Lutzomyia (Pintomyia) fischeri (Diptera: Psychodidae: Phlebotominae), a probable vector of American cutaneous leishmaniasis: detection of natural infection by Leishmania (Viannia) DNA in specimens from the municipality of Porto Alegre (RS), Brazil, using multiplex PCR assay

In order to determine natural Leishmania (Viannia) infection in Lutzomyia (Pintomyia) fischeri, a multiplex PCR methodology coupled to non-isotopic hybridization was adopted for the analysis of sand fly samples collected by CDC light traps in an endemic area of American Cutaneous Leishmaniasis (ACL) in the periurban region of the municipality of Porto Alegre, Rio Grande do Sul State, Brazil. We analyzed by PCR methodology 560 specimens of Lutzomyia (Pintomyia) fischeri (520 females and 40 males). The wild sand flies were grouped into 56 pools (52 females and 4 males) of 10 each, and positive results were detected in 2 of the 52 female pools, representing a minimum infection rate of 0.38% based on the presence of at least 1 infected insect in the pool. This result associated with some local evidence such as anthopophily, spatial distribution in accordance with the transmission area and human case incidence, suggests that L. (P.)fischeri may be considered as a secondary vector of ACL in the studied locality.     

Molecular genotyping of <Emphasis Type="Italic">Anisakis</Emphasis> Dujardin, 1845 (Nematoda: Ascaridoidea: Anisakidae) larvae from marine fish of Balinese and Javanese waters,Indonesia

Summary The genetic identification and distribution of Anisakis larvae in Indonesia is described. 110 Auxis rochei rochei and 45 Decapterus russellii were sampled from fish markets in North (Anturan) and South (Kedonganan) Bali. Nematode larvae from A. rochei rochei, Caesio cuning and Epinephelus areolatus from Kedonganan and from Coryphaena hippurus from Pelabuhan Ratu, South Java, were identified using sequence analysis of the internal transcribed spacers (ITS-1, ITS-2) and 5.8S region of rDNA. The larvae belonged to Anisakis typica with an identical sequence to this species from the spinner dolphin (Stenella longirostris) from Brazil, and to 2 further genotypes that differed from that sequence by 0.24–0.47 %. A. typica occurred in the migratory A. rochei rochei and C. hippurus, while Anisakis sp. 1 and 2 were isolated from the same fish species and the non-migratory C. cuning and E. areolatus. The latter genotype is distinguishable by 4 positions in the ITS-1 region (1.1 %), a genetic distance that indicates the presence of an Indonesian A. typica sibling species. The musculature infection in A. rochei rochei was low (2.5 %), indicating no major risk for the fish consumers. The much higher A. typica infection of fish intermediate hosts in the northern Bali coast is suggested to be dependent on the large dolphin population (nematode final hosts) in the waters off Lovina Beach (North Bali).     

Relationship between genetic polymorphisms of drug-metabolizing enzymes (CYP1A1, CYP2E1, GSTM1, and NAT2), drinking habits, histological subtypes, and p53 gene point mutations in Japanese patients with gastric cancer

Background Genetic polymorphisms of drug-metabolizing enzymes have recently been shown to affect susceptibility to chemical carcinogenesis. However, the molecular mechanisms of individual susceptibility to gastric cancer have not been fully understood. Therefore, we studied the relationship between the genetic polymorphisms of drug-metabolizing enzymes, drinking habits, histological subtypes, and p53 gene point mutations in Japanese patients with gastric cancer.Methods The genotypes of cytochromes P450 (CYP) 1A1 and 2E1, glutathione S-transferase (GST) M1, and N-acetyltransferase (NAT) were investigated by polymerase chain reaction (PCR), allele-specific PCR, or restriction fragment length polymorphism (RFLP) following PCR in 146 Japanese patients with gastric cancer (67 intestinal-type and 79 diffuse-type carcinomas) and 177 autopsied controls. In addition, p53 gene point mutations of exons 5 through 9 in gastric cancer tissues were determined.Results The frequency of either being a habitual drinker or having a CYP2E1^*1A/^*1A genotype was significantly higher in patients with intestinal-type gastric cancer than in control subjects. The difference between the frequencies of habitual drinkers with the CYP2E1^*1A/^*1A genotype and non-drinkers with the CYP2E1^*5B allele was much more significant in the intestinal-type cancer versus the control group. Among intestinal-type cancer patients with the CYP2E1^*1A/^*1A genotype, p53 point mutations were significantly more frequent in the group of habitual drinkers than in that of non-drinkers. On the other hand, the combination of GSTM1 null and CYP2E1^*1A/^*1A genotypes increased the risk for diffuse-type gastric cancer, but had no influence on the frequency of p53 gene mutations.Conclusions The present study suggests that individuals with both the CYP2E1^*1A/^*1A genotype and a history of habitual drinking have an increased risk of intestinal-type gastric cancer with a high frequency of p53 gene point mutations in the gastric mucosa.     

