PCR monitoring of Lactobacillus and Bifidobacterium dynamics in fermentations by piglet intestinal microbiota

A new group-specific primer (Lact71R), targeting the 16S-23S rDNA intergenic spacer region of Lactobacillus, was tested in its specificity to amplify rDNA of lactobacilli from piglet intestinal origin by polymerase chain reaction (PCR). Lact71R and Lab0677F, a Lactobacillus group-specific primer targeting the 16S rDNA, generated a common amplicon by PCR with DNA from Lactobacillus and Pediococcus reference strains, but not from Weissella strains. Sequence analysis of clones obtained by PCR amplification with Lact71R and Lab0677F and total DNA isolated from the ileal, caecal and colonic contents of one piglet resulted in Lactobacillus and Lactobacillus-like sequences mainly retrieved from intestinal environments. The primer pair was further validated in a culture independent PCR-analysis to monitor broad fluctuations of lactobacilli populations in fructo-oligosaccharides (FOS) fermentations by piglet intestinal microbiota. Bifidobacterium genus-specific primers were also used for PCR titre determination throughout FOS fermentations, in parallel with lactate and short chain fatty acids (SCFA) quantification. Increases between PCR titres were correlated with lactate detection in early stages of fermentation. Based on the obtained results, a simple monitoring PCR approach is proposed, foreseeing its application to the study of the dynamics of specific bacterial populations in complex environments.     

Endophytic bacterial diversity in roots of Typha angustifolia L. in the constructed Beijing Cuihu Wetland (China)

We investigated the community structure of endophytic bacteria in narrowleaf cattail (Typha angustifolia L.) roots growing in the Beijing Cuihu Wetland, China, using the 16S rDNA library technique. In total, 184 individual sequences were used to assess the diversity of endophytic bacteria. Phylogenetic analysis revealed that 161 clones (87.5%) were affiliated with Proteobacteria, other clones grouped into Cytophaga/Flexibacter/Bacteroids (3.3%), Fusobacteria (3.8%), and nearly 5% were uncultured bacteria. In Proteobacteria, the beta and gamma subgroups were the most abundant, accounting for approximately 46% and 36.6% of all Proteobacteria, respectively. The dominant genera included Rhodoferax, Pelomonas, Uliginosibacterium, Pseudomonas, Aeromonas, Rhizobium, Sulfurospirillum, Ilyobacter and Bacteroides. While some of these endophytic bacteria are capable of fixing nitrogen and can therefore improve plant growth, other endophytes may play important biological roles by removing nitrogen, phosphorus and/or organic matter from the water body and thus have the potential to enhance the phytoremediation of eutrophic water bodies. These bacteria have the potential to degrade xenobiota such as methane, methanol, methylated amines, catechol, oxochlorate, urea, cyanide, and 2,4-dichlorophenol. Hence, the use of certain endophytic bacteria in the process of phytoremediation could be a powerful approach for the restoration of eutrophic systems.     

Molecular diversity of peanut‐nodulating rhizobia in soils of Argentina

RSα sequencing is a valuable tool for identification of bacterial strains, and for evaluating the genetic structure of indigenous rhizobial populations. The purpose of this study was to evaluate, qualitatively, the presence or absence of RSα fragment in peanut‐nodulating strains isolated from plants grown at four sites in central Argentina. RSα fragment was found in only three of 26 indigenous strains, and in one of three inoculant strains analyzed. In contrast to results from studies of other symbiotic nitrogen‐fixing bacteria, such as soybean‐nodulating strains, no correlation was found between generation time and presence of RSα sequence. Phylogenetic analysis of the 16S rRNA gene sequence grouped peanut‐nodulating strains into two clusters, Bradyrhizobium japonicum vs. B. elkanii, and showed divergence among strains positive for RSα sequence. Our results confirm the genetic diversity previously reported for various peanut‐nodulating rhizobial strains, and indicate that the RSα fragment is not applicable as a marker or tool for competition assays at the field or ecological level. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Recent advances in the use of nanoparticles for allergen‐specific immunotherapy

