Effects of Naringenin on Oxidative Stress and Histopathological Changes in the Liver of Lead Acetate Administered Rats

Lead has several adverse effects on the body due to one of the environmental pollutants. We aimed to determine the effects of naringenin on the oxidative stress and the hepatic damage against lead acetate treatment in the liver of male rats. Naringenin was administered by orogastric gavage (50 mg/kg) and lead acetate was given as daily 500 parts per million in drinking water for 4 weeks. Lead and antioxidant activities were measured, and histopathological evaluation was performed in the liver. Lead concentrations, malondialdehyde, and antioxidant activity were restored by the naringenin. The grade of necrosis, hydropic degeneration, and hepatic cord disorganization was decreased by the naringenin. However, there were no differences in the degree of sinusoidal congestion, hepatic steatosis, and capsular fibrosis between lead acetate and naringenin + lead acetate groups. We can suggest that naringenin has antioxidant and chelating effects in the liver. Nevertheless, this effect is not enough against the lead acetate induced hepatic injury.     

Complete mitochondrial genome sequences of the Arctic Ocean codfishes <Emphasis Type="Italic">Arctogadus glacialis</Emphasis> and <Emphasis Type="Italic">Boreogadus saida</Emphasis> reveal oriL and tRNA gene duplications

We have determined the complete mitochondrial genome sequences of the codfishes Arctogadus glacialis and Boreogadus saida (Order Gadiformes, Family Gadidae). The 16,644 bp and 16,745 bp mtDNAs, respectively, contain the same set of 37 structural genes found in all vertebrates analyzed so far. The gene organization is conserved compared to other Gadidae species, but with one notable exception. B. saida contains heteroplasmic rearrangement-mediated duplications that include the origin of light-strand replication and nearby tRNA genes. Examination of the mitochondrial control region of A. glacialis, B. saida, and four additional representative Gadidae genera identified a highly variable domain containing tandem repeat motifs in A. glacialis. Mitogenomic phylogeny based on the complete mitochondrial genome sequence, the concatenated protein-coding genes, and the derived protein sequences strongly supports a sister taxa affiliation of A. glacialis and B. saida.     

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

Background and Aims

Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns.

Methods

Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden.

Key Results

The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden.

Conclusions

Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.Key words: Biogeography, Cypripedium calceolus, genome size, plastid microsatellites, population genetics, seed dispersal     

Dendritic Cells Loaded with Tumor Antigens and a Dual Immunostimulatory and Anti-Interleukin 10-Specific Small Interference RNA Prime T Lymphocytes against Leukemic Cells

Vaccines using dendritic cells (DCs) harboring leukemic antigens to stimulate T cells is a possible treatment of acute myeloid leukemia (AML). Limitations of breaking tolerance to leukemic cells and lack of specific activation of T cell-mediated cytotoxicity may explain the discouraging clinical results with this approach. To break self-tolerance against AML cells, we loaded DCs with AML antigens and a bifunctional small interference (si) RNA targeting interleukin (IL) 10 and simultaneously activating toll-like receptors (TLRs). In vitro, this active siRNA inhibited (P < .05) IL-10 production by silencing the IL-10 gene in DCs. The active siRNA stimulated production of tumor necrosis factor α, implying activation of TLRs. Vaccination in a nonimmunogenic rat model mimicking human AML with the loaded DCs induced a substantial and specific T-cell cytotoxicity. Leukemic rats treated with the active siRNA lived longer and had markedly less leukemic cell mass infiltrating their bone marrow compared with rats given inactive siRNA (P < .05). Furthermore, compared with inactive siRNA treatment, the active siRNA led to significantly less extramedullar leukemic dissemination, evidenced by reduced matrix metalloproteinase activity and smaller spleens. Our data demonstrate that this bifunctional siRNA may work as an immunomodulatory drug with antileukemic properties.     

