Tissue-associated self-antigens containing exosomes: Role in allograft rejection

Exosomes are extracellular vesicles that express self-antigens (SAgs) and donor human leukocyte antigens. Tissue-specific exosomes can be detected in the circulation following lung, heart, kidney and islet cell transplantations. We collected serum samples from patients who had undergone lung (n?=?30), heart (n?=?8), or kidney (n?=?15) transplantations to isolate circulating exosomes. Exosome purity was analyzed by Western blot, using CD9 exosome-specific markers. Tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV), were identified using western blot. Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2-fold) and Kα1T (37.1-fold) than stable. Exosomes isolated from heart transplant recipients diagnosed with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Further, Kidney transplant recipients diagnosed with transplant glomerulopathy had circulating exosomes with a 2-fold increased expression of fibronectin and 2.5-fold increase in Col-IV compared with stable. We conclude that circulating exosomes with tissue associated SAgs have the potential to be a noninvasive biomarker for allograft rejection.     

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung,Melanoma, and Gastrointestinal Malignancies

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.In modern oncologic practice, patients with advanced-stage non-small cell lung cancer (NSCLC),^{1, 2} melanoma,^{3, 4} and colorectal adenocarcinoma^{5, 6} are often treated with targeted therapies as standard of care or after enrollment in clinical trials. Molecular mutation analysis is the preferred testing modality to guide therapeutic decision making and/or eligibility for biological studies. Therefore, laboratory-developed mutation assays require robust workflows that produce high-quality sequence information from routine clinical specimens, namely formalin-fixed, paraffin-embedded (FFPE) samples. As molecular testing transitions from an ancillary tool to a seminal requirement for optimal oncologic patient management, multiplex sequencing assays with clearly defined content and bioinformatics workflows are essential for accurate and consistent results, reporting, and patient management.Published guidelines endorse which genes to test in a particular tumor type and provide timeframes for receipt of actionable results, but they also grant individual laboratories autonomy to perform mutation testing using any suitable validated method.² Historically at our institution, single-gene mutation analysis for clinically relevant genes was performed either in-house or at a Clinical Laboratory improvement Amendment–certified reference laboratory. Depending on the result, reflex testing was performed for additional genes per mutation frequency or designated algorithms. Unfortunately, this approach introduced considerable turn-around time delays and unnecessary cost, particularly when send-out testing was required. Therefore, we sought testing modalities that could analyze multiple clinically relevant mutations simultaneously, accurately, and expeditiously.Next-generation sequencing (NGS) technologies have revolutionized genomic medicine by allowing high-throughput, parallel sequencing of the human genome.⁷ Currently, however, a large proportion of clinical NGS endeavors are supported by larger academic institutions with shared access to established genomic and bioinformatics research infrastructures, and routine clinical implementation of NGS is complicated by mitigating factors, such as clinical performance, laboratory expertise, lengthy turn-around times, and cost.⁸ Thus, we investigated affordable methods to detect clinically relevant somatic mutations in NSCLC, melanoma, and gastrointestinal (GI) malignancies that generated high-quality sequencing data from FFPE samples, and offered manageable turn-around times. Targeted amplicon-based library preparation methods combined with parallel sequencing offered a practical solution, and recent studies have demonstrated the utility of this approach.^{9, 10}Reversible terminal dideoxynucleotide sequencing chemistry by Illumina (San Diego, CA) consistently generates accurate and reproducible sequencing data.^{11, 12} To use this chemistry for clinical testing, we purchased the bench-top NGS sequencer, the Illumina MiSeq, and paired it with the MiSeq-compatible Illumina TruSight tumor (TST) 26-target amplicon-based library preparation kit. TST targets 26 genes and 174 amplicons selected from College of American Pathologists/National Comprehensive Cancer Network guidelines, relevant publications, and late-phase pharmaceutical clinical trials (Supplemental Table S1). TST offered several advantages over other commercially available mutation testing kits, such as bidirectional targeting of the positive and negative DNA strands, full-exon coverage as opposed to hotspot analysis, and robust vendor-supplied bioinformatics techniques optimized for somatic variant detection. More important, TST library preparation is optimized for FFPE samples, multiple safeguards exist to detect FFPE variant artifacts, and deep sequencing of TST libraries consistently yields high depths of coverage of targeted regions.Somatic mutation testing for many of the TST genes has clinical utility in a wide variety of solid tumors. For example, testing for CTNNB1 exon 3 is performed clinically for diagnostic and prognostic purposes in pediatric desmoid tumors, select PIK3CA hotspot mutations are positive prognostic factors for breast carcinoma, and multiple exons in PDGFRA and KIT are routinely tested in GI stromal tumors to predict response to targeted therapies. More important for our intended validation purposes, all of the clinically relevant genes and regions mutated in NSCLC, melanoma, and colonic adenocarcinoma that were tested in our routine clinical practice were represented. In addition, we could easily incorporate the TST NGS into a 5 business day workflow model, and a cost-analysis demonstrated a reasonable cost per test.Last, TST NGS data are processed from raw sequence (FASTQ) to called variants with on-board MiSeq Reporter software version 2.3, and variant annotations can be performed with Illumina''s VariantStudio software version 2.1 software using standard desktop and laptop computers. The ease of library preparation, sequencing, and data analysis with tools provided by a single vendor best fit our clinical priorities and the resources available at our academic molecular pathology laboratory.Herein, we present our results from the clinical validation of TST NGS using 80 sequenced samples that were selected from 100 FFPE patient samples (40 NSCLCs, 30 melanomas, and 30 GI malignancies). During our validation, we achieved high depths of coverage for multiple clinically relevant variants when multiplexing 8 to 12 samples on a single MiSeq flow cell. TST NGS consistently demonstrated sensitivities comparable to reference assays, showed 100% concordance with known variants, detected novel variants in many samples, and uncovered variants missed by less-sensitive testing modalities. The TST variant-calling pipeline was robust and showed high concordance when compared with an alternative analysis pipeline, and we used an in-house custom Java program to assess laboratory-defined quality control (QC) metrics and streamline clinical reporting (developed by G.H.S., Emory University, http://github.com/ghsmith/coverageQc). More important, although the results detailed herein represent the experience of a single institution, the data and validation strategies shown herein are broadly applicable to most clinical molecular laboratories interested in offering NGS for NSCLCs, melanomas, and GI malignancies as well as many other solid tumors.     