The number of patients suffering from allergic asthma and rhinoconjunctivitis has increased dramatically within the last decades. Allergen‐specific immunotherapy (AIT) is the only available cause‐oriented therapy so far. AIT reduces symptoms, but has also a disease‐modifying effect. Disadvantages are a long‐lasting procedure, and in a few cases potential systemic adverse reactions. Encapsulation of allergens or DNA vaccines into nanostructures may provide advantages compared to the conventional AIT with noncapsulated allergen extracts: The protein/DNA molecule can be protected from degradation, higher local concentrations and targeted delivery to the site of action appear possible, and most importantly, recognition of encapsulated allergen by the immune system, especially by IgE antibodies, is prevented. AIT with nanoparticles (NPs) may offer a safer and potentially more efficient way of treatment for allergic diseases. In this review, we summarize the use of biodegradable NPs consisting of synthetic or natural polymers, liposomes, and virus‐like particles as well as nonbiodegradable NPs like dendrimers, and carbon‐ or metal‐based NPs for AIT. More or less successful applications of these NPs in prophylactic as well as therapeutic vaccination approaches in rodents or other animals as well as first human clinical trials are discussed in detail.     

Heat‐labile Escherichia coli toxin enhances the induction of allergen‐specific IgG antibodies in epicutaneous patch vaccination

Epicutaneous allergen‐specific immunotherapy (EPIT) is proposed as an alternative route for allergen‐specific immunotherapy (AIT). The induction of allergen‐specific blocking IgG antibodies represents an important mechanism underlying AIT, but has not been investigated for EPIT. Here, we compared the induction of allergen‐specific blocking IgG in outbred guinea pigs which had been immunized with recombinant birch pollen allergen Bet v 1 using patch delivery system (PDS) with or without heat‐labile toxin (LT) from Escherichia coli or subcutaneously with aluminum hydroxide (Alum)‐adsorbed rBet v 1. Only subcutaneous immunization with Alum‐adsorbed rBet v 1 and epicutaneous administration of rBet v 1 with PDS in combination with LT from E. coli induced allergen‐specific IgG antibodies blocking allergic patients' IgE, but not immunization with rBet v 1 via PDS alone. Our results suggest that patch vaccination with rBet v 1 in combination with LT may be a promising strategy for allergen‐specific immunotherapy against birch pollen allergy.     

BRCA1‐ and BRCA2‐specific in silico tools for variant interpretation in the CAGI 5 ENIGMA challenge

BRCA1 and BRCA2 (BRCA1/2) germline variants disrupting the DNA protective role of these genes increase the risk of hereditary breast and ovarian cancers. Correct identification of these variants then becomes clinically relevant, because it may increase the survival rates of the carriers. Unfortunately, we are still unable to systematically predict the impact of BRCA1/2 variants. In this article, we present a family of in silico predictors that address this problem, using a gene‐specific approach. For each protein, we have developed two tools, aimed at predicting the impact of a variant at two different levels: Functional and clinical. Testing their performance in different datasets shows that specific information compensates the small number of predictive features and the reduced training sets employed to develop our models. When applied to the variants of the BRCA1/2 (ENIGMA) challenge in the fifth Critical Assessment of Genome Interpretation (CAGI 5) we find that these methods, particularly those predicting the functional impact of variants, have a good performance, identifying the large compositional bias towards neutral variants in the CAGI sample. This performance is further improved when incorporating to our prediction protocol estimates of the impact on splicing of the target variant.     

A three‐dimensional microcomputed tomographic study of site‐specific variation in trabecular microarchitecture in the human second metacarpal

Variation in trabecular microarchitecture is widely accepted as being regulated by both functional (mechanical loading) and genetic parameters, although the relative influence of each is unclear. Studies reporting inter‐site differences in trabecular morphology (volume, number and structure) reveal a complex interaction at the gene–environment interface. We report inter‐ and intra‐site variation in trabecular anatomy using a novel model of contralateral (left vs right) and ipsilateral (head vs base) comparisons for the human second metacarpal in a sample of n = 29 historically known 19th century EuroCanadians. Measures of bone volume fraction, structure model index, connectivity, trabecular number, spacing and thickness as well as degree of anisotropy were obtained from 5‐mm volumes of interest using three‐dimensional microcomputed tomography. We hypothesized that: (i) the more diverse loading environment of metacarpal heads should produce a more robust trabecular architecture than corresponding bases within sides and (ii) the ipsilateral differences between epiphyses will be larger on the right side than on the left side, as a function of handedness. Analysis of covariance (Side × Epiphysis) with Age as covariate revealed a clear dichotomy between labile and constrained architectures within and among anatomical sites. The predicted variation in loading was accommodated by changes in trabecular volume, whereas trabecular structure did not vary significantly by side or by epiphysis within sides. Age was a significant covariate only for females. We conclude that environmental and genetic regulation of bone adaptation may act through distinct pathways and local anatomies to ensure an integrated lattice of sufficient mass to meet normal functional demands.     

