Single-cell chromatin accessibility identifies pancreatic islet cell type- and state-specific regulatory programs of diabetes risk.

Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.

Intron 1-Mediated Regulation of EGFR Expression in EGFR-Dependent Malignancies Is Mediated by AP-1 and BET Proteins.

The epidermal growth factor receptor (EGFR) is overexpressed in numerous solid tumors and is the subject of extensive therapeutic efforts. Much of the research on EGFR is focused on protein dynamics and downstream signaling; however, few studies have explored its transcriptional regulation. Here, we identified two enhancers (CE1 and CE2) present within the first intron of the EGFR gene in models of glioblastoma (GBM) and head and neck squamous cell carcinoma (HNSCC). CE1 and CE2 contain open chromatin and H3K27Ac histone marks, enhance transcription in reporter assays, and interact with the EGFR promoter. Enhancer genetic deletion by CRISPR/Cas9 significantly reduces EGFR transcript levels, with double deletion exercising an additive effect. Targeted repression of CE1 and CE2 by dCas9-KRAB demonstrates repression of transcription similar to that of genomic deletion. We identify AP-1 transcription factor family members in concert with BET bromodomain proteins as modulators of CE1 and CE2 activity in HNSCC and GBM through de novo motif identification and validate their presence. Genetic inhibition of AP-1 or pharmacologic disruption of BET/AP-1 binding results in downregulated EGFR protein and transcript levels, confirming a role for these factors in CE1 and CE2. Our results identify and characterize these novel enhancers, shedding light on the role that epigenetic mechanisms play in regulating EGFR transcription in EGFR-dependent cancers. IMPLICATIONS: We identify critical constituent enhancers present in the first intron of the EGFR gene, and provide a rationale for therapeutic targeting of EGFR intron 1 enhancers through perturbation of AP-1 and BET in EGFR-positive malignancies.

A randomized comparison of the efficacy of 2 techniques for piriformis muscle injection: ultrasound-guided versus nerve stimulator with fluoroscopic guidance.

BACKGROUND: Piriformis muscle injections are most often performed using fluoroscopic guidance; however, ultrasound (US) guidance has recently been described extensively in the literature. No direct comparisons between the 2 techniques have been performed. Our objective was to compare the efficacy and efficiency of fluoroscopic- and US-guided techniques. METHODS: A randomized, comparative trial was carried out to compare the 2 techniques. Twenty-eight patients with a diagnosis of piriformis syndrome, based on history and physical examination, who had failed conservative treatment were enrolled in the study. Patients were randomized to receive the injection either via US or fluoroscopy. Injections consisted of 10 mL of 1% lidocaine with 80 mg of triamcinalone. The primary outcome measure was numeric pain score, and secondary outcome measures included functional status as measured by the Multidimensional Pain Inventory, patient satisfaction as measured by the Patient Global Impression of Change scale, and procedure timing characteristics. Outcome data were measured preprocedure, immediately postprocedure, and 1 to 2 weeks and 3 months postprocedure. RESULTS: We found no statistically significant differences in numeric pain scores, patient satisfaction, procedure timing characteristics, or most functional outcomes when comparing the 2 techniques. Statistically significant differences between the 2 techniques were found with respect to the outcome measures of household chores and outdoor work. CONCLUSIONS: Ultrasound-guided piriformis injections provide similar outcomes to fluoroscopically guided injections without differences in imaging, needling, or overall procedural times.

Disorders of childhood growth and development: childhood obesity.

The incidence of childhood obesity in the United States is estimated at 17%, or 12 million children ages 2 to 19 years. Obesity is a multifactorial condition with syndromic and nonsyndromic variants. Genetic, social, ethnic, endocrinologic, and behavioral issues are all potential etiologic factors. Preventive efforts should begin with monitoring from birth and include breastfeeding until age 6 months, avoiding juices, and promoting fruit and vegetable consumption and adequate exercise. Childhood obesity is diagnosed based on body mass index; a child is considered overweight at the 85th to 95th percentiles and obese at or above the 95th percentile. After obesity is diagnosed, testing should include blood pressure levels, fasting lipid profile, diabetes screening, and liver function tests. The physician should obtain a detailed history of the physical activity level and food intake and assess possible complications of obesity, including depression and hypertension, annually. Lifestyle interventions with family involvement are the mainstay of management, with pharmacotherapy or bariatric surgery considered for adolescents only if intensive lifestyle modifications have failed and in the presence of comorbidities. Intervention by multiple disciplines (ie, medicine, nutrition, psychology) is recommended, and family physicians are encouraged to become more involved in encouraging physical activity and improved nutrition for children.