Mesocestoides litteratus (Batsch, 1786) (Cestoda: Cyclophyllidea: Mesocestoididae) from the red fox: Morphological and 18S rDNA characterization of European isolates

Summary Mesocestoides litteratus (Batsch, 1786) (Cestoda: Cyclophyllidea: Mesocestoidae) is a common parasite of the red fox (Vulpes vulpes) and other carnivores across Europe. There has been considerable debate as to the validity of M. litteratus and other closely related, often sympatric species of Mesocestoides. We examine isolates of M. litteratus from red foxes in the Czech Republic, Slovakia and Spain both morphometrically and by characterization of 18S rDNA. Morphometric ranges of all isolates confirmed their identity as M. litteratus and were usually within the range published formerly. The sequences of 18S rDNA of one or two isolates from each country were analysed. The sequences were the same and distinct from all published Mesocestoides 18S sequences with the exception of tetrathyridia from a lizard in the Czech Republic, which was identical to those of M. litteratus.     

Alterations of thep53, Rb andMDM2 genes in osteosarcoms

Molecular defects affecting tumor-suppressor genes are an important step in the genesis of sarcomas. For example, inheritance of a defectiveRb orp53 gene predisposes the carrier to develop osteosarcoma, among other malignancies. In this study, we have assessed the occurrence ofp53, Rb andMDM2 alterations in the same samples of osteosarcomas, along with representative samples of various other sarcomas. Point mutations of thep53 gene were found in 13 of 42 osteosarcomas and 1 of 8 leiomyosarcomas, and gross rearrangement of thep53 gene was demonstrated in 5 of 37 osteosarcomas. The retinoblastoma susceptibility gene (Rb) was either rearranged or deleted in 7 of 37 osteosarcomas, 1 of 7 soft-tissue sarcomas and 1 of 4 Ewing sarcomas. Remarkably, 5 of the osteosarcomas havingRb alterations also hadp53 mutations. Amplification and overexpression of theMDM2 oncogene may lead to increasedMDM2-p53 binding resulting in inactivation ofp53 function. A two- to threefold increase in the copy number ofMDM2 was detected in 7 of 37 samples, 5 of which were osteosarcomas. Amplification of theMDM2 gene occurred independently ofp53 mutation; one sample having threefold amplification ofMDM2 also had ap53 mutation. In summary, 34 alterations of thep53, Rb andMDM2 genes were found in 26 of 42 (62%) osteosarcomas.     

Quercetin mediated lifespan extension in Caenorhabditis elegans is modulated by age-1, daf-2, sek-1 and unc-43

The nematode Caenorhabditis elegans responds to flavonoid-rich diets with improved health and longevity. The precise mechanism(s) responsible for this remains to be identified, but is believed to be linked to the highly antioxidative properties of flavonoids. This study provides a dissection of lifespan modulation by the flavonoid quercetin. In detail, quercetin was shown not to act as a simple antimicrobial agent or exclusively via radical scavenging capacities. Likewise, lifespan extension had no effect on reproduction and body length. Furthermore, neither a caloric restriction mimetic nor a sirtuin (sir-2.1) dependence was identified as a likely mode of action. However, four genes were pinpointed to be required for the quercetin derived lifespan extension, namely age-1, daf-2, unc-43 and sek-1. The latter two have, to date, not been linked to quercetin-mediated lifespan extension. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A checklist of helminth fauna of weatherfish, <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> (Pisces,Cobitidae): state of the art,species list and perspectives of further studies