Characterization of patients referred for non‐specific intellectual disability testing: the importance of autosomal genes for diagnosis

Genetic testing for non‐specific intellectual disability (ID) presents challenges in daily clinical practice. Historically, the focus of the genetic elucidation of non‐specific ID has been on genes on the X chromosome, and recent research has brought attention to the growing contribution of autosomal genes. In addition, next‐generation sequencing (NGS) has greatly improved the ability to simultaneously analyze multiple genetic loci, making large panel testing a practical approach to testing for non‐specific ID. We performed NGS analysis of a total of 90 genes implicated in non‐specific ID. The 90 genes included 56 X‐linked genes and 34 autosomal genes. Pathogenic variants were identified in 11 of 52 (21%) patient samples. Nine of the eleven cases harbored mutations in autosomal genes including AP4B1, STXB1, SYNGAP1, TCF4 and UBE3A. Our mutation‐positive cases provide further evidence supporting the prevalence of autosomal mutations in patients referred for non‐specific ID testing and the utility of their inclusion in multi‐gene panel analysis.     

The distribution of neuron‐specific gene family member 1 in brain and germ cells: Implications for the regulation of germ‐line development by brain

Vesicular acidification at early endosomes dissociates endocytosed receptor‐ligand complexes. The ligands, receptors, or both are then directed to late endosomes for degradation or recycled back to the plasma membrane. Of neuron‐specific gene (NSG) family members, early endosomal protein neuron‐specific gene family member 1 (NSG1) is the most important in receptor recycling. In this study, we characterized chicken NSG1 (cNSG1). We found several functional sites related to endocytotic machinery in cNSG1 that were highly conserved with most other vertebrate NSG1 proteins. We examined the tissue and duration specificity and the temporal and spatial patterns of cNSG1 expression. cNSG1 expression was preferentially located in all regions of the brain, neuroendocrine glands, and spinal cord. Unexpectedly, cNSG1 expression was strongly detected during male and female germ‐line development. Expression of NSG1 in two apparently unrelated cell types such as neurons and germ cells suggests NSG1 roles in neurons and germ‐cells chemotaxis and endocytotic machinery. Developmental Dynamics 240:850–861, 2011. © 2011 Wiley‐Liss, Inc.     

Detection of ABCB5 tumour antigen‐specific CD8+ T cells in melanoma patients and implications for immunotherapy

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour‐initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti‐ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5‐reactive T cells are present in human melanoma patients and tested the applicability of ABCB5‐derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co‐cultured with ABCB5 antigen‐loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5‐reactive CD8⁺ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART‐1)‐reactive CD8⁺ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART‐1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide‐loaded autologous DC. As ABCB5‐reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5‐specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.     

Increase of allergen‐specific immunoglobulin E antibodies from 1973 to 1994 in a Finnish population and a possible relationship to Helicobacter pylori infections1

Background The prevalence of atopic diseases – hayfever, asthma and eczema – has increased over the past decades. The increase may be associated with decreased rates of infections such as measles, hepatitis A, tuberculosis, toxoplasmosis, and, as recently suggested, Helicobacter pylori gastritis. Objective Since the increase of atopy has been mainly based on clinical studies, we wanted to study the prevalence of allergen‐specific Immunoglobulin (Ig)E antibodies in two cross‐sectional, adult population‐based serum samples two decades apart. Since the sera had been tested for H. pylori antibodies, we also had a chance to look for a possible relationship between these two findings. Methods We determined the prevalence rate of allergen‐specific serum IgE antibodies against birch and timothy pollen, and cat and dog epithelium allergens by the radioallergosorbent test in a 15–54‐years‐old Finnish population using 326 sera collected in 1973 and 319 sera collected in 1994 from randomly selected subjects. Results From 1973 to 1994 allergen‐specific IgE prevalence rates and IgE antibody levels rose. In 1994, the prevalence rate of positive findings in 15–24‐year‐old population had increased from 11 to 38% (3.5‐fold increase, P = 0.0001, OR 5.12, CI 95% 2.32–11.3). In older 10‐year age groups similar trends did not reach significance, but the overall change was significant with all three cut‐off levels of allergen‐specific IgE analysed. The percentage of IgE‐positive persons rose mainly in the subgroup with no H. pylori antibodies. In 1994 21% of the H. pylori‐negative subjects had IgE antibodies compared with 5% of the H. pylori‐positive subjects (in 1973 11% in both subgroups). Conclusions IgE‐based evidence for an increase in IgE‐mediated allergy was uncovered. The increase occurred mainly in the subgroup with no antibodies to H. pylori, which support the hypothesis that H. pylori could be one of the microbes counteracting atopy.     