Treatment of meralgia paresthetica with ultrasound-guided pulsed radiofrequency ablation of the lateral femoral cutaneous nerve.

A 23-year-old female with an 18-month history of left anterolateral thigh paresthesias and burning pain consistent with meralgia paresthetica was referred to our clinic after failing trials of physical therapy, nonsteroidal anti-inflammatories, gabapentin, and amitriptyline. We performed 3 lateral femoral cutaneous nerve blocks with corticosteroid over a 4-month period; however, each block provided only temporary relief. As this pain was limiting the patient's ability to perform her functions as an active duty service member, we elected to perform a pulsed radiofrequency treatment of the lateral femoral cutaneous nerve with ultrasound guidance and nerve stimulation. After locating the lateral femoral cutaneous nerve with ultrasound and reproducing the patient's dysthesia with stimulation, pulsed radiofrequency treatment was performed at 42°C for 120 seconds. The needle was then rotated 180° and an additional cycle of pulsed radiofrequency treatment was performed followed by injection of 0.25% ropivacaine with 4 mg of dexamethasone. At 1.5 and 3 month follow-up visits, the patient reported excellent pain relief with activity and improved ability to perform her duties as an active duty service member. ▪

Biomarkers for early and late stage chronic allograft nephropathy by proteogenomic profiling of peripheral blood.

BACKGROUND: Despite significant improvements in life expectancy of kidney transplant patients due to advances in surgery and immunosuppression, Chronic Allograft Nephropathy (CAN) remains a daunting problem. A complex network of cellular mechanisms in both graft and peripheral immune compartments complicates the non-invasive diagnosis of CAN, which still requires biopsy histology. This is compounded by non-immunological factors contributing to graft injury. There is a pressing need to identify and validate minimally invasive biomarkers for CAN to serve as early predictors of graft loss and as metrics for managing long-term immunosuppression. METHODS: We used DNA microarrays, tandem mass spectroscopy proteomics and bioinformatics to identify genomic and proteomic markers of mild and moderate/severe CAN in peripheral blood of two distinct cohorts (n = 77 total) of kidney transplant patients with biopsy-documented histology. FINDINGS: Gene expression profiles reveal over 2400 genes for mild CAN, and over 700 for moderate/severe CAN. A consensus analysis reveals 393 (mild) and 63 (moderate/severe) final candidates as CAN markers with predictive accuracy of 80% (mild) and 92% (moderate/severe). Proteomic profiles show over 500 candidates each, for both stages of CAN including 302 proteins unique to mild and 509 unique to moderate/severe CAN. CONCLUSIONS: This study identifies several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAN, the most common cause of late allograft failure. These biomarkers are the necessary first step to a proteogenomic classification of CAN based on peripheral blood profiling and will be the targets of a prospective clinical validation study.

ASSOCIATION BETWEEN DE NOVO DONOR SPECIFIC HLA ANTIBODY, C4D STAINING IN RENAL GRAFT BIOPSY AND GRAFT OUTCOME: A SINGLE CENTER EXPERIENCE.