Summary  The helminth fauna of weatherfish (Misgurnus fossilis) in natural range is reviewed. Several helminth species reported in weatherfish are discussed with reference to host specificity and their geographical distribution. The current list of helminth parasites of the weatherfish includes 37 species. Most (15) are digenean trematodes, half of them being larval stages. Only one species of trematode — Allocreadium transversale is more specific parasite of weatherfish. Second largest group are Monogenea, with 10 species. Two of them (Gyrodactylus strelkovi and G. misgurni) are found only in the genus Misgurnus. Tapeworms (6 species) and Nematodes (6 species) are less numerous. No Acanthocephala, however, were ever found.     

A multidrug-resistant Pseudomonas aeruginosa isolate from a lethal case of sepsis induces necrosis of human neutrophils

A multidrug-resistant Pseudomonas aeruginosa (r-Pa) was isolated from a lethal case of sepsis in a bone marrow transplant recipient. Genotypic analysis of P. aeruginosa isolates demonstrated that sepsis was secondary to gut colonization. The interactions between r-Pa and patient's neutrophils were studied. The results indicate that: (1) the patient's neutrophil killing activity and nitric oxide production against r-Pa or drug sensitive P. aeruginosa (s-Pa) were profoundly impaired; (2) r-Pa cells, but not s-Pa cells or their filtered culture supernatants, induced necrosis of healthy donor neutrophils. Neutrophil necrosis emerges as a remarkable event in the pathogenesis of P. aeruginosa sepsis.     

D6S265*15 marks a DRB1*15, DQB1*0602 haplotype associated with attenuated protection from type 1 diabetes mellitus

Aims/hypothesis The HLA class II DQB1*0602 allele confers strong dominant protection against type 1 diabetes but protection is not absolute. The aim of this study was to identify markers within the HLA region that differentiate DQB1*0602 haplotypes and show different associations with disease risk. Methods We defined alleles at eight microsatellite markers spanning the HLA region in a case-control cohort from Sweden. Results We found that allele 15 at marker D6S265 (109 kb centromeric of HLA-A) was over-represented among patients carrying DRB1*15, DQB1*0602. A detailed haplotype analysis showed that DRB1*15, DQB1*0602 haplotypes carrying D6S265*15 have a ten-fold higher odds ratio (OR) than those carrying other alleles and thus confer reduced protection [OR D6S265*15=0.186 (95% CI 0.074, 0.472) vs OR D6S265*15−=0.017 (95% CI 0.005, 0.062), p<0.001]. Conclusions/interpretation Our data support the existence of a locus that modifies the protective effect associated with DQB1*0602. Typing for allele D6S265*15 can identify a less protective DQB1*0602 haplotype, thereby allowing a more accurate prediction of type 1 diabetes risk. Electronic Supplementary Material Supplementary material is available for this article at * For full lists of members of these study groups, see Electronic Supplementary Material.     

Campylobacter pylori in alcoholic hemorrhagic “gastritis”

Previously we have shown that alcohol- associated subepithelial hemorrhages histologically represent localized superficial mucosal hemorrhage, with edema in the surrounding mucosa. We studied the relationship between Campylobacter pylori (CP)and histology in gastric subepithelial hemorrhages from 20 actively drinking alcoholic patients. Biopsies of the hemorrhagic lesions and adjacent mucosa 1 and 3 cm away were taken with a jumbo forceps. Biopsy slides were coded and randomized before histologic scoring and examination for CP. CP was present in 15/20 (75%)biopsies of subepithelial hemorrhages and in 32/40 (80%)biopsies from surrounding mucosa. The mean hemorrhage score was not significantly different in biopsies of subepithelial hemorrhages with and without CP (2.7±0.4 vs 3.2±0.4)and the edema scores in adjacent, nonhemorrhagic mucosa were similar in specimens with and without CP (2.0±0.3 vs 1.6±0.5).The inflammatory cell density was significantly greater in CP-positive biopsies than in CP-negative specimens (2.0±0.2 vs 0.5±0.2, P<0.05)with the mononuclear cell and neutrophil scores contributing equally to the overall inflammatory cell score. Almost a quarter of CP-positive specimens (11/47)had no inflammation. The mean score for lining epithelial abnormalities was also significantly higher in biopsy specimens positive for CP (1.7±0.2 vs 0.5±0.3, P<0.05).In conclusion, CP is present in 75% of alcohol- associated subepithelial hemorrhages, but its prevalence is similar in adjacent, nonhemorrhagic mucosa. No relationship exists between the presence of CP and the characteristic histologic abnormalities of these subepithelial hemorrhages.Supported in part by DK grant AM 17328 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases.     

