HIV‐1 vaccine clinical trials: the Brazilian experience

Although the development of an effective HIV‐1 vaccine has proved very challenging for more than two decades, it remains the best hope to control the HIV pandemic. Since Brazil has particular epidemiological features, as well as adequate policies and infrastructure, the country has been an interesting site for HIV vaccine trials. Since 1995, eight trials were performed in Brazil enrolling over 2000 subjects. Peptide vaccine candidates were initially designed to elicit neutralising antibodies as an attempt to provide sterilising immunity against HIV‐1. This strategy, however, has proved extremely difficult, and candidates were poorly immunogenic. Therefore, the next vaccine candidates focused mainly on the induction of cell mediated immune responses that would limit AIDS progression and transmission by suppressing viremia. Such candidates were naked DNA or viral vectors in either prophylactic or therapeutic approaches. Even though several candidates were immunogenic, protective immune responses against HIV‐1 remain to be achieved. However, several studies with non‐human primates and human elite controllers demonstrate that effective immune responses against HIV‐1 may be elicited, supporting the belief that an HIV‐1 vaccine is possible. Much has been learned, and now the development of an effective HIV‐1 vaccine requires resetting priorities with focus on basic research, considering the merits of neutralising antibodies and CMI, as well as the role of innate immunity on HIV‐1 protection. In this new perspective, large‐scale trials should be replaced by smaller preliminary efficacy studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A single hERG mutation underlying a spectrum of acquired and congenital long QT syndrome phenotypes

The long QT syndrome (LQTS) is a multi-factorial disorder that predisposes to life-threatening arrhythmias. Both hereditary and acquired subforms have been identified. Here, we present clinical and biophysical evidence that the hERG mutation c.1039 C > T (p.Pro347Ser or P347S) is responsible for both the acquired and the congenital phenotype. In one case the genotype remained silent for years until the administration of several QT-prolonging drugs resulted into a full-blown phenotype, that was reversible upon cessation of these compounds. On the other hand the mutation was responsible for a symptomatic congenital LQTS in a Dutch family, displaying a substantial heterogeneity of the clinical symptoms. Biophysical characterization of the p.Pro347Ser potassium channels using whole-cell patch clamp experiments revealed a novel pathogenic mechanism of reciprocal changes in the inactivation kinetics combined with a dominant-negative reduction of the functional expression in the heterozygous situation, yielding a modest genetic predisposition for LQTS. Our data show that in the context of the multi-factorial aetiology underlying LQTS a modest reduction of the repolarizing power can give rise to a spectrum of phenotypes originating from one mutation. This observation increases the complexity of genotype-phenotype correlations in more lenient manifestations of the disease and underscores the difficulty of predicting the expressivity of the LQTS especially for mutations with a more subtle impact such as p.Pro347Ser.     

Further immunohistochemical characterization of <Emphasis Type="Italic">BRD1</Emphasis> a new susceptibility gene for schizophrenia and bipolar affective disorder

We have recently shown that the gene BRD1 is associated with schizophrenia and bipolar affective disorder and that the BRD1 protein (BRD1) which is expressed in neurons may occur in a short and a long variant. The aim of the study was to generate polyclonal antibodies against new BRD1 epitopes enabling discrimination between the long and short BRD1 variants, and elucidate the BRD1 distribution in several human tissues, including the CNS. Polyclonal rabbit antibodies were raised against three different BRD1 epitopes. One (67) was specific for the long BRD1 variant, whereas the two others (63/64 and 65/66) like the original monoclonal mouse antibody (K22) were predicted to stain both variants. Immunohistochemical staining procedures were subsequently performed on paraffin-embedded human cerebral cortex and microarray slides containing 30 different human tissues. Western blotting confirmed the predicted specificity of the developed antibodies. K22, 63/64 and 65/66 displayed a similar neuronal staining pattern characterized by a distinct but weak nuclear staining, while the surrounding cytoplasm and proximal dendrites were more intensely stained. Interestingly, staining with 67 generated in contrast primarily an intense nuclear staining. The new antibodies resulted, furthermore, in a prominent neuroglial reaction characterized by staining of cell bodies, nuclei and glial processes. The tissue microarray analysis revealed that BRD1 was widely distributed in human tissues. The particular expression profile, e.g., the degree of nuclear and/or cytoplasmatic staining, seemed, however, to be highly tissue dependent. These results suggest a general role of BRD1 in the cell and stress that the two BRD1 variants may play different roles in the etiology of psychiatric disease.     

