Hormone,vitamin and contaminant status during the moulting/fasting period in ringed seals (Pusa [Phoca] hispida) from Svalbard

This study investigates the potential effects of moulting, and the concomitant period of fasting undertaken by ringed seals, on hormone, vitamin and contaminant status in adult animals in a population from Svalbard, Norway, which has relatively low contaminant levels. Concentrations of circulating total and free thyroxine and triiodothyronine, circulating and hepatic vitamin A, hepatic persistent organic pollutants and their circulating hydroxyl metabolites were higher in moulting seals compared to pre-moulting seals. The opposite trend was observed for body condition, circulating calcitriol levels and hepatic mRNA expression of thyroid hormone receptor β. No differences were observed for circulating or hepatic vitamin E levels or hepatic mRNA expressions for deioidinase 1 or 2, or retinoic acid receptor α between the two seal groups. The observed differences are likely the result of increased metabolic rates required during moulting to maintain thermal balance and replace the pelage, in combination with mobilization of lipid soluble compounds from blubber stores during the fasting period that is associated with moulting. The present study shows that contaminant levels and their relationships with physiological or endogenous variables can be highly confounded by moulting/fasting status. Thus, moulting status and body condition should be taken into consideration when using variables related to thyroid, calcium or vitamin A homeostasis as biomarkers for contaminant effects.     

Second cancers following the diagnosis of Merkel cell carcinoma: A nationwide cohort study

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin. MCCs and some other skin cancers, such as basal cell carcinomas, frequently harbour Merkel cell polyomavirus DNA. The purpose of the study was to investigate the frequency of second cancers following the diagnosis of MCC. We studied the incidence of second primary cancers after the diagnosis of MCC from the files of the Finnish Cancer Registry in 1979–2006. Among the 172 MCC patients identified a total of 34 second primary cancers were detected in 30 individuals after the diagnosis of MCC. Female MCC patients were diagnosed with 25 subsequent cancers (SIR, 2.35; 95% CI, 1.52–3.47; p < 0.001) and male patients with 9 cancers (SIR, 2.32, 95% CI, 1.06–4.40; p < 0.05). The MCC patients had an increased risk for a subsequent cancer (any site) compared to age-, gender- and calendar period-matched general population (standardized incidence ratio [SIR] 2.34; 95% confidence interval [CI], 1.62–3.27). The risks for basal cell carcinoma of the skin (O = 11), SIR, 3.48; 95% CI, 1.74–6.22 and chronic lymphocytic leukemia (O = 2), SIR, 17.9; 95% CI, 2.16–64.6 were significantly elevated. The SIRs for an overall second primary cancer risk did not change markedly with time since the diagnosis of MCC. We conclude that patients diagnosed with MCC have an increased risk for a second cancer. This risk may in part result from shared etiological factors between MCC and other tumour types, such as immunosuppression or possibly Merkel cell polyomavirus infection.     

Women With Hypertensive Pregnancies Have Difficulties in Regaining Pre‐pregnancy Weight and Show Metabolic Disturbances

The aim was to determine maternal weight gain and body composition during pregnancy and 3 months postpartum in women with uncomplicated singleton and twin pregnancies and in women with gestational diabetes (GDM) and gestational hypertension (GH). This prospective study includes four groups of subjects: those with an uncomplicated pregnancy (n = 32), those with a diagnosis of GH (n = 28), those with a diagnosis of GDM (n = 52), and those with twin pregnancy (n = 11). Their body compositions were estimated by a bioimpedance analysis and fasting lipids and glucose levels were determined during the pregnancy and 3 months after pregnancy. Women with GDM were 11.7 kg heavier than the reference group before pregnancy, whereas weight before pregnancy was not different in other investigated groups. Weight loss after delivery was attenuated in GH group. Percentage body fat remained elevated in women with GDM (34.1 ± 7.0%) and hypertension (31.5 ± 6.4%) at 3 months after pregnancy. Also their total cholesterol and low‐density lipoprotein (LDL)‐cholesterol levels as well as fasting glucose remained elevated in comparison to values of the reference group. In conclusion, women with hypertensive pregnancies, though not overweight before pregnancy, gain and retain excess gestational weight and this leads to metabolic abnormalities similar to those seen in women GDM. Thus, postpartum period appears to be critical for weight management and interventional programs are called for.     

