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Is Barrett's esophagus the precursor of most adenocarcinomas of the esophagus and cardia? A biochemical study.

OBJECTIVE: To obtain biochemical evidence that Barrett's esophagus (BE) is the precursor of most adenocarcinomas (Adc) of the esophagus and cardia. SUMMARY BACKGROUND DATA: Based on morphologic data, BE was previously proposed as the precursor of most Adc of the esophagus. This hypothesis would receive strong support if biochemical evidence were found to demonstrate a pattern common to BE and Adc of the esophagus and cardia. METHODS: We studied the presence of intestinal-type proteins sucrase-isomaltase (SI) and crypt Cell Antigen (CCAg) in BE, Barrett's Adc, and esophageal-cardial Adc without BE. In each case specimens were collected from normal esophagus, stomach, tumor, and BE mucosa when present. To study related conditions, five specimens of peptic esophagitis and of squamous cell carcinoma were also analyzed. An indirect immunofluorescence technique was employed and sections were analyzed with laser confocal microscopy imaging. RESULTS: Most Barrett's mucosa specimens stained positively for SI (93%) and CCAg (89%). These proteins were detected in BE independently of the type of metaplasia, the coexistence of dysplasia, or the presence of associated Adc. SI and CCAg were present in 25 (96%) and 24 (92%) of the cases of Adc respectively. No statistical difference was detected in SI and CCAg expression between Adc samples with and without BE, between BE and Adc samples with or without BE, and between tumors located in the esophagus versus the cardia. No staining for these proteins was detected in stomach or esophageal mucosa, in submucosal glands of the esophagus, in peptic esophagitis or squamous cell carcinoma. CONCLUSION: These data show that BE and Adc of the esophagus and cardia have a similar phenotype and support the hypothesis that most of these tumors probably originate from preexisting BE.

Adenocarcinoma↗

Polymorphism in the interleukin-4 promoter affects acquisition of human immunodeficiency virus type 1 syncytium-inducing phenotype.

The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4(+) cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 -589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.

Alleles↗

Evidence for a role of T-helper type 2 cytokines in the acquisition of human immunodeficiency virus syncytium-inducing phenotype.

BACKGROUND: The objective of the present study was to investigate the relationship between the serum cytokine pattern of T-helper (Th) response and the acquisition of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants in HIV-1-seropositive patients treated with antiretroviral drugs. METHODS: Serum cytokines of Th1/Th2 responses were analysed in a case-control study of 20 individuals selected from a cohort of HIV-1-infected patients without SI variants at entry, who developed or did not develop SI virus during a prospective follow-up. A group of 10 patients with SI variants at study entry was also evaluated. Serum concentration of interferon (IFN) gamma, interleukin (IL) 2, IL-4 and IL-10 was evaluated by mean of a commercial enzyme immunoassay. RESULTS: Despite close matching for immunological (CD4+ cell count) and virological (p24 antigen, serum HIV viraemia) parameters, SI-converting patients showed at baseline significantly lower serum levels of IL-2 and higher concentrations of IL-4 than those who remained persistently negative for SI variants. Shortly after the phenotype conversion, SI-converting patients were characterized by significantly high serum concentration of IL-4 and by low levels of IFN-gamma (Th2-like pattern). Patients with SI phenotype at study entry featured lower mean levels of both IL-4 and IL-10, but mean IFN-gamma and IL-2 values were higher, although the clinical and immunological baseline was also poorer and no statistical analysis could be applied. CONCLUSION: Serum cytokine pattern of Th1/Th2 response differs between patients with NSI and SI phenotype. Our data strongly suggest that the Th2 cytokine pattern could be associated with the acquisition of the SI HIV-1 phenotype.

Case-Control Studies↗

Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture.