The second wind phenomenon in very young McArdle’s patients

We investigated the phenomenon of second wind in four patients with McArdle’s disease: a brother and sister (aged 4 and 12 years respectively) and two unrelated patients, a boy of 14 and a 17-year-old girl. We also studied the siblings’ healthy 6-year-old sister. Each patient performed a 15-min exercise test at a constant workload and a subsequent graded exercise test until exhaustion. Overall the healthy girl and the youngest McArdle’s patient, the 4-year-old boy, did not show a second wind phenomenon. Further, the peak cardio-respiratory capacity of the young McArdle’s boy was normal for his age (32.3mL02/kg/min) and he did not report any function limitations during physical education classes.     

Physical fitness effect on bone mass is mediated by the independent association between lean mass and bone mass through adolescence: a cross-sectional study

We studied 278 adolescents (169 females) aged 13.0–18.5 years to elucidate whether an independent effect of physical fitness and lean mass in the differences between male and female bones can be detected. Lean and fat masses and bone mineral content (BMC) were measured with DXA. Physical fitness was evaluated with six different tests included in the EUROFIT test battery (flexibility, isometric, dynamic and endurance strength, speed, and cardiovascular fitness). To test the independent relationship between physical fitness and bone mass, multiple regression analysis was applied, including lean mass, age, and Tanner development as covariates. The males had a 43% lower fat mass and 40% and 16% higher lean mass and total BMC compared with the females (all P < 0.05). After adjustment for differences in body size and lean mass, the females exhibited a 7.4% higher BMC than the males (P < 0.05). The multiple regression analysis showed that lean mass had an independent relationship with bone mass (P < 0.001), explaining 67% of the total variance in whole-body BMC. In males, change in R ² was 0.658 for hand grip and 0.035–0.151 for the rest of physical fitness-related variables; but 0.019–0.042 in females (all P–0.001); however, the independent relationships between physical fitness and bone disappeared after controlling for lean mass. In conclusion, it is likely the differences between male and female in bone mass could be explained by differences in lean mass and physical fitness.     

The K153R variant in the myostatin gene and sarcopenia at the end of the human lifespan

We studied the A55T, E164K, I225T, K153R and P198A variants in the myostatin (GDF8) gene, muscle strength and mass, and physical function during daily living in 41 nonagenarians [33 women, age range, 90, 97]. No participant carried a mutant allele of the aforementioned variants, except three participants (all women), who carried the R allele of the K153R polymorphism, with one of them (woman aged 96 years) being homozygous. Overall, in KR women muscle phenotype values (1RM leg press and estimated muscle mass) were low-to-normal compared to the whole group (∼25th–50th percentile), and their functional capacity (Barthel and Tinetti tests) was normal. In the woman bearing the RR genotype, values of muscle mass and functional capacity were below the 25th percentile. She is the first RR Caucasian whose phenotype has been characterised specifically. In summary, heterozygosity for the GDF8 K153R polymorphism does not seem to exert a negative influence on the muscle phenotypes of women who are at the end of the human lifespan, yet homozygosity might do so. More research on larger cohorts of nonagenarians is needed to corroborate the present findings.     

Cardiorespiratory fitness relates more strongly than physical activity to cardiovascular disease risk factors in healthy children and adolescents: the European Youth Heart Study.

