The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.

A new risk model to predict time to first treatment in chronic lymphocytic leukemia based on heavy chain immunoparesis and summated free light chain.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is frequently accompanied by immune dysregulation. AIMS: In this multicenter prospective study, we investigated whether heavy + light chains (HLC: IgGκ, IgGλ, IgAκ, IgAκ, IgMκ, IgMλ) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) could be used as novel prognostic markers of immunoparesis in 105 treatment-naïve patients with CLL. RESULTS: Heavy + light chains immunoparesis of ≥1, ≥2, and ≥3 isotypes was evident in 74 (70%), 58 (55%), and 36 (34%) patients, respectively. Severe HLC immunoparesis was identified in 40 (38%) patients. Of the IgG subclasses, IgG1 and IgG2 were most frequently suppressed, affecting 46 (44%) and 36 (34%) patients, respectively; 63 (60%) patients had low levels of at least one IgG subclass. In multivariate analysis, severe HLC immunoparesis (hazard ratio [HR]: 36.5; P = .010) and ΣFLC ≥ 70 mg/L (HR: 13.2; P = .004) were the only factors independently associated with time to first treatment (TTFT). A risk model including these variables identified patients with 0, 1, and 2 risk factors and significantly different TTFT (P < .001). Patients with two factors represented an ultra-high-risk group with a median TTFT of only 1.3 months. CONCLUSION: The above findings demonstrate the potential for the use of HLC immunoparesis, together with sFLC measurements, as future prognostic biomarkers in CLL.

Ubiquitin-dependent recruitment of the Bloom syndrome helicase upon replication stress is required to suppress homologous recombination.

Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.

Cystatin C and Cardiac Measures in Children and Adolescents With CKD.

BACKGROUND: Cardiovascular disease (CVD) is highly prevalent among children with chronic kidney disease (CKD). Cystatin C is an established marker of kidney function and an emerging biomarker for CVD events. We quantified the relationship between cystatin C level and cardiac structure and function over time among children with CKD and assessed whether cystatin C level and diastolic function retained an association after accounting for kidney function. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 678 children and adolescents with mild to moderate CKD enrolled in the CKD in Children (CKiD) Study with 1,228 echocardiographically obtained cardiac structure and function measurements. PREDICTOR: Serum cystatin C (mg/L) measured annually. OUTCOMES: Cardiac structure (left ventricular mass index [g/m2.7]) and cardiac function (shortening fraction; E/A, E'/A', E/E' ratios) measured every other year. MEASUREMENTS: Demographics and anthropometrics, measured glomerular filtration rate (mGFR), heart rate, blood pressure, hemoglobin z score, serum albumin level, and calcium-phosphorus product. RESULTS: Independent of time, each 1-mg/L increase in cystatin C level was independently associated with a concurrent 7.7% (95% CI, 5.3%-10.0%) increase in left ventricular mass index, a -4.7% (95% CI, -7.0% to -2.4%) change in E/A ratio, a -6.6% (95% CI, -9.0% to -4.2%) change in E'/A' ratio, and a 2.5% (95% CI, 0.3%-4.7%) increase in E/E' ratio. mGFR was also independently associated with E'/A' ratio. When cystatin C level and mGFR were included in the same model, cystatin C level remained independently associated with E'/A' ratio, whereas mGFR was not. LIMITATIONS: 24% of the cohort was missing data for outcomes of interest or measurements; study population includes only children and adolescents with mild to moderate CKD. CONCLUSIONS: In this study of children and adolescents with mild to moderate CKD, cystatin C level was independently associated with cardiac structure and diastolic function. Cystatin C level remained able to predict diastolic function decline via E'/A' ratio even after adjusting for mGFR, suggesting that cystatin C level may have an independent role in CVD risk stratification among children and adolescents with CKD.

The maternal childbirth experience more than a decade after delivery.