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain

Colidiarrhea and colienterotoxemia caused by F4⁺ and/or F18⁺ enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac⁺ non-ETEC vaccine candidate strain against challenge infection with F4ac⁺ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac⁺ non-ETEC strain (at day 0), and challenged with F4ac⁺ ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3⁺, CD45RA⁺, CD45RC⁺, CD21⁺, IgA⁺ and myeloid SWC3⁺ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3⁺, CD45RC⁺ and SWC3⁺ cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21⁺ cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac⁺ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.Key words: nonspecific/specific immunization, E. coli, gut immune cells, pigs.Porcine colidiarrhea and colienterotoxemia induced with F4⁺ and/or F18⁺ enterotoxigenic Escherichia coli (ETEC) strains are economically the most significant diseases of swine which account for moderate to high mortality rates and growth retardation, causing death of 5 million pigs per year in the World. Protection from ETEC is a constant challenge due to high genetic flexibility of this widespread bacterial organism.The virulence characteristics of ETEC are strongly dependent on the production of fimbrial adhesins and enterotoxins (Nagy and Fekete, 1999).The ability of adhesion of ETEC to intestinal wall is mainly due to the production of fimbriae. Enterotoxins produced by adherent ETEC strains act locally on enterocytes and stimulate increased water and electrolyte secretion and decreased fluid absorption. Several types of porcine ETEC are known today, including ETEC strain producing F18 fimbriae with their variants “ab” and “ac” (Bretschinger et al., 1990; Nagy and Fekete, 1999; Zang et al., 2007). The ETEC strains causing diarrhea mostly express F4 or F18 adhesins (Fairbrother et al., 2005; Zang et al., 2007). Nearly all known E. coli enterotoxin genes are produced by ETEC strains expressing either F4 or F18 fimbria. Zhang et al. (2007) have conclude that the dominant pathotypes causing diarrhea in weaned pigs are porcine ETEC strains expressing either F4 fimbria and heat-labile (LT) / heat stable (STb) toxins or LT/STb/EAST1 toxins, or F18 fimbria and STa/STb/Stx2e toxins. However, F18ab is more frequently associated with Shiga like toxin 2e, whereas F18ac is more frequently associated with enterotoxin STI (Cheng et al., 2005). Olasz et al. (2005) showed that the 200-kb plasmid, called pF18, contained the genes responsible for F18 fimbriae production.The curing of F18⁺ ETEC strain, i.e. loss of plasmid carrying the heat stable toxin genes in bacterial mutants has been performed by the plasmid transformation and conjugation following co-culturing of donor and recipient strains as reported earlier (Olasz et al., 2005). New vaccination strategies include the oral immunization of pigs with live avirulent nontoxigenic E. coli strains carrying the fimbrial adhesins F4 and/or F18 (Fairbrother et al., 2005).Receptors for F18ab and F18ac variants are increasingly produced up to the weaning age and the fimbriae F18ac seem to have more receptors around the ileal Peyer''s patches (Nagy et al., 1992). The colonization of the small intestine by an F18⁺ ETEC strain causes enterotoxemia. The typical clinical symptoms of the disease are neurological signs such as ataxia, convulsions and paralysis (Vögeli et al., 1996). It is well known that enterotoxic colibacillosis produces significant losses in two different age groups of pigs: first among newborn pigs and later at the postweaning age (Nagy and Fekete, 1999). The disease usually starts a few days after lacteal protection completely ceases (within the first 2 weeks after weaning), especially when weaning occurs at 3–4 weeks of age.Thus, the success of a vaccine against porcine colidiarrhea and colienterotoxemia depends upon applying it in the most efficient form at the optimal time and matching the right protective antigens with the type of virulence factors of ETEC present in the given animal population (Nagy and Fekete, 2005).The gut mucosal immune system contains specialized lymphoid tissues where environmental antigens are presented inducing B- and T-cell responses (Stokes et al., 1994). These responses are regulated by T cells and cytokines and they lead to plasma cell differentiation and the secretion of IgA antibodies onto intestinal mucosal surfaces.The aggregated lymphoid tissue such as Peyer’s patches and solitary lymphoid cells in the lamina propria both play important roles in the induction and regulation of immune responses in the gut associated lymphoid tissues (GALT) (Lacković et al.,1997b). Such organization of the GALT may provide immune protection at mucosal surfaces where the infection actually occurs (McGhee et al., 1992). Bertschinger et al. (2000) demonstrated the protective effects of a live oral vaccine containing F18 fimbria against porcine postweaning diarrhea and oedema disease.In this study we have examined the distribution and quantitative patterns of the subsets of T and B cells as well as of macrophages and secretory IgA⁺ plasma cells within GALT compartments of 4 weeks old pigs perorally immunized with an attenuated F18ac⁺ non-ETEC vaccine candidate strain against porcine colienterotoxemia. Additionally, we have evaluated adjuvanticity of levamisole in the combination with the experimental vaccine and its immunostimulatory effect when applied as an immune response modifier (IRM). Levamisole (2,3,5,6-tetrahydro-6-phenylimidazole thiazole), was originally described as a highly effective anti-helminthic compound (Thienpont et al., 1966). Subsequent studies have established its ability to restore and enhance depressed immune responses in domestic food animals and to act as an effective adjuvant for parenteral and oral vaccines (Mulcahy and Quinn, 1986; Jenkins and Hurdle, 1989; Božić et al., 2002).     