In 69 renal transplant recipients (RTR), all had a functioning graft (SCr < 2.0) at one year. After one year, transplant dysfunction was observed and these 69 RTR were biopsied, tested for C4d deposition and donor specific antibodies (DSA). Of these 69 RTR, 29 (42%) showed C4d negativity, 27 (39%) were C4d positive and 13 (19%) were not diagnostic. Forty-nine (71%) recipients had HLA antibodies and 41 (59%) had DSA. The proportion of C4d positivity was significantly higher in patients with DSA (HLA Class I only, II only, and I & II) in comparison to patients without post-transplant HLA antibodies. The incidence of graft failure (including current SCr > 4.0) in RTR with HLA Class II antibodies (Class II only or I & II) was significantly higher than in RTR without post-transplant HLA antibodies (P=0.03).Even after amelioration of rejection, the RTR with Class II DSA group continued to fail beyond 2 years after transplantation when compared with the other 2 groups (None/NDSA or HLA Class I only), however, the difference in graft survival between HLA Class II and None/NDSA groups did not reach statistical significance (log-rank P=0.32). Significant association between C4d staining, post-transplant HLA Class II antibodies and graft failure strongly suggests the importance of post-transplant HLA antibodies. HLA Class II DSAs may be an indicator of chronic allograft nephropathy (CAN) proceeding to graft loss. We propose that amelioration of CAN graft loss may be affected by monitoring and identification of DSA with appropriate immunosuppression of these antibodies.

The evolving notion of "senior" kidney transplant recipients.

The maximum age of recipients expected to benefit with a kidney transplant has increased in the past three decades. In 1980, patients older than age 50 were not listed for a transplant. In 2004, almost 90% aged 50-60 yr with end-stage renal disease were listed, and some were even older than age 80. We summarize previous articles to illustrate how the notion of "senior" has evolved for kidney transplantation, and using data reported to the Organ Procurement Transplant Network, describe characteristics, treatments and outcomes in recipients older than 50 yr. Fractions of male, white, non-obese, unsensitized recipients and use of expanded criteria donors increased in cohorts with increasing recipient age. The percentage of recipients with hypertension or diabetes decreased, but the percentage with cancer increased. The fraction spared steroids increased with increasing age, but other aspects of immunosuppression were not remarkably different. No differences in early outcomes were notable, and elderly recipients likely did not return to dialysis. However, both graft and patient survival rates decreased with increasing age. Although a small fraction was selected, and survival rates were lower, patients older than 80 yr received kidney transplants.

High seroprevalence of anti-HTLV-I/II antibodies among solid organ donors necessitates confirmatory testing.

BACKGROUND: Human T-cell lymphotrophic virus (HTLV) type I has been linked to adult T-cell leukemia/lymphoma (ATL) and HTLV-I associated myelopathy (HAM). Transmission of HTLV by blood and organ transplantation has been documented, with some infections leading to clinical disease. Organ donors are tested for anti-HTLV antibodies and donor suitability is determined primarily by results from enzyme immunoassays (EIA). Confirmatory testing is not routinely performed, and the number of false positive organ donors is unknown. METHODS: In order to investigate the contemporary seroprevalence of anti-HTLV I/II antibodies among solid organ donors and determine the number of false positive samples, we tested 1,408 specimens from prospective organ donors in 2002 and 2003. All specimens were tested for anti-HTLV antibodies by a commercial EIA. Repeatedly reactive specimens underwent confirmatory testing using a commercial Western blot. RESULTS: There were 22 repeatedly EIA reactive donor specimens (1.56%). Five specimens did not undergo further testing because of case shutdown or insufficient sample quantity. HTLV I/II western blot confirmed six positives, whereas five were negative and six were indeterminate. The majority of confirmed specimens were positive for antibodies to HTLV-II. CONCLUSIONS: Our data shows that 29% of initially reactive specimens were false positives. With the increasing demand for organs, the unnecessary rejection of organs that are falsely positive for HTLV antibodies becomes of tremendous importance and stresses the need for timely confirmatory testing for HTLV.

Cerebellar herniation after cervical transforaminal epidural injection.