We previously demonstrated a correlation between the presence of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants showing tropism for cell line H9 and the occurrence of rapid CD4 cell decline and progression to AIDS. In contrast, in stable asymptomatic individuals, we detected only isolates with low replication rates that were non-syncytium-inducing (NSI) and nontropic for the H9 cell line. Here, we investigated the monocytotropism of established HIV-1 isolates with a panel of isolates and with biological HIV-1 clones with distinct phenotypes. Moreover, the prevalence and biological phenotypes of monocytotropic HIV-1 variants in the course of HIV-1 infection were analyzed in comparative primary isolation studies on peripheral blood lymphocytes (PBL) and monocyte-derived macrophages (MDM). In cell-free infection studies with MDM from eight blood donors, 13 of 17 NSI isolates but only 4 of 14 SI isolates were able to infect MDM. NSI isolates also infected significantly more different donors than SI variants (median, 3 of 8 versus 0 of 8). This enhanced monocytotropism of NSI isolates was confirmed in experiments with biological HIV-1 clones with distinct phenotypes recovered from the same donor. To investigate the prevalence and biological phenotypes of monocytotropic variants in different stages of HIV-1 infection, sequential isolates from peripheral blood mononuclear cell samples from nine asymptomatic individuals, five of whom progressed to AIDS and seven of whom had a known time of seroconversion, were recovered by cocultivation with both PBL and MDM. Monocytotropic variants were obtained from 37 of 42 time points. All monocytotropic variants were NSI in PBL culture and non-T-cell-line tropic, even when SI, T-cell-line-tropic HIV-1 variants could be recovered from the same patient sample by cocultivation with PBL. We conclude that monocytotropic HIV-1 variants mostly have an NSI phenotype in PBL and, in contrast to SI variants, are present at all stages of HIV-1 infection. These results suggest an important role for monocytotropic variants in the persistence of HIV-1 infection.

Acquired Immunodeficiency Syndrome↗

Sonographic assessment and grading of spleen size.

Ultrasonography was used to evaluate spleen size in patients with various clinical conditions including those of the liver, blood, collagen, or autoimmune disease. To express spleen size, a spleen index (SI), the product of the transverse diameter and its perpendicular diameter measured on the maximum cross-sectional image of the spleen, was used. SI correlated well with the volumes of resected spleens. Splenomegaly was present in high percentages of patients with liver, blood, collagen, and autoimmune disease, even though a majority of these spleens were not large enough to palpate. By grading the SI, characteristic distributions of SI were obtained for patients with different types of diseases. Obtaining and grading the SI by the use of ultrasound appears to be a significant supplemental aid for evaluating spleen size, especially in patients whose spleens are not palpable.

Adolescent↗

MRI signal characteristics of progressive massive fibrosis in silicosis.

AIM: To determine the magnetic resonance imaging (MRI) signal characteristics of progressive massive fibrosis (PMF) in silicosis. SUBJECTS AND METHODS: We evaluated prospectively the MR appearances in 17 patients with 34 PMF lesions on the basis of pre-contrast signal intensity (SI) and SI pattern and post-contrast enhancement pattern, using a 0.5-T unit. There were 13 PMF lesions in six patients who had silicosis and 21 PMF lesions in 11 patients who had silicotuberculosis. The SI pattern on T2-weighted image (WI) was classified into four types and the pattern of contrast enhancement on T1-WI was classified into three types. MR appearances of PMF lesions were correlated with the findings of computed tomography (CT). RESULTS: The commonest signal intensity characteristic was isointensity (70%) on T1-WI and hypointensity (68%) on T2-WI when compared with skeletal muscle. For signal pattern on T2-WI, a type with only internal high SI areas (46% in silicosis group, 38% in silicotuberculosis group) was most frequent. All of these areas corresponded to the low density areas at CT, suggestive of necrosis. After intravenous contrast medium enhancement, rim enhancement (54% in silicosis group, 52% in silicotuberculosis group) was most frequent, followed by no enhancement. CONCLUSION: The most common MRI appearance of PMF was isointensity on T1-WI and hypointensity on T2-WI when compared with skeletal muscle, with internal high SI areas on T2-WI and either rim enhancement or no enhancement.

Aged↗

The degree of fat saturation does not alter glycemic, insulinemic or satiety responses to a starchy staple in healthy men.

Inclusion of fat reduces the glycemic response to a carbohydate meal, although the effect of different types of fat on glycemic, insulinemic and satiety responses is unclear. Ten healthy men received 50-g carbohydrate portions of mashed potato with isoenergetic amounts of butter (saturated fatty acid), Sunola oil (monounsaturated fatty acid) or sunflower oil (PUFA) and two 50-g glucose loads on separate days. Capillary blood was collected at regular intervals for 2 h. Satiety ratings were assessed by use of a rating scale. The glycemic index (GI), insulin index (II) and satiety index (SI) scores were calculated. Energy intakes from a meal consumed ad libitum at 2 h and for the remainder of the day were quantified. The GI values ranged from 68 +/- 8 to 74 +/- 10 and the II values ranged from 113 +/- 10 to 122 +/- 17, but there was no effect of fat type. SI scores and subsequent energy intake did not differ among the test meals. Substitution of unsaturated fats for saturated fatty acids had no acute benefits on postprandial glycemia, insulin demand or short-term satiety in young men.

