David L. Perkins

Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing

The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

Rituximab in the treatment of refractory pulmonary sarcoidosis

To the Editor: nnSarcoidosis is a chronic disease characterised by granulomatous depositions that can occur in virtually any organ system [1]. Currently, there is no US Food and Drug Administration (FDA)-approved therapy for sarcoidosis; however, corticosteroids have proven efficacious and are a commonly used treatment [2]. In patients with chronic or pulmonary disease who do not respond to corticosteroids, or in whom steroid use is contraindicated, agents such as methotrexate, azathioprine and tumour necrosis factor (TNF)-α antagonists may be effective [3, 4]. However, a need persists for patients who fail to respond to current options.nnSarcoidosis is a T-cell-mediated disease; however, humoral mechanisms may play a role in its pathogenesis [5]. Sarcoidosis is often associated with hypergammaglobulinaemia, autoantibody production and circulating immune complexes [6].nnB-cell-targeted therapies have shown positive results in many T-cell-mediated autoimmune diseases. Rituximab is a chimeric monoclonal antibody that causes depletion of CD20+ B-cells [7]. Rituximab is FDA approved for the treatment of rheumatoid arthritis, granulomatosis with polyangiitis (Wegener’s) and microscopic polyangiitis, and is also being studied in Sjogren’s syndrome, systemic lupus erythematosus and vasculitis [8]. There have been case reports of the effectiveness of rituximab for sarcoidosis [9–11]. Given the evidence for humoral involvement in sarcoidosis pathogenesis, this study sought to evaluate the utility of B-cell depletion using rituximab in patients with refractory pulmonary sarcoidosis.nnThis was a prospective, open-label, phase I/II trial. The study was approved by the …

Tocilizumab in the treatment of rheumatoid arthritis and beyond.

Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by joint pain, swelling, stiffness, and progressive destruction of the small joints of the hands and feet. Treatment of RA has improved over the past decade. With multiple cytokines well-known now to play a role in the pathogenesis of RA, including tumor necrosis factor alpha, interleukin (IL)-1β, and IL-6, many targeted biological treatments against these cytokines have emerged, changing the treatment of this disease. Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody against the IL-6 receptor and has been approved in many countries, including the United States, for the treatment of moderate to severe RA in patients who have not adequately responded to one or more disease-modifying antirheumatic drugs (DMARDs) or cannot tolerate other approved drug classes for RA. The aim of this review is to discuss the role of IL-6 in RA, and to provide an overview of the mode of action, pharmacokinetics, and safety of TCZ. Furthermore, efficacy studies of TCZ as both monotherapy and combination therapy will be evaluated. There have been several important clinical trials evaluating the efficacy and safety of TCZ in RA patients; this review summarizes this data from 14 key trials with emphasis on Phase III trials. Review of these trials provides strong evidence that its use, both as monotherapy and in combination with methotrexate or other DMARDs, is an effective treatment in reducing the signs and symptoms of RA. TCZ showed tolerable safety but care is required for its use since there are some important safety concerns including elevated liver enzymes, elevated low-density lipoprotein, infections, and gastrointestinal perforations. Additionally, given the efficacy of TCZ in the treatment of RA, this review discusses how TCZ may be beneficial in the treatment of other autoimmune diseases, spinal disease, cardiovascular disease, organ transplantation, and malignancies where elevated levels of IL-6 may play a role in the pathogenesis of these diseases.

CD45 ligation expands Tregs by promoting interactions with DCs.

Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs.

A diverse virome in kidney transplant patients contains multiple viral subtypes with distinct polymorphisms

Recent studies have established that the human urine contains a complex microbiome, including a virome about which little is known. Following immunosuppression in kidney transplant patients, BK polyomavirus (BKV) has been shown to induce nephropathy (BKVN), decreasing graft survival. In this study we investigated the urine virome profile of BKV+ and BKV− kidney transplant recipients. Virus-like particles were stained to confirm the presence of VLP in the urine samples. Metagenomic DNA was purified, and the virome profile was analyzed using metagenomic shotgun sequencing. While the BK virus was predominant in the BKV+ group, it was also found in the BKV− group patients. Additional viruses were also detected in all patients, notably including JC virus (JCV) and Torque teno virus (TTV) and interestingly, we detected multiple subtypes of the BKV, JCV and TTV. Analysis of the BKV subtypes showed that nucleotide polymorphisms were detected in the VP1, VP2 and Large T Antigen proteins, suggesting potential functional effects for enhanced pathogenicity. Our results demonstrate a complex urinary virome in kidney transplant patients with multiple viruses with several distinct subtypes warranting further analysis of virus subtypes in immunosuppressed hosts.

Effect of the Obesity Epidemic on Kidney Transplantation: Obesity Is Independent of Diabetes as a Risk Factor for Adverse Renal Transplant Outcomes