Identification of Novel ��-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn?????) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn????? and Ac-α-syn?????) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).     

Time-dependent changes in the serum levels of prolactin, nesfatin-1 and ghrelin as a marker of epileptic attacks young male patients

A relationship between hormones and seizures has been reported in animals and humans. Therefore, the purpose of this study was to investigate the association between serum levels of prolactin, nesfatin-1 and ghrelin measured different times after a seizure or non-epileptic event and compared with controls. The study included a total of 70 subjects, and of whom 18 patients had secondary generalized epilepsy (SGE), 16 patients had primary generalized epilepsy (PGE), 16 patients exhibited paroxysmal event (psychogenic) and 20 healthy males were control subjects. The first sample was taken within 5 min of a seizure, with further samples taken after 1, 24, and 48 h so long as the patient did not exhibit further clinically observable seizures; blood samples were taken once from control subjects. Prolactin was measured immediately using TOSOH Bioscience hormone assays. Nesfatin-1 and ghrelin peptides were measured using a commercial immunoassay kit. Patients suffering from focal epilepsy with secondary generalization and primary generalized epilepsy presented with significantly higher levels of serum prolactin and nesfatin-1 and lower ghrelin levels 5 min, 1 and 24 h after a seizure than patients presenting with paroxysmal events (psychogenic) and control subjects; the data were similar but not statistically significant after 48 h. The present study suggests that increased serum prolactin and nesfatin-1 concentrations, decreased ghrelin concentrations could be used as markers to identify patients that have suffered a recent epileptic seizure or other paroxysmal event (psychogenic).     

Visualization of CD44 and CD133 in Normal Pancreas and Pancreatic Ductal Adenocarcinomas: Non-overlapping Membrane Expression in Cell Populations Positive for Both Markers

Tumor-initiating cells of pancreatic ductal adenocarcinoma (PDAC) have been isolated based on expression of either CD133 or CD44. The authors aimed to visualize pancreatic cells simultaneously expressing both these cell surface markers by employing the same antibodies commonly used in cell-sorting studies. Normal and diseased pancreatic tissue, including 51 PDAC cases, were analyzed. CD44 and CD133 expression was determined by immunohistochemical double staining on formalin-fixed material and subcellular protein distribution evaluated by immunofluorescence/confocal microscopy. In the normal pancreas, CD44 and CD133 were coexpressed in the centroacinar regions but in non-overlapping subcellular compartments. As expected, CD44 was found mainly basolaterally, whereas CD133 was present on the apical/endoluminal membrane. This was also the case in chronically inflamed/atrophic pancreatic tissue and in PDAC. In some malignant ducts, CD44 was found at the apical cell membrane adjacent to but never overlapping with CD133 expression. CD44 level was significantly associated with the patient’s lymph node status. In conclusion, a CD44+/CD133+ cell population does exist in the normal and neoplastic pancreas. The preferentially centroacinar localization of the doubly positive cells in the normal parenchyma suggests that this population could be of particular interest in attempts to identify tumor-initiating cells in PDAC. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

An exploratory trial of cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses

Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E(2) following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8(+) T cells (-24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8(+) T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3(+) CD4(+) CD25(+) CD127(lo/-) Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.     

Orthostatic responses in adolescent chronic fatigue syndrome: contributions from expectancies as well as gravity

Background

Orthostatic intolerance is common in chronic fatigue syndrome (CFS), and several studies have documented an abnormal sympathetic predominance in the autonomic cardiovascular response to gravitational stimuli. The aim of this study was to explore whether the expectancies towards standing are contributors to autonomic responses in addition to the gravitational stimulus itself.

Methods

A total of 30 CFS patients (12–18 years of age) and 39 healthy controls underwent 20° head-up tilt test and a motor imagery protocol of standing upright. Beat-to-beat cardiovascular variables were recorded.

Results

At supine rest, CFS patients had significantly higher heart rate, diastolic blood pressure, and mean arterial blood pressure, and lower stroke index and heart rate variability (HRV) indices. The response to 20° head-up tilt was identical in the two groups. The response to imaginary upright position was characterized by a stronger increase of HRV indices of sympathetic predominance (power in the low-frequency range as well as the ratio low-frequency: high-frequency power) among CFS patients.

Conclusions

These results suggest that in CFS patients expectancies towards orthostatic challenge might be additional determinants of autonomic cardiovascular modulation along with the gravitational stimulus per se.