Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3-M-38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala-Ala-Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38 and p3-Ub-M-38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H-2b) mice. The cytotoxicity of T cells was detected by LDH-release method and by enzyme-linked immunospot assay for epitope-specific cells secreting interferon-gamma. The results showed that DNA immunization with p3-M-38 vaccine could induce epitope-specific CD8 CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein-targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C-terminus and protein-targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi-epitope DNA vaccines against tuberculosis.     

Performance of the CKD-EPI creatinine-cystatin C glomerular filtration rate estimation equations in a multiethnic Asian population

INTRODUCTION

Clinical practice guidelines recommend using creatinine-based equations to estimate glomerular filtration rates (GFRs). While these equations were formulated for Caucasian-American populations and have adjustment coefficients for African-American populations, they are not validated for other ethnicities. The Chronic Kidney Disease-Epidemiology Collaborative Group (CKD-EPI) recently developed a new equation that uses both creatinine and cystatin C. We aimed to assess the accuracy of this equation in estimating the GFRs of participants (healthy and with chronic kidney disease [CKD]) from a multiethnic Asian population.

METHODS

Serum samples from the Asian Kidney Disease Study and the Singapore Kidney Function Study were used. GFR was measured using plasma clearance of ^99mTc-DTPA. GFR was estimated using the CKD-EPI equations. The performance of GFR estimation equations were examined using median and interquartile range values, and the percentage difference from the measured GFR.

RESULTS

The study comprised 335 participants (69.3% with CKD; 38.5% Chinese, 29.6% Malays, 23.6% Indians, 8.3% others), with a mean age of 53.5 ± 15.1 years. Mean standardised serum creatinine was 127 ± 86 µmol/L, while mean standardised serum cystatin C and mean measured GFR were 1.43 ± 0.74 mg/L and 67 ± 33 mL/min/1.73 m², respectively. The creatinine-cystatin C CKD-EPI equation performed the best, with an estimated GFR of 67 ± 35 mL/min/1.73 m².

CONCLUSION

The new creatinine-cystatin C equation estimated GFR with little bias, and had increased precision and accuracy in our multiethnic Asian population. This two-biomarker equation may increase the accuracy of population studies on CKD, without the need to consider ethnicity.     

A comparison of the thresholding strategies of micro-CT for periodontal bone loss: a pilot study

Objectives:

Micro-CT provides three-dimensional details and has been widely used for biomedical assessments. This study aimed to determine the most appropriate threshold method for quantitatively assessing the dynamics of periodontal destruction.

Methods:

Inflammation was induced by submerging a silk ligature in the sulcus of the maxillary second molars of rats, and the animals were killed prior to ligature placement and after 7 and 21 days. The maxillae were examined for the bone resorptive activities by micro-CT, histology and tartrate-resistant acid phosphatase staining. The imaging threshold was determined by CT phantom, global and local algorithms. A bone fraction measurement from each threshold-determining technique was compared with histomorphometry. The reliability and reproducibility were examined by the intraclass correlation coefficient (ICC) and the coefficient of variation.

Results:

Significant reduction of inflammatory infiltration (p < 0.01) and active osteoclastic resorption (p < 0.05) from Day 7 to Day 21 were noted. High inter- and intraexaminer agreement were demonstrated in both histomorphometric and micro-CT assessments (ICC > 0.98). The algorithm-based technique demonstrated stronger correlation to histomorphometry than phantom-based thresholds, and the highest agreement was presented by the local algorithm (ICC > 0.96). This, however, was considerably computationally expensive.

Conclusions:

The local threshold-determining algorithm is suggested for examining inflammation-induced bone loss. Further investigation will be aimed at enhancing computational efficiency.     

Synthesis of cobalamin coenzymes by human lymphocytes in vitro and the effect of folates and metabolic inhibitors