Background Obesity is a growing epidemic in most developed countries including the United States resulting in an increased number of obese patients with end-stage renal disease. A previous study has shown that obese patients with end-stage renal disease have a survival benefit with transplantation compared with dialysis. However, due to serious comorbidities, many centers place restrictions on the selection of obese patients for transplantation. Further, due to obese patients having an increased risk of diabetes, it is unclear whether obesity can be an independent risk, independent of diabetes for increasing adverse renal transplant outcomes. Methods To investigate the role of obesity in kidney transplantation, we used the Scientific Registry of Transplant Recipients database. After filtering for subjects that had the full set of covariates including age, gender, graft type, ethnicity, diabetes, peripheral vascular disease, dialysis time and time period of transplantation for our analysis, 191,091 subjects were included in the analyses. Using multivariate logistic regression analyses adjusted for covariates we determined whether obesity is an independent risk factor for adverse outcomes such as delayed graft function, acute rejection, urine protein and graft failure. Cox regression modeling was used to determine hazard ratios of graft failure. Results Using multivariate model analyses, we found that obese patients have significantly increased risk of adverse transplant outcomes, including delayed graft function, graft failure, urine protein and acute rejection. Cox regression modeling hazard ratios showed that obesity also increased risk of graft failure. Life-table survival curves showed that obesity may be a risk factor independent of diabetes mellitus for a shorter time to graft failure. Conclusions A key observation in our study is that the risks for adverse outcome of obesity are progressive with increasing body mass index. Furthermore, pre-obese overweight recipients compared with normal weight recipients also had increased risks of adverse outcomes related to kidney transplantation.

WEVOTE: Weighted Voting Taxonomic Identification Method of Microbial Sequences.

Background Metagenome shotgun sequencing presents opportunities to identify organisms that may prevent or promote disease. The analysis of sample diversity is achieved by taxonomic identification of metagenomic reads followed by generating an abundance profile. Numerous tools have been developed based on different design principles. Tools achieving high precision can lack sensitivity in some applications. Conversely, tools with high sensitivity can suffer from low precision and require long computation time. Methods In this paper, we present WEVOTE (WEighted VOting Taxonomic idEntification), a method that classifies metagenome shotgun sequencing DNA reads based on an ensemble of existing methods using k-mer-based, marker-based, and naive-similarity based approaches. Our evaluation on fourteen benchmarking datasets shows that WEVOTE improves the classification precision by reducing false positive annotations while preserving a high level of sensitivity. Conclusions WEVOTE is an efficient and automated tool that combines multiple individual taxonomic identification methods to produce more precise and sensitive microbial profiles. WEVOTE is developed primarily to identify reads generated by MetaGenome Shotgun sequencing. It is expandable and has the potential to incorporate additional tools to produce a more accurate taxonomic profile. WEVOTE was implemented using C++ and shell scripting and is available at www.github.com/aametwally/WEVOTE.

Urinary microbiome of kidney transplant patients reveals dysbiosis with potential for antibiotic resistance

&NA; Recent studies have established that a complex community of microbes colonize the human urinary tract; however, their role in kidney transplant patients treated with prophylactic antibiotics remains poorly investigated. Our aim was to investigate the urinary microbiome of kidney transplant recipients. Urine samples from 21 patients after kidney transplantation and 8 healthy controls were collected. All patients received prophylactic treatment with the antibiotic combination trimethoprim‐sulfamethoxazole. Metagenomic DNA was isolated from urine samples, sequenced using shotgun sequencing approach on Illumina HiSeq 2000 platform, and analyzed for microbial taxonomic and functional annotations. Our results demonstrate that the urine microbiome of kidney transplants was markedly different at all taxonomic levels from phyla to species, had decreased microbial diversity, and increased abundance of potentially pathogenic species compared with healthy controls. Specifically, at the phylum level, we detected a significant decrease in Actinobacteria and increase in Firmicutes due to increases in Enterococcus faecalis. In addition, there was an increase in the Proteobacteria due to increases in Escherichia coli. Analysis of predicted functions of the urinary metagenome revealed increased abundance of enzymes in the folate pathway including dihydrofolate synthase that are not inhibited by trimethoprim‐sulfamethoxazole, but can augment folate metabolism. This report characterizes the urinary microbiome of kidney transplants using shotgun metagenomics approach. Our results indicate that the urinary microbiota may be modified in the context of prophylactic antibiotics, indicating that a therapeutic intervention may shift the urinary microbiota to select bacterial species with increased resistance to antibiotics. The evaluation and development of optimal prophylactic regimens that do not promote antibiotic resistance is an important future goal.

The Potential for Emerging Microbiome-Mediated Therapeutics in Asthma

Purpose of ReviewIn terms of immune regulating functions, analysis of the microbiome has led the development of therapeutic strategies that may be applicable to asthma management. This review summarizes the current literature on the gut and lung microbiota in asthma pathogenesis with a focus on the roles of innate molecules and new microbiome-mediated therapeutics.Recent FindingsRecent clinical and basic studies to date have identified several possible therapeutics that can target innate immunity and the microbiota in asthma. Some of these drugs have shown beneficial effects in the treatment of certain asthma phenotypes and for protection against asthma during early life.SummaryCurrent clinical evidence does not support the use of these therapies for effective treatment of asthma. The integration of the data regarding microbiota with technologic advances, such as next generation sequencing and omics offers promise. Combining comprehensive bioinformatics, new molecules and approaches may shape future asthma treatment.

Detection of Differential Abundance Intervals in Longitudinal Metagenomic Data Using Negative Binomial Smoothing Spline ANOVA

Metagenomic longitudinal studies have become a widely-used study design to investigate the dynamics of the microbial ecological systems and their temporal effects. One of the important questions to be addressed in longitudinal studies is the identification of time intervals when microbial features show changes in their abundance. We propose a statistical method that is based on a semi-parametric Smoothing Spline ANOVA and negative binomial distribution to model the time-course of the features between two phenotypes. We demonstrate the superior performance of our proposed method compared to the two currently existing methods using simulated data. We present the analysis results of our proposed method in an analysis of a longitudinal dataset that investigates the association between the development of type 1 diabetes in infants and the gut microbiome. The identified significant species and their specific time intervals reveal new information that can be used in improving intervention or treatment plans.