Multi-Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge of Cynomolgus Macaques Immunized with “Hyperattenuated” SIV Constructs

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Δ5-CMV and Δ6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10⁸ 50% tissue culture infective doses (TCID₅₀) of Δ5-CMV or Δ6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Δ5-CMV vaccine group compared to the Δ6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Δ5-CMV-vaccinated animals (3.7 × 10⁵ copies/ml) was ∼1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 × 10⁴ copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.To date, the most promising approach to inducing sterilizing immunity in the macaque model has been through the use of live attenuated virus (LAV) vaccines based on simian immunodeficiency virus (SIV). A major advantage of an attenuated virus strategy for the development of a human immunodeficiency virus (HIV) vaccine is the ability of attenuated viruses to induce broad and persistent immunity (29, 51). In particular, SIV strains engineered with deletions of nef (SIVΔnef) have afforded the most significant protection upon challenge with pathogenic SIV (13, 14, 29, 60, 65, 72). Numerous SIV-derived live attenuated vaccine models have been developed, many of which employ deletions in the viral accessory genes (3, 12, 14, 15, 25, 29, 30, 53, 64, 72). In many cases, vaccinations have been shown to substantially decrease viral burden during the acute phase of infection, maintain low to undetectable levels of virus during the chronic phase of infection, and limit the progression to AIDS. Although promising, a major caveat to the live attenuated virus vaccine approach is the potential for compensatory reversion and the observations that incompletely attenuated viruses may harbor residual pathogenicity (5, 10, 14). Even SIV constructs containing multiple deletions in nef, vpr, and the negative regulatory element (NRE) can cause AIDS-like disease in adult macaques and particularly in neonates (4, 5, 27, 53). This may be analogous to some human long-term nonprogressors infected by nef-deleted HIV variants in whom a slowly increasing viral burden has been accompanied by disease progression (22, 34, 37). Additional mutations can be engineered into vaccine vectors to generate highly attenuated viruses, but this often comes at the expense of their protective efficacy (8, 23, 30).We previously made two series of novel live attenuated SIV vaccine models (25) in which the simplified SIV constructs retain all the structural viral proteins but have inactivating mutations for all viral accessory genes. These constructs retain significant antigenicity, without the pathogenic effects associated with accessory viral factors, thus limiting or eliminating the potential for reversion (25).Whether administered parenterally or mucosally, conventional challenge trials in macaques have often utilized artificially high single-dose inocula in an effort to ensure that most, if not all, of the naive or placebo-immunized animal subjects become infected following a single exposure. The rationale for using a single massive challenge has been reconsidered in light of the possibility that vaccines with protective efficacy under physiologic challenge conditions may not identified. This practice is now being replaced by an approach designed to better approximate the relatively low in vivo acquisition rates following a single sexual exposure to HIV (21, 45, 69) and should provide a more realistic assessment of vaccine efficacy in “real-world” situations. Importantly, recent studies using this approach have demonstrated viremia of magnitude and kinetics comparable to that seen following single high-dose mucosal inocula (47), and this approach has been used successfully in more recent challenge trials (31, 70). Here we are assessing the safety, immunogenicity, and protective efficacy of two hyperattenuated SIV vaccine candidates following a multi-low-dose intrarectal challenge with highly pathogenic SIVmac239 in the cynomolgus macaque model.SIV-specific humoral immune responses were assessed at various time points postvaccination and postchallenge by Western blotting. Cellular immunogenicity was monitored by evaluation of peripheral T-cell responses (via gamma interferon [IFN-γ] enzyme-linked immunospot [ELISPOT] assay) following stimulation with peptide pools spanning the entire SIVmac239 proteome. The protective efficacy of the different vaccine candidates was assessed by classical endpoints, such as quantitative analysis of plasma viral load, quantitative immunophenotyping of lymphocytes, and clinical markers of disease progression. Even using extremely attenuated SIV constructs with only minimal evidence of replication, a modest immune response that can impact long-term disease progression is generated.     