Bidirectional Glenn shunt for right ventricular endomyocardial fibrosis

A 25-year-old man in New York Heart Association functional class IV with right ventricular endomyocardial fibrosis received a palliative bidirectional Glenn shunt. Despite a stormy postoperative convalescence the bidirectional Glenn shunt provided good long-term palliation.     

Long noncoding RNAs could be potential key players in the pathophysiology of Sjögren's syndrome

Long noncoding RNAs (lncRNAs) are a recently discovered class of noncoding functional RNAs encoded by metazoan genomes. Recent studies suggest a larger regulatory role for lncRNAs in critical biological and disease processes. Mounting evidence on the role of lncRNAs in regulating key processes of the immune system prompted us to hypothesize the role of lncRNAs as key regulators of the pathophysiology of Sjögren's syndrome (SS). We used two similar approaches based on reanalysis of microarray expression datasets and curation of lncRNA‐protein coding gene interactions from literature to derive support for our hypothesis. We also discuss potential caveats to our approach and suggest approaches to validate the hypothesis. Our analysis suggests the potential larger and hitherto unknown role of lncRNA regulatory networks in modulating the expression of key genes involved in the pathogenesis of SS and thereby modulating the pathophysiology of SS.     

Loss of PTEN promotes podocyte cytoskeletal rearrangement,aggravating diabetic nephropathy

In diabetic nephropathy (DN), podocyte cytoskeletal rearrangement occurs followed by podocyte effacement and the development of proteinuria. PTEN (phosphatase and tensin homologue) is a ubiquitously expressed phosphatase that plays a critical role in cell proliferation, cytoskeletal rearrangement, and motility. In mouse models of diabetes mellitus, PTEN expression is reportedly decreased in mesangial cells, contributing to expansion of the mesangial matrix, but how PTEN in the podocyte influences the development of DN is unknown. We observed that PTEN expression is down‐regulated in the podocytes of diabetic db/db mice and patients with DN. In cultured podocytes, PTEN inhibition caused actin cytoskeletal rearrangement and this response was associated with unbalanced activation of the small GTPases Rac1/Cdc42 and RhoA. In mice treated with PTEN inhibitor, actin cytoskeletal rearrangement occurred in podocytes and was accompanied by increased albumin excretion. We also created mice with an inducible deletion of PTEN selectively in podocytes. These mice exhibited increased albumin excretion and moderate foot process effacement. When the mice were challenged with a high fat diet, podocyte‐specific knockout of PTEN resulted in substantially increased proteinuria and glomeruloclerosis compared to control mice fed a high fat diet or mice with PTEN deletion fed a normal diet. These results indicate that PTEN is involved in the regulation of cytoskeletal rearrangement in podocytes and that loss of PTEN predisposes to the development of proteinuria and DN. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Antibiotic resistance among gram-negative bacteria of lower respiratory tract secretions in hospitalized patients