BACKGROUND: Maternal satisfaction with the birth experience is multidimensional and influenced by many factors, including mode of delivery. To date, few studies have investigated maternal satisfaction outside of the immediate postpartum period. OBJECTIVE: This study investigated whether differences in satisfaction based on mode of delivery are observed more than a decade after delivery. STUDY DESIGN: This was a planned, supplementary analysis of data collected for the Mothers' Outcomes after Delivery study, a longitudinal cohort study of pelvic floor disorders in parous women and their association with mode of delivery. Obstetric and demographic data were obtained through patient surveys and obstetrical chart review. Maternal satisfaction with childbirth experience was assessed via the Salmon questionnaire, administered to Mothers' Outcomes after Delivery study participants >10 years from their first delivery. This validated questionnaire yields 3 scores: fulfillment, distress, and difficulty. These 3 scores were compared by mode of delivery (cesarean prior to labor, cesarean during labor, spontaneous vaginal delivery, and operative vaginal delivery). In addition, the impact of race, age, education level, parity, episiotomy, labor induction, and duration of second stage of labor on maternal satisfaction were examined. RESULTS: Among 576 women, 10.1-17.5 years from delivery, significant differences in satisfaction scores were noted by delivery mode. Salmon scale scores differed between women delivering by cesarean and those delivering vaginally: women delivering vaginally reported greater fulfillment (0.40 [-0.37 to 0.92] vs 0.15 [-0.88 to 0.66], P < .001) and less distress (-0.34 [-0.88 to 0.38] vs 0.20 [-0.70 to 0.93], P < .001) than those who delivered by cesarean. Women who delivered by cesarean prior to labor reported the greatest median fulfillment scores and the lowest median difficulty scores. Median distress scores were lowest among those who delivered by spontaneous vaginal birth. Among women who underwent cesarean delivery, labor induction and prolonged second stage were associated with higher difficulty scores. These factors did not affect satisfaction scores among women who delivered vaginally. Among women who delivered vaginally, operative vaginal delivery was associated with less favorable scores across all 3 scores. CONCLUSION: Maternal satisfaction with childbirth is influenced by mode of delivery. The birth experience leaves an impression on women more than a decade after delivery.

Population-Level Persistence of Immunity 2 Years After the PsA-TT Mass-Vaccination Campaign in Mali.

BACKGROUND: In 2010, Africa's first preventive meningococcal mass vaccination campaign was launched using a newly developed Neisseria meningitidis group A (NmA) polysaccharide-tetanus toxoid conjugate vaccine, PsA-TT (MenAfriVac), designed specifically for the meningitis belt. Given PsA-TT's recent introduction, the duration of protection against meningococcal group A is unknown. METHODS: We conducted a household-based, age-stratified seroprevalence survey in Bamako, Mali, in 2012, 2 years after the vaccination campaign targeted all 1- to 29-year-olds. Randomly selected participants who had been eligible for PsA-TT provided a blood sample and responded to a questionnaire. Sera were analyzed to assess NmA-specific serum bactericidal antibody titers using rabbit complement (rSBA) and NmA-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay. The proportion of participants putatively protected and the age group- and sex-specific rSBA geometric mean titers (GMTs) and IgG geometric mean concentrations (GMCs) were determined. RESULTS: Two years postvaccination, nearly all of the 800 participants (99.0%; 95% confidence interval [CI], 98.3%-99.7%) maintained NmA-specific rSBA titers ≥8, the accepted threshold for protection; 98.6% (95% CI, 97.8%-99.4%) had titers ≥128, and 89.5% (95% CI, 87.4%-91.6%) had titers ≥1024. The rSBA GMTs were significantly higher in females than in males aged <18 years at vaccination (P < .0001). NmA-specific IgG levels ≥2 µg/mL were found in 88.5% (95% CI, 86.3%-90.7%) of participants. CONCLUSIONS: Two years after PsA-TT introduction, a very high proportion of the population targeted for vaccination maintains high antibody titers against NmA. Assessing the duration of protection provided by PsA-TT is a priority for implementing evidence-based vaccination strategies. Representative, population-based seroprevalence studies complement clinical trials and provide this key evidence.

Adjusted-Dose Enoxaparin for VTE Prevention in the Morbidly Obese.