Microemulsion electrokinetic chromatographic analysis of some polar compounds

This study presents the optimization of a microemulsion electrokinetic chromatographic (MEEKC) electrolyte solution by using UV detection and with the method, simultaneous separations of chemically, biochemically and pharmaceutically related anionic and cationic compounds. Representatives of the compound groups were from isoflavonoids, benzodiazepines, metanephrines, diuretics and peptide hormones. The MEEKC separations under basic conditions were first optimized using a two-component isoflavonoid mixture as the sample and an electrolyte containing 10 mM tetraborate as the main buffer (pH 9.5). The stable microemulsion phase was adjusted with various amounts of octane, 1-butanol and sodium dodecyl sulfate (SDS). An only acidified electrolyte solution used in the study was made of phosphoric acid (pH 1.8) containing octane, SDS and ethyl acetate. The analyses with isoflavonoids showed that electrophoretic mobilities of the investigated compounds were highly related to the concentrations of SDS and 1-butanol with linear and parabolic correlation, respectively. However, addition of octane gave linear correlation only at low concentrations. In most cases four to six structurally related compounds and even 13 diuretics with various polar properties were separated from each other in basic microemulsion medium. The acidified MEEKC electrolyte gave good resolution for anionic metanephrines.     

Differential processing of leading- and lagging-strand ends at Saccharomyces cerevisiae telomeres revealed by the absence of Rad27p nuclease

Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres. Furthermore, the lengths of telomeric repeats become very heterogeneous. These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends. To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity. The results show that double-mutant strains died more rapidly than strains lacking only telomerase components. Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27delta cells. The results from studies of the replication intermediates of a linear plasmid in rad27delta cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis. These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.     

Development of a bilayered living skin construct for clinical applications

An in vitro construct of human skin (living skin equivalent, LSE) has been engineered using serially passaged human epidermal keratinocytes and human dermal fibroblasts with a matrix of type I collagen. Cells are obtained from neonatal foreskin. LSE is cast, cultured, and shipped in a single culture insert. The size and shape of theinsert determines thesize and shape of the LSE. The dermal matrix consists of dermal fibroblasts within a condensed collagen lattice. The overlying epidermis is developed at the air-liquid interface to generate a protective cornified layer. Serum was not necessaryfor development of the epidermis. LSE for graft (Graftskin) has handling characteristics similar to split-thickness skin allowing it to be meshed, stapled, and sutured. LSE was cryopreserved using 65% glycerol an rapid freezing. Viability and in vivo performance on athymic mice were similar to fresh LSE. Cells derived from human eccrine gland were able to invade and form tubules rudimentary appendages may be possible.     

Characteristics of phenotypic antibiotic resistance of Helicobacter pylori and its correlation with genotypic antibiotic resistance: A retrospective study in Ningxia

Background

Geographic differences exist in the antibiotic resistance patterns of Helicobacter pylori. Personalized treatment regimens based on local or individual resistance data are essential. We evaluated the current status of H. pylori resistance in Ningxia, analyzed resistance-related factors, and assessed the concordance of phenotypic and genotypic resistance.

Methods

Strains were isolated from the gastric mucosa of patients infected with H. pylori in Ningxia and relevant clinical information was collected. Phenotypic antibiotic susceptibility assays (Kirby–Bauer disk diffusion) and antibiotic resistance gene detection (Sanger sequencing) were performed.

Results

We isolated 1955 H. pylori strains. The resistance rates of H. pylori to amoxicillin, levofloxacin, clarithromycin, and metronidazole were 0.9%, 42.4%, 40.4%, and 94.2%, respectively. Only five tetracycline-resistant and one furazolidone-resistant strain were identified. Overall, 3.3% of the strains were sensitive to all six antibiotics. Multidrug-resistant strains accounted for 22.9%, of which less than 20% were from Wuzhong. Strains isolated from women and patients with nonulcerative disease had higher rates of resistance to levofloxacin and clarithromycin. Higher rates of resistance to metronidazole, levofloxacin, and clarithromycin were observed in the older age group than in the younger age group. The kappa coefficients of phenotypic resistance and genotypic resistance for levofloxacin and clarithromycin were 0.830 and 0.809, respectively, whereas the remaining antibiotics showed poor agreement.

Conclusion

H. pylori antibiotic resistance is severe in Ningxia. Therefore, furazolidone, amoxicillin, and tetracycline are better choices for the empirical therapy of H. pylori infection in this region. Host sex, age, and the presence of ulcerative diseases may affect antibiotic resistance of the bacteria. Personalized therapy based on genetic testing for levofloxacin and clarithromycin resistance may be a future direction for the eradication therapy of H. pylori infection in Ningxia.