BACKGROUND: Physical activity and cardiorespiratory fitness are closely related to health variables in adults, especially those considered to be among risk factors for cardiovascular diseases. The possible tracking of cardiovascular disease risk factors from childhood to adulthood makes it important to increase our understanding of the complex relationships between physical activity, cardiorespiratory fitness and cardiovascular risk factors early in life. DESIGN: A cross-sectional, school-based study on healthy children and adolescents, aged 9-10 years (295 girls, 295 boys) and 15-16 years (302 girls, 233 boys) was performed during a school year in Sweden and Estonia, as part of the European Youth Heart Study. METHODS: Total physical activity, and minutes spent in inactivity and activity of moderate or higher intensity were measured by accelerometry. A maximal ergometer bike test was used for estimation of cardiorespiratory fitness. The risk factors included blood pressure and fasting blood levels of insulin, glucose, triglycerides, total cholesterol and high-density lipoprotein cholesterol. RESULTS: Canonical correlations between physical activity and cardiorespiratory fitness versus cardiovascular disease risk factors showed significant associations in both age and sex groups (rc=0.46-0.61, P<0.0001). The cardiorespiratory fitness was found to be the strongest contributor to these relationships. In girls high values of the physical activity variables were also associated with a favourable cardiovascular profile. CONCLUSIONS: Cardiorespiratory fitness relates more strongly to cardiovascular risk factors than components of objectively measured physical activity in children and adolescents. Physical activity becomes more important in the 15-year-old adolescents, indicating that these modifiable lifestyle factors increase in importance with age.     

From the Cover: A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrus australiasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr. Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

Citrus Huanglongbing (HLB), also known as citrus greening, is caused by the vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas). It is the most destructive disease threatening citrus industries worldwide (1, 2), and thus far, no cure has been discovered. Current management strategies include insecticide application to control the transmission vector Asian citrus psyllids (ACP) and antibiotics treatment to inhibit CLas (3), but neither of these could control HLB effectively. Since the first report of HLB in Florida in 2005, citrus acreage and production in Florida decreased by 38% and 74%, respectively (2, 4). The disease has spread to most citrus-producing states, including Texas and California. Along with drastic losses in fruit production, increasing chemical applications to control the vector and the bacteria have raised costs significantly, making citrus production for growers unsustainable. In severely affected areas, such as Florida, effective therapy is demanded because disease eradication is impractical. In recently impacted areas, such as California, the focus remains on preventing new infections. Hence, innovative therapeutic and preventive strategies to combat this lethal citrus disease are urgently needed to ensure the survival of the citrus industry.One of the most effective and ecofriendly strategies to combat pathogen infection is to utilize existing plant innate immunity–related genes from disease resistant or tolerant varieties for plant protection. Upon pathogen infection, plant defense response genes undergo expression reprogramming to trigger plant innate immunity. Plant endogenous small RNAs play a pivotal role in this regulatory process (5, 6). In addition, primary pathogen infection or application of some phytohormone analogs and chemicals, such as salicylic acid (SA) analogs, could induce systemic acquired resistance or defense priming in plants, which can promote faster and stronger host immune responses upon subsequent pathogen challenges (7, 8).Although all commercially important citrus varieties are susceptible to HLB (9, 10), HLB tolerance has been observed in some hybrids [e.g., US-942 and Sydney hybrid 72 (11, 12)] and close citrus relatives (e.g., Microcitrus australiasica, Eremocitrus glauca, and Poncirus trifoliata) (13). By comparative expression analysis of small RNAs and messenger RNAs (mRNAs) between HLB-sensitive cultivars and HLB-tolerant citrus hybrids and relatives (11, 12), we identified a list of candidate natural defense genes potentially responsible for HLB tolerance (14). One of the candidate regulators is a novel antimicrobial peptide (AMP), which we named “stable antimicrobial peptide” (SAMP). Here, we demonstrate that SAMP not only has the antimicrobial activity but also has the priming activity and can induce citrus systemic defense responses. This dual-functional SAMP can reduce CLas titer and suppress disease symptoms in HLB-positive trees and activate plant systemic defense responses against new infection.