Background: Venous thromboembolism (VTE) is a major health problem and common cause of morbidity and mortality in hospitalized patients. While trials in both surgical and medically ill patients have demonstrated efficacy and safety of enoxaparin for VTE prophylaxis (VTEP), they failed to adequately represent morbidly obese (body mass index > 40 kg/m2) patients. Objective: To assess the impact of a weight-adjusted enoxaparin dosing algorithm on anti-factor Xa levels, thrombosis, and bleeding in morbidly obese patients. Methods: A retrospective chart review was conducted, which included morbidly obese patients receiving VTEP with adjusted-dose enoxaparin. Patients received enoxaparin 0.5 mg/kg subcutaneously once or twice daily based on VTE risk. An anti-factor Xa level was drawn 3 to 5 hours after 2 or more consecutive doses. The primary outcome was the percentage of patients achieving target anti-factor Xa levels, defined as 0.2 to 0.6 IU/mL. Secondary outcomes included the incidence of symptomatic VTE and major bleeding. Results: Of the 182 charts reviewed, 141 anti-factor Xa levels from 130 patients met inclusion criteria. The study population was 44% male, and the median body mass index was 45.6 kg/m2. A total of 120 anti-factor Xa levels (85.1%) were within the target prophylactic range. Sixteen anti-factor Xa levels (11.3%) were below target range, and 5 (3.4%) were above range. The only significant difference among the 3 groups was baseline renal function (P = .035). There were 2 thromboembolic events and 1 major bleed in the study population. Conclusion: A weight-based VTEP dosing strategy for morbidly obese patients is effective without an apparent increase in adverse events.

Mediator of DNA damage checkpoint 1 (MDC1) regulates mitotic progression.

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053-32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.

Isolating polysaccharide IgG pneumococcal antibody responses by pre-adsorption of conjugate vaccine serotypes: A modified approach for the conjugate vaccine era.

BACKGROUND: Assessment of pure polysaccharide response to the 23-valent pneumococcal polysaccharide vaccine (PPV23) can be biased by previous exposure to the conjugate vaccine (PCV). We applied pre-analytical modification to the existing ELISA by pre-incubating serum with PCV. METHODS: PCV-adsorbed and non-adsorbed sera were prepared before measuring the concentration of anti-pneumococcal capsular polysaccharide (PCP) IgG antibodies by the whole pneumococcal ELISA. Paired pre and post-pneumococcal vaccination sera from 73 subjects were analyzed and the baseline anti-PCP IgG for each sample was subtracted from the post-vaccination value to measure vaccine responses. Absolute change in titers and fold changes were then compared between both methods. RESULTS: In the PCV-vaccinated group (n = 28), pre-adsorption with PCV significantly reduced the vaccine responses compared to non-adsorbed sera [median increase in anti-PCP titers: 27.55 mg/l and 45.98 mg/l, respectively]. In addition, the median fold change dropped significantly from 3.026 to 2.313. In PPV23-vaccinated immunocompetent subjects (n = 28) there was a significant difference in anti-PCP responses with PCV adsorption [median values: 73.71 mg/l without and 51.04 mg/l with adsorption]. All the antibody deficiency patients (n = 17) displayed poor PPV23 responses. Although PPV23 responsiveness was not statistically different between both methods, we have observed a trend for lower anti-PCP IgG titers in PCV-adsorbed sera compared to non-adsorbed ones. Serotype-specific IgG analysis using a multiplexed bead-based immunoassay performed on 10 paired samples confirmed that the adsorption observed is specific to PCV serotypes. CONCLUSION: Pre-analytical modification to the conventional ELISA by removing the PCV-specific serotypes may differentiate true polysaccharide response from recall response induced by previous PCV vaccination.

Testing the Stability of Plasma Protein and Whole Blood RNA in Archived Blood of Loggerhead Sea Turtles, Caretta caretta.

