Mitochondrial compromise in 3‐year old patas monkeys exposed in utero to human‐equivalent antiretroviral therapies

Antiretroviral (ARV) drug therapy, given during pregnancy for prevention of mother‐to‐child transmission of human immunodeficiency virus 1 (HIV‐1), induces fetal mitochondrial dysfunction in some children. However, the persistence/reversibility of that dysfunction is unclear. Here we have followed Erythrocebus patas (patas) monkey offspring for up to 3 years of age (similar in development to a 15‐year old human) after exposure of the dams to human‐equivalent in utero ARV exposure protocols. Pregnant patas dams (3–5/exposure group) were given ARV drug combinations that included zidovudine (AZT)/lamivudine (3TC)/abacavir (ABC), or AZT/3TC/nevirapine (NVP), for the last 10 weeks (50%) of gestation. Infants kept for 1 and 3 years also received drug for the first 6 weeks of life. In offpsring at birth, 1 and 3 years of age mitochondrial morphology, examined by electron microscopy (EM), was compromised compared to the unexposed controls. Mitochondrial DNA (mtDNA), measured by hybrid capture chemiluminescence assay (HCCA) was depleted in hearts of patas exposed to AZT/3TC/NVP at all ages (P < 0.05), but not in those exposed to AZT/3TC/ABC at any age. Compared to unexposed controls, mitochondrial reserve capacity oxygen consumption rate (OCR by Seahorse) in cultured bone marrow mesenchymal fibroblasts from 3‐year‐old patas offspring was ～50% reduced in AZT/3TC/ABC‐exposed patas (P < 0.01), but not in AZT/3TC/NVP‐exposed patas. Overall the data show that 3‐year‐old patas sustain persistent mitochondrial dysfunction as a result of perinatal ARV drug exposure. Environ. Mol. Mutagen. 57:526–534, 2016. © 2016 Wiley Periodicals, Inc.     

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor

Virus Genes - Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus...     

Cisplatin-DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin

In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.     

Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods,and use in human biomonitoring

A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N(2)-yl)-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, approximately 1.5 adducts/10(9) nucleotides using 20 micro g DNA) and a high signal-to-noise ratio (> or =100). The CIA BPDE-DNA standard curve gave 50% inhibition at 0.60 +/- 0.08 fmol BPdG (mean +/- SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-(3)H]BPDE was assayed by radiolabeling, (32)P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-(3)H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose-response was obtained with all assays. The BPDE-DNA CIA was further validated in MCL-5 cells exposed to 4 micro M BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE-DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH-DNA adducts/10(8) nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH-DNA adduct values obtained were highly correlated (r(2) = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.     

Thoracic outlet decompression for subclavian vein thrombosis: experience in 71 patients

HYPOTHESIS: There is a difference in outcomes when patients have neurogenic thoracic outlet syndrome in addition to subclavian vein thrombosis. METHODS: Analysis of a prospectively developed database, medical record review, and a patient questionnaire were used to summarize clinical experience from December 1990 to December 2001 on the basis of the patient's original evaluation. Patients were stratified on the presence (group 1) or absence (group 2) of additional neurogenic pathologic features. RESULTS: Of 928 patients evaluated for thoracic outlet syndrome, 71 underwent 73 operative procedures for subclavian vein obstruction. Men predominated (55%), and the mean age was 32 years. Group 1 (41%) had more preoperative disability, a higher incidence of persistent pain (24%), and less likelihood of returning to full activity compared with group 2 (67% vs 93%; P = .01). Catheter-directed thrombolysis was used in 65% of veins. Preoperative balloon angioplasty was used selectively (34%), and only 4% required stents. Supraclavicular decompression and venolysis were usually delayed 3 weeks to allow for healing of the venous endothelium. Complications included wound infection (3%) and postoperative hematoma (8%). CONCLUSIONS: Patients with isolated subclavian vein obstruction have a more favorable outcome relative to those with combined neurogenic and venous pathologic features. Decompression following thrombolysis should be delayed to reduce the incidence of postoperative complications.     

Postoperative tonsillectomy bleed: coblation versus noncoblation

OBJECTIVE: To examine the incidence of postoperative bleeding after coblation and noncoblation tonsillectomy and to use postoperative bleeding as an outcome measure to determine the presence of a learning curve with this new surgical technique. STUDY DESIGN: A retrospective review of records from January 1999 to April 2003 to determine type of tonsillectomy performed and the presence of postoperative bleeding. A chi-square analysis was used to determine a statistical difference between the postoperative bleed rate of coblation and noncoblation procedures. The examined time period was divided into 3-month intervals, and the coblation postoperative bleeds were tallied for each interval. The Cochraine-Armitage test of linear trend was used to assess change in the postoperative bleeds. RESULTS: One thousand seven hundred sixty-two tonsillectomies were performed. The postoperative bleed rate for noncoblation tonsillectomy was 6.1% (74/1,216). The bleeding rate for coblation tonsillotomy was 5.9% (18/303) and 5.4% (13/239) for coblation tonsillectomy. There was no statistical difference (P = .93) between bleed rates for coblation versus noncoblation techniques. There was no difference in the need for operative intervention to control postoperative bleeding: 16.2% (12/74) for noncoblation technique and 25.85 (8/31) for coblation procedures (P = .25). The postoperative coblation bleed rates for the 3-month periods did not reveal an increasing or decreasing trend in the postoperative bleed rate (P = .49). CONCLUSION: Coblation is a safe procedure for performing tonsil surgery with no significant difference in postoperative bleeding from previous techniques and no increased need for operative intervention to control postoperative bleeding. A learning curve could not be identified when using postoperative bleeding as an outcome measure for coblation tonsillectomy.     