Adult↗

CXCR4 mediates entry and productive infection of syncytia-inducing (X4) HIV-1 strains in primary macrophages.

CCR5 and CXCR4 are the main coreceptors for non-syncytia-inducing (NSI) and syncytia-inducing (SI) HIV-1 strains, respectively. NSI HIV-1 isolates do not infect either human lymphoid or monocytoid cell lines, and this inability correlates with the absence of CCR5 expression in these cell types. The ability of SI HIV-1 isolates to infect human primary macrophages has been disputed. Here, we report that CXCR4 is expressed in primary blood-derived human mononuclear phagocytes at all stages of differentiation, although the maturation process correlates with downregulation of CXCR4 mRNA. Infection experiments with the SI molecular clone NL4-3 tagged with a mutant of the green fluorescent protein established that both monocytes and attached macrophages are susceptible to infection with CXCR4-restricted HIV-1 strains. NL4-3 entry into primary macrophages could be blocked by SDF-1alpha in a dose-dependent manner, or by the anti-CXCR4 monoclonal antibody 12G5. HIV-1 entry led to productive infection. No evidence of postentry defects or nuclear import delay for CXCR4-restricted HIV-1 strains was detected using a quantitative real-time PCR assay measuring HIV-1 DNA entry into the nucleus. Macrophages infected by HIV-1 and expressing virus were maintained in culture for long periods of time (up to 5 months). These results demonstrate that CXCR4 is the main HIV-1 SI coreceptor in human primary macrophages and underline the importance of the macrophage as a long-living viral reservoir for HIV-1.

Cells, Cultured↗

The First Tri- and Tetraalkoxysilanes with Four Different Substituents.

Unsymmetrically substituted tri- and tetraalkoxysilanes were surprisingly found to be configurationally stable and easy to prepare. It was found that compounds of type MeSi(OR)(2)(OR'), MeSi(OR)(OR')(OR"), Si(OR)(2)(OR')(2), Si(OR)(2)(OR')(OR"), Si(OR)(3)(OR'), and even Si(OR)(OR')(OR")(OR) could be obtained by sequential addition of a variety of chiral and achiral alcohols to methyltrichlorosilane or tetrachlorosilane in the presence of pyridine. In the case of MeSi(OR)(OR')(OR") and Si(OR)(OR')(OR")(OR), two types of compounds that have never been prepared before, were obtained in good yield.

Journal Article↗

Calcium currents in rat myoballs and their inhibition by insulin.

Two Ca2+ currents were found in myoballs prepared from primary culture of hindlimb muscles from rat embryos. One whole cell current, not described previously, was early, fast, and transient. It depended on the presence of Na+ in the bathing solution and was blocked by tetrodotoxin. Despite this behavior suggesting that it might be via a Na+ channel, its reversal potential exceeded 80 mV compared to 46 mV for the Na+ equilibrium potential. It was increased by increased Ca2+ concentration in the bathing solution and eliminated by Co2+ and by diltiazem. The other Ca2+ current resembled the slow inward Ca2+ current ICa(si), described in other cell types. Insulin decreased both Ca2+ currents; even at 64 pM insulin reduced ICa(si). When outward K+ currents were prevented, the myoball action potential was altered greatly, owing to the unopposed effect of ICa(si). Its duration was on the order of seconds. Insulin, in a concentration-dependent manner, beginning at 60 pM, reduced the duration of this action potential more effectively than nimodipine. This is the most sensitive response to insulin observed in skeletal muscle.

