased to 0.12 and 0.63 circ/s, respectively. One of the two patient died of congestive heart failure 2 months after the study. The Doppler pattern of left ventricular filling in the three remaining patients showed a decrease in the ratio of peak flow velocity in early diastole to that in late diastole, with an increase in deceleration time. CONCLUSION: Although cardiac involvement in patients with CPEO is generally considered to be limited to the cardiac conduction system, left ventricular dysfunction may be present and should receive more attention in the management of patients with CPEO.

Angiogenesis in hepatocellular carcinoma as evaluated by alpha smooth muscle actin immunohistochemistry.

BACKGROUND/AIMS: Angiogenesis has been known to be associated with tumor development. In this study, neovascularization in small hepatocellular carcinoma was investigated by evaluation of intratumoral arteriole counts, using alpha smooth muscle actin antibody immunohistochemistry. METHODOLOGY: Surgical specimens from 38 patients with small hepatocellular carcinoma were immunostained for alpha smooth muscle actin and proliferating cell nuclear antigen. The correlation between intratumoral arteriole density and clinicopathological factors including angiographic findings, proliferative activity, and patient prognosis were analyzed. RESULTS: Significant difference in intratumoral arteriole density were observed between well-differentiated hepatocellular carcinoma and poorly differentiated hepatocellular carcinoma (P = 0.004) or moderately differentiated hepatocellular carcinoma and poorly differentiated hepatocellular carcinoma (P = 0.011). The mean intratumoral arteriole count in the tumors showing angiographic hypervascularity was significantly higher than that in the tumors without angiographic hypervascularity (P = 0.011). A significant and positive correlation was found between proliferating cell nuclear antigen labeling index and intratumoral arteriole density (r = 0.5232, P = 0.001). A high intratumoral arteriole density in tumor was significantly correlated with shorter patients survival (P = 0.018). Cox's multivariate regression analysis showed that the intratumoral arteriole density was independent prognostic factors (P = 0.0306). CONCLUSIONS: Intratumoral arteriole density was found to be significantly associated with histological grade, proliferative activity, and patient survival. It also reflected the angiographic findings. Alpha smooth muscle actin antibody immunohistochemistry would provide a simple and biologically significant method which is usable to screen neovascularization and arterial blood supply in hepatocellular carcinoma, and may have predicting utility for patients outcome. This technique is applicable to routine paraffin sections, and may be useful as an adjunct to surgical pathology of hepatocellular carcinoma.

Plasma levels of brain natriuretic peptide as an index for evaluation of cardiac function in female gene carriers of Duchenne muscular dystrophy.

The level of plasma brain natriuretic peptide (BNP) was elevated in 8 of 15 female gene carriers of Duchenne muscular dystrophy (DMD), and the level correlated with indices of cardiac function. In one of these carriers, whose clinical course was followed for one year, the plasma BNP level was elevated before the development of cardiac symptoms, further increased with the evolution of cardiac symptoms, and then decreased after treatment for cardiac failure. These results suggest that the plasma BNP level may be useful for the early detection of cardiac dysfunction and for evaluating the efficacy of cardiac treatment in female DMD carriers.