Use of cross-sectional imaging in predicting surgical location of parotid neoplasms

OBJECTIVE: The purpose of this study was to determine the diagnostic accuracy of using the retromandibular vein as seen on cross-sectional imaging to help differentiate superficial lobe from deep lobe tumors. METHODS: Of the patients who had parotid neoplasms between January 1997 and July 2002, we were able to identify 44 patients with preoperative imaging studies that were available for evaluation. The films were reviewed by a single head and neck radiologist to determine whether the neoplasms involved the superficial, deep, or both lobes of the parotid gland (total). The lateral margin of the retromandibular vein was used as a marker for the facial nerve, since the nerve is not always visible on CT and MRI scans. The radiologist's findings were then compared with the findings during surgery. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of predicting the location of neoplasms were then calculated. RESULTS: For lesions in the superficial lobe, cross-sectional imaging was able to predict the location of the neoplasm with a sensitivity of 0.91 (95% CI, 0.70-0.98), specificity of 0.86 (95% CI, 0.63-0.96), PPV of 0.88 (95% CI, 0.67-0.97), and NPV of 0.90 (95% CI, 0.67-0.98). For lesions in both lobes (total), cross-sectional imaging was able to predict the location of the neoplasm with a sensitivity of 0.94 (95% CI, 0.68-0.99), specificity of 0.89 (95% CI, 0.71-0.97), PPV of 0.83 (95% CI, 0.58-0.96), and NPV of 0.96 (95% CI, 0.78-0.99). CONCLUSION: Use of the retromandibular vein as a marker for the facial nerve is a sensitive method for identifying the location of parotid gland neoplasms on cross-sectional imaging. This supports the accuracy of using preoperative imaging to detect the position of parotid neoplasms with respect to the facial nerve.     

Identification of tamoxifen-DNA adducts in monkeys treated with tamoxifen

The risk of developing endometrial cancer is increased in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention. We have detected previously TAM-DNA adducts in the endometrium of women receiving TAM (Shibutani et al., Carcinogenesis, 21: 1461-1467, 2000). To investigate the genotoxic damage induced by TAM in the uterus and other tissues of primates, we gave adult female cynomolgus monkeys six times the human-equivalent dose of TAM (2 mg/kg body weight/day) for 30 days. DNA samples were prepared from the uterus, ovary, liver, kidney, and brain cortex of three TAM-exposed monkeys and one control monkey and were analyzed as coded specimens. To identify the TAM-DNA adducts, we established a new high-performance liquid chromatography gradient system for (32)P-postlabeling/high-performance liquid chromatography analysis, which can resolve the trans- and cis-diastereoisomers of alpha-(N(2)-deoxyguanosinyl)TAM (dG-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethylTAM, and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. Trans-forms of dG-N(2)-TAM and dG-N(2)-N-desTAM adducts were detected in the livers of all three TAM-fed monkeys at levels of 2.7 adducts/10(8) nucleotides and 1.7 adducts/10(8) nucleotides, respectively. The levels of dG-N(2)-TAM adducts observed in the uterus of one monkey and in the ovaries of two monkeys were approximately 10-fold lower than those observed in the livers. TAM exposure also induced dG-N(2)-TAM adduct in the brain cortex of all three monkeys with a value of 1.5 adducts/10(8) nucleotides. No TAM-DNA adducts were detected in the kidneys or in any tissues obtained from the unexposed monkey. Our results suggest that women receiving TAM may form genotoxic damage in many organs, including the reproductive organs.     

Tocilizumab controls bone turnover in early polymyalgia rheumatica

ObjectivesThis study explores changes in the bone homeostasis by testing the N-terminal collagen type I extension propeptide (PINP) marker for osteo-formation and the carboxy-terminal region of collagen type I (CTX-I) marker for osteo-resorption in patients taking tocilizumab for polymyalgia rheumatica (PMR).MethodsTwenty patients were included in the prospective open-label TENOR study (Clinicaltrials.gov NCT01713842) and received three monthly tocilizumab infusions, followed by corticosteroids starting at week (W) 12. PINP and CTX-I were tested at inclusion (W0), after tocilizumab but before steroid initiation (W12), at the end of the protocol (W24) and were compared to healthy controls. Information regarding disease activity, bone mineral density using scanographic bone attenuation correlation (SBAC), inflammatory parameters and interleukin (IL)-6 levels were collected during the follow-up of the patients.ResultsPMR patients were characterised by a reduction in bone mineral density and a higher level of CTX-I relative to healthy controls matched in age and sex at baseline. PINP levels increased at W12 (P < 0.001, versus W0) following tocilizumab introduction and CTX-I levels decreased at W24 and after steroid initiation (P = 0.001, versus W0). Such modifications explain the altered correlation observed between PINP and CTX-I at W0 (r = 0.255 at W0 versus r = 0.641 in healthy controls) and its correction after treatment (r = 0.760 at W12 and r = 0.767 at W24). Finally, greater changes in PINP were observed in patients whose circulating IL-6 levels decreased after tocilizumab therapy.ConclusionsControl of bone turnover, in part through the inhibition of the IL-6 axis, is observed during tocilizumab and subsequent steroid treatment of PMR.     

Incidence and Mortality of Physician-Diagnosed Primary Sjögren Syndrome: Time Trends Over a 40-Year Period in a Population-Based US Cohort

Objective

To estimate the incidence and mortality rates, and their evolution over time, of physician-diagnosed primary Sjögren syndrome (pSS) in residents of Olmsted County, Minnesota.