Action Potentials↗

Determinants involved in the affinity of alpha-conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors from muscle contain two functionally active and pharmacologically distinct acetylcholine-binding sites located at the alpha/gamma and alpha/delta subunit interfaces. The alpha-conotoxins are competitive antagonists of nicotinic receptors and can be highly site-selective, displaying greater than 10,000-fold differences in affinities for the two acetylcholine-binding sites on a single nicotinic receptor. The higher affinity site for alpha-conotoxins GI, MI, and SI is the alpha/delta site on mouse muscle-derived BC3H-1 receptors. However, alpha-conotoxins GI and MI exhibit higher affinity for the other site (alpha/gamma site) on nicotinic receptors from Torpedo californica electric organ. alpha-Conotoxin SI does not distinguish between the two acetylcholine-binding sites on Torpedo receptors. In this study, alpha-conotoxins [K10H]SI and [K10N]SI displayed wild-type affinity for the two acetylcholine-binding sites on BC3H-1 receptors but a 10-20-fold decrease in apparent affinity at one of the two acetylcholine-binding sites on Torpedo receptors. alpha-Conotoxin [P9K]SI displayed a selective and dramatic increase in the apparent affinity for the alpha/delta site of BC3H-1 receptors and for the alpha/gamma site of Torpedo receptors. alpha-Conotoxin [R9A]GI displayed a reduction in affinity for both acetylcholine-binding sites on BC3H-1 receptors, although the extent of its selectivity for the alpha/delta site was retained. alpha-Conotoxin [R9A]GI also displayed a loss of affinity for the two acetylcholine-binding sites on Torpedo receptors, but its site-selectivity was apparently abolished. These results indicate that positions 9 and 10 in alpha-conotoxins GI and SI are involved in complex species- and subunit-dependent interactions with nicotinic receptors.

Animals↗

Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay.

Syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) are evolutionary variants that are associated with rapid CD4+ cell loss and rapid disease progression. The heteroduplex tracking assay (HTA) was used to detect evolutionary V3 variants by amplifying the V3 sequences from viral RNA derived from 50 samples of patient plasma. For this V3-specific HTA (V3-HTA), heteroduplexes were formed between the patient V3 sequences and a probe with the subtype B consensus V3 sequence. Evolution was then measured by divergence from the consensus. The presence of evolutionary variants was correlated with SI detection data on the same samples from the MT-2 cell culture assay. Evolutionary variants were correlated with the SI phenotype in 88% of the samples, and 96% of the SI samples contained evolutionary variants. In most cases the evolutionary V3 variants represented discrete clonal outgrowths of virus. Sequence analysis of the six discordant samples that did not show this correlation indicated that three non-syncytium-inducing (NSI) samples had V3 sequences that had evolved away from the consensus sequence but not toward an SI genotype. A fourth sample showed little evolution away from the consensus but was SI, which indicates that not all SI variants require basic substitutions in V3. The other two samples had SI-like genotypes and NSI phenotypes, suggesting that V3-HTA was able to detect SI emergence in these samples in the absence of their detection in vitro. V3-HTA was also used to confirm SI variant selection in MT-2 cells and to examine the possibility of variant selection during virus culture in peripheral blood cells.

Acquired Immunodeficiency Syndrome↗

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.

Animals↗

Gastrointestinal stromal tumours (GISTs): a clinicopathological and molecular study of 66 cases.

AIMS: Predicting the clinical behaviour of gastrointestinal stromal tumours (GISTs) is difficult and criteria delineating benign from malignant cases are not firmly established. The aims of this study were to define the clinicopathological and molecular features of 66 GISTs, and to determine whether any specific parameters were associated with patient outcome. METHODS: Archival cases of GIST from two major teaching hospitals in Western Australia were studied. Inclusion criteria for the study were: (1) appropriate morphology, (2) CD117 positivity, (3) adequacy of pathological material for study, and (4) exclusion of other tumour types on the basis of immunophenotypic and/or ultrastructural features. Expression of CD117, CD34, S100 protein, keratin (using broad spectrum MNF116), alpha-smooth muscle actin (SMA) was determined by immunohistochemistry. PCR and single strand conformation polymorphism (PCR-SSCP) analysis were used to screen for mutations in exons 11 and 9 of c-kit. RESULTS: There were equal numbers of males and females with a mean age at diagnosis of 60 years, followed up for a mean of 54 months. Thirteen patients (21%) had died of GIST by the end of the study. Tumours were mostly located in the stomach (67%) and small intestine (SI; 25%). The cell types were pure spindle (68%), pure epithelioid (12%) and mixed epithelioid/spindle (20%). c-kit mutations were found in 69% of GISTs, with the large majority (91%) occurring in exon 11. Size > or = 10 cm, tumour necrosis and pure epithelioid cell morphology each were the only factors significantly associated with adverse survival (p=0.038, and p=0.047 and p=0.028, respectively). Mitotic activity > or = 5/50 HPF showed a definite trend association with adverse survival, but unlike some other studies, did not achieve statistical significance (p=0.067). c-kit mutations were more frequent in small intestinal GISTs (p=0.05) and in those with pure spindle cell morphology (p=0.023) but were not associated with patient outcome. CONCLUSION: In this study, size > or = 10cm, necrosis and/or pure epithelioid cell morphology correlated significantly with adverse survival. Mitotic activity showed a strong association with survival but this did not reach statistical significance. c-kit mutations occurred mainly in GISTs of the SI, and in purely spindle cell tumours. While the mutation status did not associate with patient outcome in this series, this remains a controversial issue, and further studies are needed to assess whether the type of mutation affects response to tyrosine kinase inhibitor therapy in metastatic GISTs. CD117 staining of any mesenchymal lesion of the gastrointestinal tract should be mandatory for accurate classification. PCR-SSCP analysis is a fast, sensitive and relatively inexpensive method of analysing c-kit mutations, which may be important prognostically and also of therapeutic relevance in the assessment of new tyrosine kinase inhibitor therapies.