Data on antibiotic resistance pattern of gram-negative bacterial isolates of lower respiratory tract secretions of hospitalized patients were fed into WHONET computer and analyzed for the year 1999. Out of 860 samples, 269 (31.2%) were culture positive. Gram-negative bacteria (GNB) accounted for 238 (88.4%) positive samples. Non-fermenting gram-negative bacteria (NFGNB) were found in 34% samples, the other common ones being Klebsiella spp (29.8%) and Pseudomonas spp (17.2%). GNB isolates from tracheal aspirates and sputum were 132 (55.4%) and 106 (44.5%) respectively. Adults (32.7%) and elderly patients (24.3%) recorded higher isolation of GNB as compared to pediatric patients (1.6%). The highest mean resistance among predominant GNB in both tracheal aspirate (96.6%) and sputum (86.9%) was noted to ampicillin while the lowest mean resistance in tracheal aspirate (28%) and sputum (14.3%) was to amikacin. NFGNB of tracheal aspirates and sputum showed highest resistance of 50% and 32% to amikacin, respectively. Pseudomonas spp showed the highest variation in the resistance pattern between tracheal aspirates and sputum samples. Overall mean resistance was highest among tracheal aspirate isolates compared to sputum isolates.     

Diagnostic challenges of an unusually large schwannoma of the mandible: Report of a case

Schwannomas are slow-growing, benign neoplasms arising from the Schwann cells and are commonly reported as peripheral tumors in the head and neck region. Central intramandibular schwannomas are extremely rare lesions. We report a case of intramandibular schwannoma in a 70?year old male patient. Panoramic radiography revealed a large, multilocular radiolucent lesion with distinct borders involving the right mandibular body and ramus. A complete excision was achieved by removing the tumor followed by reconstruction of the mandible. The clinical, radiological, and histopathological features are discussed within the context of this case.     

Nerve sparing can preserve orgasmic function in most men after robotic-assisted laparoscopic radical prostatectomy

Study Type – Therapy (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Orgasm has a major influence on patients’ satisfaction with the overall sexual experience, and alternations in orgasm are associated with significant reductions in emotional and physical satisfaction, which in turn may lead to sexual avoidance behaviour, disharmonious relationships and relationship breakdowns. Studies have found a reduction in orgasmic function after retropublic radical prostatectomy. While open radical prostatectomy inevitably damages some pelvic neuronal circuitry, which will thus impact on orgasmic responses, there is a paucity of data investigating the effect on robotic assisted radical prostatectomy on this. To our knowledge this study represents the largest analysis of orgasmic function in the robotic prostatectomy literature, and therefore would be of value to surgeons in counseling candidates for RALP about orgasmic outcomes. In our series, young men (age ≤60 years) and those who underwent bilateral nerve sparing approaches had a better recovery of their premorbid orgasmic function when compared to older men or men with no nerve sparing.

OBJECTIVE

• ? To investigate orgasmic outcomes in patients undergoing robotic‐assisted laparoscopic radical prostatectomy (RALP) and the effects of age and nerve sparing on these outcomes.

PATIENTS AND METHODS

• ? Between January 2005 and June 2007, 708 patients underwent RALP at our institution.
• ? We analysed postoperative potency and orgasmic outcomes in the 408 men, of the 708, who were potent, able to achieve orgasm preoperatively and available for follow‐up.

RESULTS

• ? Of men aged ≤60 years, 88.4% (198/224) were able to achieve orgasm postoperatively in comparison to 82.6% (152/184) of older men (P < 0.001).
• ? Of patients who received bilateral nerve sparing (BNS) during surgery, 273/301 (90.7%) were able to achieve orgasm postoperatively compared with 46/56 (82.1%) patients who received unilateral nerve sparing and 31/51 (60.8%) men who received non‐nerve‐sparing surgery (P < 0.001).
• ? In men ≤60 years who also underwent BNS, decreased sensation of orgasm was present in 3.2% of men, and postoperative orgasmic rates were significantly better than men ≤60 years who underwent unilateral or no nerve sparing (92.9% vs 83.3% vs 65.4%, respectively; P < 0.001).
• ? Potency rates were also significantly higher in men ≤60 years and in those who underwent BNS.

CONCLUSIONS

• ? Age and nerve sparing influence recovery of orgasm and erectile function after RALP.
• ? Men ≤60 years old and those who undergo BNS are most likely to maintain normal sexual function.

     

Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38kDa antigen in pulmonary tuberculosis

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%.