OBJECTIVE: The purpose of this study is to inform readers of potential catastrophic complications associated with performing cervical transforaminal epidural steroid injections. CASE REPORT: A 31-year-old man presented to the pain clinic with a history of cervical radicular pain and right upper-extremity radicular symptoms. He was referred from the orthopedic spine clinic for evaluation for epidural steroid injection, having failed conservative treatment consisting of nonsteroidal anti-inflammatory drugs, muscle relaxants, oral steroids, and physical therapy. After undergoing a transforaminal epidural steroid injection at the right C8 nerve root, he developed a cerebellar infarct and brainstem herniation. He survived but has residual deficits of persistent diplopia on right lateral gaze and difficulties with short-term memory loss and concentration. CONCLUSIONS: Although transforaminal epidural steroid injections are an efficacious treatment for radicular syndromes, there can be catastrophic complications. In light of the growing body of similar case reports, further investigation is warranted to establish a safe protocol for the use of this modality.

Prevalence of antibodies to Trypanosoma cruzi among solid organ donors in Southern California: a population at risk.

Trypanosoma cruzi, a parasite that causes Chagas' disease, is endemic in parts of Mexico, South America, and Central America. Transmission of T. cruzi infection by solid organ transplantation has been reported in Latin America and recently in the United States. To determine the prevalence of T. cruzi antibodies in Southern California organ donors, 404 samples from deceased organ donors between May 2002 to April 2004 were screened using a qualitative enzyme-linked immunosorbent assay (EIA) and confirmed with an immunofluorescence assay (IFA) available through the Centers for Disease Control (CDC). Six donors were initially reactive by EIA. Three donors were repeatedly reactive after repeat testing and were sent to the CDC for confirmation. One donor (0.25%) had an IFA-confirmed reactivity to anti-T. cruzi antibodies. In areas where there is a high number of immigrants from T. cruzi endemic countries, screening for anti-T. cruzi donor antibodies may be beneficial.

A multicenter, prospective study of C2-monitored cyclosporine microemulsion in a U.S. population of de novo renal transplant recipients.

BACKGROUND: Monitoring cyclosporine microemulsion (CsA-ME; Neoral) exposure 2 hours postdose (C2) has been reported to optimize the efficacy and safety of CsA-ME therapy. The addition of induction therapy to a maintenance regimen including CsA-ME C2 monitoring has not been evaluated. METHODS: In all, 123 adult renal transplant recipients were recruited at 14 U.S. centers for this 6-month study. CsA-ME dose was to be titrated to attain C2 targets of 1700 and 1500 ng/ml during posttransplant months 1 and 2, respectively. After 2 months, patients were randomized to one of two groups with different, decreasing C2 targets. Basiliximab, mycophenolate mofetil, and corticosteroids completed the study immunosuppression. RESULTS: Of the 119 evaluable patients, 76% were male, 22% African American, and 66% deceased donor recipients. Biopsy-proven acute rejection occurred in 10 patients (9.3%); there were two failed grafts and one death. Serum creatinine and calculated GFR values suggest good renal function, with month 6 medians of 1.5 ng/ml and 67 ml/min/1.73 m. Safety and tolerability assessments revealed no unexpected outcomes. Observed C2 levels were generally lower than protocol targets, particularly in the first weeks posttransplantation. CONCLUSIONS: The striking efficacy and outcomes may have been achieved in this study with lower C2 levels of CsA-ME because of the addition of basiliximab induction.

A prospective, randomized trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantation: results at 1 year.

BACKGROUND: This is the 1-year report of a randomized, multicenter, clinical trial comparing the combination of sirolimus or mycophenolate mofetil (MMF) with tacrolimus-based immunosuppression in kidney transplantation. METHODS: Prior to transplantation, recipients were randomized to receive tacrolimus plus corticosteroids with either sirolimus (n=185) or MMF (n=176). The incidence of biopsy-confirmed acute rejection at 6 months was the primary endpoint of the study. Patient and graft survival, renal function, study drug dosing and discontinuations were evaluated at 1 year. RESULTS: At 1 year, there was no difference in patient survival (95.7% sirolimus vs. 97.2% MMF; P=0.45) or graft survival (90.8% sirolimus vs. 94.3% MMF; P=0.22). Patients without delayed graft function (DGF) receiving MMF had significantly better graft survival (99% vs. 93%; P=0.01). Patients receiving a transplant from a live donor had a trend towards better graft survival with MMF as compared to sirolimus (98% vs. 91%; P=0.07). Patients receiving sirolimus had a significantly higher incidence of study drug discontinuation (26.5% vs. 14.8% MMF; P=0.006). Patients receiving MMF had significantly better renal function as shown by median serum creatinine levels (1.3 mg/dL vs. 1.5 mg/dL; P=0.03) and a trend towards higher calculated creatinine clearance (CrCl), (58.4 ml/min vs. 54.3 ml/min; P=0.06). More patients in the sirolimus group had a serum creatinine >2.0 mg/dL, (20.4% vs. 11.0%; P=0.02). CONCLUSIONS: Tacrolimus is safe and effective in live and deceased donor kidney transplantation when given in combination with sirolimus or MMF. Patient and graft survival were excellent in both arms. Renal function is superior for patients treated with tacrolimus + MMF combination.