Upregulation of peroxisome proliferator-activated receptor gamma coactivator gene (<Emphasis Type="BoldItalic">PGC1A</Emphasis>) during weight loss is related to insulin sensitivity but not to energy expenditure

Aims/hypothesis We investigated whether skeletal muscle peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1A; also known as PPARGC1A) and its target mitofusin-2 (MFN2), as well as carnitine palmitoyltransferase-1 (CPT1; also known as carnitine palmitoyltransferase 1A [liver] [CPT1A]) and uncoupling protein (UCP)3, are involved in the improvement of insulin resistance and/or in the modification of energy expenditure during surgically induced massive weight loss. Materials and methods Seventeen morbidly obese women (mean BMI: 45.9 ± 4 kg/m²) were investigated before, and 3 and 12 months after, Roux-en-Y gastric bypass (RYGB). We evaluated insulin sensitivity by the euglycaemic–hyperinsulinaemic clamp, energy expenditure and substrate oxidation by indirect calorimetry, and muscle mRNA expression by PCR. Results Post-operatively, PGC1A was enhanced at 3 (p = 0.02) and 12 months (p = 0.03) as was MFN2 (p = 0.008 and p = 0.03 at 3 and 12 months respectively), whereas UCP3 was reduced (p = 0.03) at 12 months. CPT1 did not change. The expression of PGC1A and MFN2 were strongly (p < 0.0001) related. Insulin sensitivity, which increased after surgery (p = 0.002 at 3, p = 0.003 at 12 months), was significantly related to PGC1A and MFN2, but only MFN2 showed an independent influence in a multiple regression analysis. Energy expenditure was reduced at 3 months post-operatively (p = 0.001 vs before RYGB), remaining unchanged thereafter until 12 months. CPT1 and UCP3 were not significantly related to the modifications of energy expenditure or of lipid oxidation rate. Conclusions/interpretation Weight loss upregulates PGC1A, which in turn stimulates MFN2 expression. MFN2 expression significantly and independently contributes to the improvement of insulin sensitivity. UCP3 and CPT1 do not seem to influence energy expenditure after RYGB. G. Gastaldi and A. Russell share first authorship.     

Association of tannase-producing Staphylococcus lugdunensis with colon cancer and characterization of a novel tannase gene

Background The relationship between Streptococcus (St.) bovis endocarditis and colon cancer is well known. In St. bovis, the biotype I strain (formerly, St. gallolyticus) produces tannase that degrades tannins. The aim of this study was to investigate the association of tannase-producing bacteria with colon cancer, and to identify the major tannase-producing bacteria and the gene involved. Methods Tannase-producing bacteria were isolated in tannic acid-treated selective agar medium from feces and rectal swabs of 357 patients who underwent colon endoscopy from 1999 to 2004. Results Tannase-producing bacteria were isolated more frequently from the colon cancer group (24.3%) than from the adenoma or normal groups (14.4%; P < 0.05). S. gallolyticus, Staphylococcus (S.) lugdunensis, Lactobacillus (L.) plantarum, and L. pentosus were all identified as tannase-producing bacteria. Of these, S. lugdunensis was significantly isolated from the advanced-stage cancer group (22.2%; P < 0.001) more than from the early-stage cancer (8.6%) or adenoma (4.9%) groups. The gene (tanA) for tannase in S. lugdunensis was cloned and sequenced. The tanA gene was associated with all S. lugdunensis but not with other bacteria by Southern blotting and polymerase chain reaction. Conclusions Tannase-producing S. lugdunensis is associated with advanced-stage colon cancer, and the tanA gene is a useful marker for the detection of S. lugdunensis.     