Adult↗

Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS.

OBJECTIVE: To investigate the relation between detection of syncytium-inducing (SI), human immunodeficiency virus type 1 (HIV-1) variants, rate of CD4+ cell decline, and clinical progression. DESIGN: Prospective study during a 2.5-year follow-up period; cohort study with pairwise matched controls. SETTING: The Amsterdam cohort study on the course of HIV-1 infection in homosexual men. PARTICIPANTS: Asymptomatic HIV-1 infected men (n = 225) were tested for the presence of SI variants and were studied prospectively for CD4+ cell decline and clinical progression. In addition, 45 men with a defined moment of appearance of SI variants and 45 matched controls without SI variants were compared for CD4+ cell decline. MEASUREMENTS: Syncytium-inducing variants were detected by cocultivation of peripheral blood mononuclear cells with the MT-2 T-cell line. RESULTS: During a 30-month period, 70.8% of the men with SI variants progressed to AIDS, compared with 15.8% of men without SI variants at entry (P < 0.0001). Multivariable Cox proportional hazard analysis, controlling for CD4+ cell count and HIV-p24 antigenemia, showed a relative hazard for SI variants of 6.7 (95% Cl, 3.5 to 12.7). In the matched control study, before the appearance of SI variants, CD4+ cell counts of 45 men with SI variants and their controls were compared. Syncytium-inducing variants emerged at a mean CD4+ cell count of 0.48 x 10(9)/L(Cl, 0.42 to 0.54), coinciding with the onset of a threefold increased rate of CD4+ cell decline. Men developing AIDS with SI variants had decreased CD4+ cell counts (0.08 x 10(9)/L; 95% Cl, 0.05 to 0.12) at the time of diagnosis compared with persons progressing to AIDS without SI variants (0.25 x 10(9)/L 95% Cl, 0.15 to 0.41) (P = 0.0035]. CONCLUSIONS: The HIV-1 biological phenotype is a practical, binary marker for progression to AIDS, which is independent of decreased CD4+ cell counts and antigenemia. Appearance of SI variants, occurring 2 years before progression to AIDS on the average, is predictive for a significantly increased rate of CD4+ cell decline.

Acquired Immunodeficiency Syndrome↗

The silicon content of beer and its bioavailability in healthy volunteers.

Dietary Si, as soluble orthosilicic acid (OSA), may be important for the growth and development of bone and connective tissue. Beer appears to be a major contributor to Si intake, although the Si content of beer and its bioavailability in human subjects have not been well established. Here we investigated the Si content of different beers and then estimated Si absorption from beer in healthy volunteers. The Si content of seventy-six different beers was estimated using inductively coupled plasma optical emission spectrometry and one of the beers, used in the ingestion study, was ultrafiltered to determine OSA content. Next, following the ingestion of 0.6 litres beer (22.5 mg Si; 4.6 % (v/v) ethanol), serum and urinary Si levels were measured in nine healthy volunteers over a 6 h period. A solution of OSA was similarly investigated as a positive control and water and 4.6 % ethanol as negative controls. The mean Si level of beer was 19.2 (sd 6.6) mg/l; the median Si level was 18.0 mg/l. There was no significant difference in the Si levels of the different beers by geographical origin or type of beer. Serum and urinary Si levels increased considerably following the ingestion of beer or a solution of OSA but not with the ingestion of either 4.6 % ethanol or water. The ultrafilterability of Si from beer (about 80 %) and its absorption in volunteers (about 55 %) was comparable with that of a solution of OSA suggesting that Si in beer is present chiefly in a monomeric form and is readily bioavailable.

Adult↗