Everolimus versus mycophenolate mofetil in the prevention of rejection in de novo renal transplant recipients: a 3-year randomized, multicenter, phase III study.

BACKGROUND: This 36-month, randomized, parallel-group study compared safety and efficacy of two doses of everolimus with mycophenolate mofetil (MMF) in de novo renal-transplant recipients. METHODS: Renal-allograft recipients received 1.5 mg/day or 3 mg/day of everolimus or 2 g/day of MMF, plus full-dose cyclosporine (CsA) and corticosteroids after randomization. For at least their first year, patients received study medication according to a double-blinded, double-dummy design. Concerns over nephrotoxicity led to a protocol amendment to an open-label design with reduced CsA troughs. RESULTS: Incidences of primary efficacy failure at 36 months (biopsy-proven acute rejection, graft loss, death, or loss to follow-up) were everolimus 1.5 mg/day, 33.7% (65/193); everolimus 3 mg/day, 34.0% (66/194); and MMF, 31.1% (61/196) (P=0.810). Antibody-treated acute rejection at 36 months was significantly lower with everolimus 1.5 mg (9.8%) than MMF (18.4%, P=0.014). Discontinuation for adverse events was more frequent with everolimus and hemolytic uremic syndrome, lymphoproliferative disease, and proteinuria, and higher serum creatinine occurred at increased frequency relative to the MMF arm. Creatinine levels in the everolimus arms were stable in follow-up: the mean rise in creatinine over the first 6 months of the open-label phase was 3 micromol/L or greater with everolimus and 7 micromol/L with MMF. However, serum creatinine levels were lower in the MMF group throughout. Death and graft loss were higher in the everolimus arms (not significant). CONCLUSIONS: As part of triple-drug immunosuppression, everolimus (1.5 or 3 mg/day) was as efficacious as MMF, although the side-effect profile featured increased adverse events. Nephrotoxicity/calcineurin-inhibitor-related adverse events will require judicious lowering of CsA exposure with monitoring of everolimus troughs.

Evaluation of the L2 spinal nerve root infiltration as a diagnostic tool for discogenic low back pain.

BACKGROUND: To assess whether unilateral L2 infiltration with local anesthetic can be used to identify patients who will have negative discograms and thus eliminate the need for the discogram. Discogenic low-back pain is considered to have afferent pathways in the sinuvertebral nerves, mainly originating from the ventral rami of the spinal nerves. There is evidence that pain arising from the lower lumbar intervertebral discs may be transmitted through the sympathetic afferent fibers contained in the L2 spinal nerve root. Provocative discography, within the context of other clinical data, is the current "gold standard" by which to diagnose discogenic low-back pain, but a far more invasive procedure than L2 infiltration. OBJECTIVE: To evaluate the correlation between unilateral second lumbar (L2) spinal nerve root infiltration with local anesthetic and provocative discography in patients with chronic low back pain. STUDY DESIGN: A prospective, observational study. METHODS: All patients scheduled for discography were asked to participate in having local anesthetic infiltration of the L2 spinal nerve root at least two weeks prior to discography, until forty subjects were enrolled. Discography was performed after the patient's pain level returned to baseline. RESULTS: Local anesthetic infiltration of the L2 spinal nerve root was predictive of provocative discography results in only 46.5% of the subjects (26% true positives, and 20.5% true negatives). In 53.5% of the subjects, L2 infiltration was not predictive of discography results (20.5% false positives, and 33% false negatives). CONCLUSIONS: The results showed that unilateral L2 infiltration is not predictive of discogenic low-back pain when compared to discography, the current "gold-standard" for diagnosis.