Long-Term Rebamipide Therapy Improves Helicobacter pylori-Associated Chronic Gastritis

We investigated an antiinflammatory effect of rebamipide {2-{4-chlorobenzoylamino}-3-[2(1H)-quinolinon-4-yl] propionic acid}, a gastroprotective agent, in H. pylori-associated gastritis. Eighty-six patients with H. pylori-positive chronic gastritis were enrolled: 53 were treated with rebamipide (300 mg daily for 12 months) and 33 served as controls. Significant decreases in mononuclear cell infiltration into the antrum and corpus were noted in the rebamipide treatment group (before vs after, 1.42 ± 0.15 vs 1.02 ± 0.15; P < 0.01 and 1.60 ± 0.15 vs 1.21 ± 0.14; P < 0.05, respectively). Levels of infiltrating neutrophil were also decreased in the antrum (before vs after, 0.98 ± 0.14 vs 0.70 ± 0.13; P < 0.05) and were associated with a decrease in iNOS production. Sera from patients treated with rebamipide showed a significant decrease in gastrin (276.3 ± 58.3 pg/ml vs 173.0 ± 34.2 pg/ml; P < 0.05), whereas no change was observed in the control group. These suggest that long-term rebamipide treatment improved histologic gastritis and decreased serum gastrin levels in H. pylori-associated gastritis.     

An Immortalized Myocyte Cell Line, HL-1, Expresses a Functional δ -Opioid Receptor

The present study characterizes opioid receptors in an immortalized myocyte cell line, HL-1. Displacement of [³H]bremazocine by selective ligands for the mu (μ), delta (δ), and kappa (κ) receptors revealed that only the δ -selective ligands could fully displace specific [³H]bremazocine binding, indicating the presence of only the δ -receptor in these cells. Saturation binding studies with the δ -antagonist naltrindole afforded a B_maxof 32 fmols/mg protein and a K_Dvalue for [³H]naltrindole of 0.46 n . The binding affinities of variousδ ligands for the receptor in HL-1 cell membranes obtained from competition binding assays were similar to those obtained using membranes from a neuroblastoma×glioma cell line, NG108-15. Finally, various δ -agonists were found to stimulate the binding of [³⁵S]GTP γ S, confirming coupling of the cardiac δ -receptor to G-protein. DADLE (D-Ala-D-Leu-enkephalin) was found to be the most efficacious in this assay, stimulating the binding of [³⁵S]GTP γ S to 27% above basal level. The above results indicate that the HL-1 cell line contains a functionally coupled δ -opioid receptor and therefore provides an in vitro model by which to study the direct effects of opioids on cardiac opioid receptors.     

Altered gene expression with abnormal patterning of the telencephalon in embryos of diabetic Albino Swiss mice

Aims/hypothesis Several studies have shown that maternal diabetes increases the risk of congenital malformations in various organ systems including the neural tube. The present study analysed molecular and morphological changes in the forebrain of embryos from diabetic Albino Swiss mice.Methods Maternal diabetes-induced morphological changes in the forebrain were examined histologically. Cell proliferation index was assayed by BrdU labelling. In situ hybridisation and quantitative real-time PCR were used to analyse the expression of genes coding for sonic hedgehog (Shh), Nkx2.1, brain factor-1 (BF-1) and bone morphogenetic protein-4 (Bmp4) that control forebrain patterning.Results There were no distinguishable abnormalities in the forebrain of embryos from diabetic pregnancies on embryonic day 0.5. At embryonic day 11.5, embryos of diabetic pregnancies displayed a fusion and thickening of the ventral telencephalic neuroepithelium and a partial absence of the dorsal telencephalon, indicating a severe patterning defect in the dorsoventral axis of the telencephalon. The cell proliferation index was also higher in the ventral telencephalon of these embryos. Molecular analyses indicated that expression of Shh, Nkx2.1 and BF-1 was increased and their expression domains expanded dorsally in the ventral telencephalon in embryos of diabetic mice at embryonic day 11.5. The expression of Bmp4 was reduced in the dorsal forebrain of these embryos. At embryonic day 8.5, only Shh expression was increased.Conclusions/interpretation Altered expression of various genes involved in dorsoventral patterning of the forebrain is associated with forebrain malformations in embryos of diabetic mice.Abbreviations Shh sonic hedgehog - Bmp4 bone morphogenetic protein-4 - BF-1 brain factor-1 - BMPs bone morphogenetic proteins - E embryonic day     

Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) species associated with Pinus species in northern Turkey

Summary  A survey for Bursaphelenchus nematodes, associated with different conifer trees, was conducted in several forest areas in the northern regions of Turkey. Only pine trees (Pinus nigra, P. pinaster and P. sylvestris) yielded Bursaphelenchus specimens. Nematodes were identified using several morphological diagnostic characters of the genus (male spicule structure, number of lateral incisures, number and distribution of the male papillae, presence of female vulval flap), and confirmed by using RFLP analysis of the internal transcriber spacer (ITS) regions of ribosomal DNA. Three different species were identified from several sampled areas, namely B. mucronatus, B. pinophilus and B. sexdentati, representing a first report of the last two species for Turkey. The association of B. pinophilus with black pine (P. nigra) is herein reported for the first time.     

Human glutathione S-transferase A1, T1, M1, and P1 polymorphisms and susceptibility to prostate cancer in the Japanese population

Purpose: The incidence of prostate cancer is increasing in low-risk populations such as Japanese. One of the causes of this increase is considered to be associated with the Western diet, especially the high intake of red meat and fat. Glutathione S-transferase (GST) A1, T1, M1, and P1 are phase II enzymes that are important for activation and detoxification of chemical carcinogens.Methods: In this study, 190 Japanese male patients with prostate cancer and 294 healthy controls, frequency-matched for age, were compared for frequencies of GSTA1, GSTT1, GSTM1, and GSTP1 genotypes.Results: Among smokers, the frequency of the GSTA1*A/*B or *B/*B genotype in patients with prostate cancer (27.8%) showed a statistically significant increase compared with the control group frequency (18.2%; odds ratio [OR] =1.72; 95% CI, 1.01–2.94). In addition, the frequency of GSTT1 nondeletion genotype was associated with prostate cancer among smokers (OR =1.68; 95% CI, 1.06–2.68). The OR of carrying the combined genotyping of GSTA1*A/*B or *B/*B and GSTT1 nondeletion was 2.08 (95% CI, 1.14–3.80) with the combined genotyping of GSTA1*A/*A and GSTT1 null as a reference. On the other hand, no significant associations were observed for genotypes of GSTM1 and GSTP1 I105V.Conclusions: These findings suggest that the GSTA1 and GSTT1 polymorphisms are associated with prostate cancer susceptibility, especially among smokers.     

Furazolidone, Co-amoxiclav, Colloidal Bismuth Subcitrate, and Esomeprazole for Patients Who Failed to Eradicate Helicobacter pylori with Triple Therapy

There is increasing evidence of Helicobacter pylori (H. pylori) resistance to the classical triple therapy consisting of a proton-pump inhibitor and clarithromycin with either amoxicillin or metronidazole. This study is aimed at establishing the efficacy and safety of a 14-day regimen to eradicate H. pylori in patients who have failed with the classical triple therapy given for 14 days. One hundred seventy-six patients diagnosed to have H. pylori infection were given triple therapy for 14 days. Fifty-two patients who failed to respond as evident from positive 14C-urea breath test (UBT) done 4–6 weeks after the completion of triple therapy were offered a combination regimen comprised of furazolidone 200 mg b.i.d, co-amoxiclav 1 g b.i.d., colloidal bismuth subcitrate 240 mg b.i.d., and esomeprazole 40 mg b.i.d. for 14 days. The mean age of these patients was 41 ± 13 years (range 20–67). Thirty-four were males. To document eradication of H. pylori, UBT was repeated 4 weeks after the completion of treatment. On an intention-to-treat analysis, the eradication rate was 81% (42 out of 52) whereas on per-protocol basis, the eradication rate was 82.4% (42 out of 51). In conclusion, this new regimen represents a suitable second-line therapy. This study was conducted under ClinicalTrials.gov number NCT00520949.