The uptake of 57Co-cyanocobalamin (CN-Cbl) and its conversion to 5- deoxyadenosylcobalamin (Ado-Cbl), methylcobalamin (Me-Cbl), and hydroxocobalamin (OH-Cbl) has been studied in phytohemagglutinin (PHA)- transformed lymphocytes from normal subjects and patients with patients with pernicious anemia. Uptake and conversion were much greater by PHA- stimulated lymphocytes than by mature non-transformed lymphocytes. In normal cells, uptake of 57Co-CN-Cbl and synthesis of the cobalamin coenzymes were approximately linear between 3 and 48 hr incubation. Ado- Cbl was the major cobalamin formed, and after 72 hr the cells contained about twice as much Ado-Cbl as Me-Cbl. Uptake by lymphocytes from patients with untreated pernicious anemia (PA) was greater than that by normal lymphocytes, but the proportions of Ado-Cbl and Me-Cbl synthesized by each were similar. Folic acid and methyltetrahydrofolate enhanced synthesis of Me-Cbl both in normal and in PA cells, while methotrexate and 5-fluorouracil depressed it. This depression was overcome by 5-formyltetrahydrofolate, suggesting that an uninterrupted folate cycle may play an important role in Me-Cbl synthesis.     

镁及镁合金在仿生体液中的腐蚀降解行为

目的:观察纯镁及镁锌系列合金在仿生体液中的腐蚀行为,分析其是否具有生物临床应用价值。方法:实验采用纯镁(99.9%)、镁锌锆(ZK60)、镁锌锆钇(Mg-5.6Zn-0.55Zr-0.9Y)3种合金材料,将试样分别放入仿生溶液中浸泡10d,仿生溶液恒温(37.0±0.5)℃。用BP211D电子天平测量了试样在仿生体液中的腐蚀失重,用LK98BII型电化学系统测量了试样在仿生体液中腐蚀时的Tafel曲线,同时观察仿生溶液pH值的变化结果:①在仿生体液中242h后,纯镁、镁锌锆和镁锌锆钇损失量分别为0.9%,3.1%和抗蚀性1.7%。②在实验条件下,腐蚀电流密度纯镁为2.03mA/mm2,镁锌锆为10.14mA/mm2,镁锌锆钇为4.42mA/mm2。③随着镁合金在仿生体液中浸泡时间延长,溶液的pH值增高,电势也随着pH值的增加而减小,镁及镁合金的腐蚀速率会降低。结论:①合金中杂质元素越少,耐腐蚀性能越好,选择纯镁或含Y的镁合金作为镁基生物材料的耐蚀性较好。②在镁锌合金中添加钇后其耐体液腐蚀性能得到了改善。     

International variation in GP treatment strategies for subclinical hypothyroidism in older adults: a case-based survey

Background

There is limited evidence about the impact of treatment for subclinical hypothyroidism, especially among older people.

Aim

To investigate the variation in GP treatment strategies for older patients with subclinical hypothyroidism depending on country and patient characteristics.

Design and setting

Case-based survey of GPs in the Netherlands, Germany, England, Ireland, Switzerland, and New Zealand.

Method

The treatment strategy of GPs (treatment yes/no, starting-dose thyroxine) was assessed for eight cases presenting a woman with subclinical hypothyroidism. The cases differed in the patient characteristics of age (70 versus 85 years), vitality status (vital versus vulnerable), and thyroid-stimulating hormone (TSH) concentration (6 versus 15 mU/L).

Results

A total of 526 GPs participated (the Netherlands n = 129, Germany n = 61, England n = 22, Ireland n = 21, Switzerland n = 262, New Zealand n = 31; overall response 19%). Across countries, differences in treatment strategy were observed. GPs from the Netherlands (mean treatment percentage 34%), England (40%), and New Zealand (39%) were less inclined to start treatment than GPs in Germany (73%), Ireland (62%), and Switzerland (52%) (P = 0.05). Overall, GPs were less inclined to start treatment in 85-year-old than in 70-year-old females (pooled odds ratio [OR] 0.74 [95% confidence interval [CI] = 0.63 to 0.87]). Females with a TSH of 15 mU/L were more likely to get treated than those with a TSH of 6 mU/L (pooled OR 9.49 [95% CI = 5.81 to 15.5]).

Conclusion

GP treatment strategies of older people with subclinical hypothyroidism vary largely by country and patient characteristics. This variation underlines the need for a new generation of international guidelines based on the outcomes of randomised clinical trials set within primary care.     