Improved detection of HCV Infection in hemodialysis patients using a new HCV RNA qualitative assay: experience of a transplant center.

BACKGROUND: Hepatitis C virus (HCV) is frequently a silent infection in hemodialysis (HD) patients with a prevalence of 8-10%. Improving HCV detection in this population prior to transplantation is critical both for infection control and optimal patient care. OBJECTIVES: To assess the current HCV testing practice of the National Institute for Transplantation (PCR testing of enzyme immunoassay (EIA) positive HD patients) by evaluating a subset of EIA positive and EIA negative samples with the VERSANT HCV RNA Qualitative Assay based on transcription mediated amplification (HCV Qual (TMA)) (sensitivity < or = 9.6 IU/ml) and in-house PCR (HCV Qual (PCR)) (sensitivity approximately 149 IU/ml). STUDY DESIGN: 2321 HD patients were screened by Abbott HCV EIA 2.0. A subset of 80/169 E IA positive samples and 100/2152 EIA negative samples were tested by both assays. TMA/PCR discordant samples were genotyped. RESULTS: PCR and TMA gave concordant results in 67/80 (83.8%) of EIA positive samples. 11/80 (14.7%) were reactive by HCV Qual (TMA), but not by HCV Qual (PCR); 2/80 (2.7%) were reactive by HCV Qual (PCR), but not by HCV Qual (TMA). 2/100 (2%) EIA negative samples were reactive and 95/100 (95%) were non-reactive by both assays. Three (3%) were only HCV Qual (TMA) reactive. 11/14 TMA+/PCR-samples with sufficient volume were genotyped. CONCLUSIONS: HCV Qual (TMA) identified active HCV infection in more EIA positive and EIA negative patients than HCV Qual (PCR) and should be part of our testing algorithm.

Randomized trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantation: results at 6 months.

BACKGROUND: This is the first report of a randomized, multicenter, clinical trial comparing the combination of sirolimus or mycophenolate mofetil (MMF) with tacrolimus-based immunosuppression in kidney transplantation. Results at 6 months of follow-up are presented. METHODS: Before transplantation, patients were randomized to receive tacrolimus plus corticosteroids with sirolimus (n=185) or MMF (n=176). The primary endpoint of the study was the incidence of biopsy-confirmed acute rejection. Patient and graft survival, renal function, and composite endpoints also were evaluated. Safety was assessed by monitoring laboratory parameters and adverse events. RESULTS: By 6 months of follow-up, the incidence of biopsy-confirmed acute rejection was similar in both treatment groups (13.0% tacrolimus+sirolimus vs. 11.4% tacrolimus+MMF; P=0.64 log-rank). Patient survival (97.3% tacrolimus+sirolimus vs. 97.7% tacrolimus+MMF) and graft survival (93.0% tacrolimus+sirolimus vs. 95.5% tacrolimus+MMF) were equivalent (P=0.53, overall survival log-rank). There was a significantly higher incidence of study drug discontinuation in patients receiving sirolimus (21.1% vs. 10.8%; P=0.008). Renal function was significantly better in the MMF-treatment group (serum creatinine 1.44+/-0.45 mg/dL vs. 1.77+/-1.42 mg/dL; P=0.018). Hyperlipidemia was significantly more prevalent in the sirolimus-treatment group. Diastolic blood pressure was significantly higher in sirolimus-treated patients. There were significantly more leukopenia and gastrointestinal adverse events in the MMF-treatment group. The incidence of posttransplant diabetes mellitus was 7.6% in the sirolimus group and 7.7% in the MMF group. CONCLUSION: Tacrolimus is equally effective in renal transplantation when combined with sirolimus or MMF. The tacrolimus-MMF combination may be superior in terms of improved renal function and improved cardiovascular risk factors including hyperlipidemia and hypertension.