A population biological approach to understanding the maintenance and loss of the T‐cell repertoire during aging

The adaptive immune system requires a diverse T‐cell repertoire to be able to respond to a wide variety of pathogens. Worryingly, the repertoire diversity declines dramatically in old age. As thymic output generates novel T cells, the conventional view holds that a decrease in this output with age is responsible for the loss in the repertoire. However, many additional factors affect the repertoire such as homeostatic turnover and antigen‐dependent expansion in response to infection. Mathematical models taking a population biology perspective are important tools for understanding how the interplay between these factors affects the immune repertoire. These models suggest that thymic decline is not a major factor but rather that some combination of virus‐induced proliferation and T‐cell‐intrinsic genetic or epigenetic changes gives rise to the oligoclonal expansions that cause the decline in T‐cell diversity. We also discuss consequences for strategies to rejuvenate the immune repertoire in old age.     

The wrist is not the brain: Estimation of sleep by clinical and consumer wearable actigraphy devices is impacted by multiple patient‐ and device‐specific factors

Clinical actigraphy devices provide adequate estimates of some sleep measures across large groups. In practice, providers are asked to apply clinical or consumer wearable data to individual patient assessments. Inter‐individual variability in device performance will impact such patient‐specific interpretation. We assessed two devices, clinical and consumer, to determine the magnitude and predictors of this individual‐level variability. One hundred and two patients (55 [53.9%] female; 56.4 [±16.3] years old) undergoing polysomnography wore Jawbone UP3 and/or Actiwatch2. Device total sleep time, sleep efficiency, wake after sleep onset and sleep latency were compared with polysomnography. Demographics, sleep architecture and clinical measures were compared to device performance. Actiwatch overestimated total sleep time by 27.2 min (95% confidence limits [CL], 138.3 min over to 84.0 under), overestimated sleep efficiency by 6.8% (95% CL, 34.1% over to 20.5% under), overestimated sleep onset latency by 2.6 min (95% CL, 63.3 over to 58.2 under) and underestimated wake after sleep onset by 50.7 min (95% CL, 162.5 under to 61.2 over). Jawbone overestimated total sleep time by 59.1 min (95% CL, 208.6 min over to 90.5 under) and overestimated sleep efficiency by 14.9% (95% CL, 52.6% over to 22.7% under). In multivariate models, age, sleep onset latency, wake after sleep onset, % N1 and apnea–hypopnea index explained only some of the variance in device performance. Gender also affected performance. Actiwatch and Jawbone mis‐estimate sleep measures with very wide confidence limits and accuracy varies with multiple patient‐level characteristics. Given these large individual inaccuracies, data from these devices must be applied only with extreme caution in clinical practice.     

Analysis of the functional WT1‐specific T‐cell repertoire in healthy donors reveals a discrepancy between CD4+ and CD8+ memory formation

The Wilms’ tumour‐1 (WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1‐specific T‐cell repertoire because self‐derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1‐specific T cells from the peripheral blood. Remarkably, we detected WT1‐specific CD4⁺ and CD8⁺ T‐cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4⁺ T‐cell compartment, whereas WT1‐specific CD8⁺ T cells almost universally displayed a naive phenotype. Moreover, memory CD4⁺ and naive CD8⁺ T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4⁺ and CD8⁺ T‐cell lineages with respect to memory formation in response to a self‐derived antigen. Nonetheless, WT1‐specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self‐reactive T cells for immunotherapeutic purposes.