Genetic control of the circulating concentration of transforming growth factor type beta1

The concentration of transforming growth factor beta (TGF-beta) in plasma has been correlated with the development of several diseases, including atherosclerosis and certain forms of cancer. However, the mechanisms that control the concentration of TGF-beta in plasma are poorly understood. In a study of 170 pairs of female twins (average age 57.7 years) we show that the concentration of active plus acid- activatable latent TGF-beta1 [(a+l) TGF-beta therefore is predominantly under genetic control (heritability estimate 0.54). Single strand conformation polymorphism (SSCP) mapping of the TGF-beta1 gene promoter has identified two single base substitution polymorphisms. The two polymorphisms (G-->A at position -800 bp and C-->T at position -509 bp) are in linkage disequilibrium (correlation coefficient Delta = 0.215, P < 0.01). The C-509T polymorphism is significantly associated with the plasma concentration of (a+l) TGF-beta1, explaining 8.2% of the additive genetic variance of (a+l) TGF-beta1 concentration. It is therefore possible that predisposition to atherosclerosis, bone diseases or various forms of cancer may be correlated with the presence of particular alleles at the TGFB1 locus.      

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana. HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Plant innate immunity activation relies on detection of invasion pattern (IP) molecules that are perceived by plant cells (1, 2). Non–self-recognition IPs include essential components of whole classes of microorganisms, such as fragments of flagellin, peptidoglycans, mc-3-OH-FAs from bacteria or fragments of chitin, and β-glucans from fungi and oomycetes, respectively (3, 4). Apoplastic IPs are sensed by plant plasma membrane–localized receptor kinases (RKs) or receptor-like proteins (RLPs) that function as pattern recognition receptors (PRRs) (5, 6). Activation of the immune response requires the recruitment of regulatory receptor kinases and receptor-like cytoplasmic kinases (RLCKs) by PRRs (7). Early cellular immune signaling of pattern-triggered immunity (PTI) includes ion-flux changes at the plasma membrane, rise in cytosolic Ca²⁺ levels, production of extracellular reactive oxygen species (ROS), and activation of mitogen-activated protein kinases (MAPKs) and/or Ca²⁺-dependent protein kinases (3, 8–10). Biosynthesis and mobilization of plant hormones, including salicylic acid, jasmonic acid, ethylene, abscisic acid and brassinosteroids, ultimately modulate plant resistance to phytopathogens (11–14).Rhamnolipids (RLs) are extracellular amphiphilic metabolites produced by several bacteria, especially Pseudomonas and Burkholderia species (15–17). Acting as wetting agents, RLs are essential for bacterial surface dissemination called swarming motility and for normal biofilm development (18–20). These glycolipids are produced from l-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) precursors (15, 21). HAAs are synthesized by dimerization of (R)-3-hydroxyalkanoyl-CoA in Pseudomonas, forming congeners through the RhlA enzyme (21). The opportunistic plant pathogen Pseudomonas aeruginosa and the phytopathogen Pseudomonas syringae produce extracellular HAAs (16, 22–24). In P. syringae, HAA synthesis is coordinately regulated with the late-stage flagellar gene encoding flagellin (22). HAA and RL production is finely tuned and modulates the behavior of swarming migrating bacterial cells by acting as self-produced negative and positive chemotactic-like stimuli (25). RLs contribute to the alteration of the bacterial outer membrane composition, by shedding flagellin from the flagella (26) and by releasing lipopolysaccharides (LPS), resulting in an increased hydrophobicity of the bacterial cell surface (27). In mammalian cells, RLs produced by Burkholderia plantarii exhibit endotoxin-like properties similar to LPS, leading to the production of proinflammatory cytokines in human mononuclear cells (28, 29). They also subvert the host innate immune response through manipulation of the human beta-defensin-2 expression (30). Moreover, RLs from Burkholderia pseudomallei induce interferon gamma (IFN-γ)–dependent host immune response in goat (31).In plants, RLs induce defense responses and resistance to biotrophic and necrotrophic pathogens (32, 33). They also contribute to the biocontrol activity of the plant beneficial bacterium P. aeruginosa PNA1 against oomycetes (17). Recently, it was reported that the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29) mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) sensing in Arabidopsis thaliana (hereafter, Arabidopsis) and that bacterial compounds comprising mc-3-OH-acyl building blocks including LPS and RLs do not stimulate LORE-dependent responses (34).Here, we show that the lipidic secretome produced by P. aeruginosa (RL secretome), mostly composed of RLs and HAAs, induces Arabidopsis immunity. HAAs are perceived through the RK LORE. We demonstrate that, albeit not being sensed by LORE, RLs trigger an immune response characterized by an atypical defense signature. Altogether, our results demonstrate that RLs and their precursors produced by Pseudomonas bacteria stimulate the plant immune response by two distinct mechanisms.