Sample storage conditions can affect accuracy and reproducibility of biological measurements. Storing samples rapidly at the lowest available temperatures is considered ideal but is not always feasible when sampling in remote and logistically challenging field conditions, as is often the case with sea turtles. The objective of this study was to examine the stability of plasma proteins and quality of whole blood RNA from loggerhead sea turtle samples collected as part of an eighteen-year-long curated specimen collection. These biological variables are often used to assess sea turtle health; therefore, it is necessary to maintain the integrity of these components during storage. Protein electrophoresis was conducted on heparinized plasma from individual turtles collected in 2018 (n = 3), 2008 (n = 3), and 2001 (n = 3). Plasma was also pooled from four turtles sampled in 2018 and subjected to various storage temperatures. Whole blood was collected in blood collection tubes containing sodium heparin or PAXgene tubes with an RNA preservative. These were subjected to different storage treatments that can possibly occur during logistically difficult field sampling. Following various treatments, plasma proteins showed minor differences across collection years and no differences among storage treatments were observed, even when exposed to 38°C for three hours. RNA quality was assessed from whole blood using an RNA integrity number (RIN). RINs were poor from sodium heparin tubes that were frozen and from PAXgene tubes after an extended thaw. High-quality RNA was obtained from sodium heparin tubes that were never frozen and from PAXgene tubes with freezing delayed by up to 11 days. Overall, these results indicate that plasma proteins remain stable over time and when exposed to undesirable storage conditions, and RNA degrades rapidly in sea turtle blood after freezing and when not properly preserved. These aspects are important to consider when planning sampling protocols and logistics for optimal long-term sample preservation.

Effects of age on optical coherence tomography measurements of healthy retinal nerve fiber layer, macula, and optic nerve head.

PURPOSE: To determine the effects of age on global and sectoral peripapillary retinal nerve fiber layer (RNFL), macular thicknesses, and optic nerve head (ONH) parameters in healthy subjects using optical coherence tomography (OCT). DESIGN: Retrospective, cross-sectional observational study. PARTICIPANTS: A total of 226 eyes from 124 healthy subjects were included. METHODS: Healthy subjects were scanned using the Fast RNFL, Fast Macula, and Fast ONH scan patterns on a Stratus OCT (Carl Zeiss Meditec, Dublin, CA). All global and sectoral RNFL and macular parameters and global ONH parameters were modeled in terms of age using linear mixed effects models. Normalized slopes were also calculated by dividing the slopes by the mean value of the OCT parameter for interparameter comparison. MAIN OUTCOME MEASURES: Slope of each OCT parameter across age. RESULTS: All global and sectoral RNFL thickness parameters statistically significantly decreased with increasing age, except for the temporal quadrant and clock hours 8 to 10, which were not statistically different from a slope of zero. Highest absolute slopes were in the inferior and superior quadrant RNFL and clock hour 1 (superior nasal). Normalized slopes showed a similar rate in all sectors except for the temporal clock hours (8-10). All macular thickness parameters statistically significantly decreased with increasing age, except for the central fovea sector, which had a slight positive slope that was not statistically significant. The nasal outer sector had the greatest absolute slope. Normalized macular slope in the outer ring was similar to the normalized slopes in the RNFL. Normalized inner ring had shallower slope than the outer ring with a similar rate in all quadrants. Disc area remained nearly constant across the ages, but cup area increased and rim area decreased with age, both of which were statistically significant. CONCLUSIONS: Global and regional changes caused by the effects of age on RNFL, macula, and ONH OCT measurements should be considered when assessing eyes over time. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Automated assessment of the optic nerve head on stereo disc photographs.

PURPOSE: To develop automated software for optic nerve head (ONH) quantitative assessment from stereoscopic disc photographs and to evaluate its performance in comparison with human expert assessment. METHODS: A fully automated system, including three-dimensional ONH modeling, disc margin detection, cup margin detection, and calculation of stereometric ONH parameters, was developed and tested. One eye each from 54 subjects (23 healthy, 17 suspected glaucoma, and 14 glaucoma) was enrolled. The majority opinion of three experts defined disc and cup margins on the disc photographs was used for comparison. Seven ONH parameters, disc area, rim area, rim volume, cup area, cup volume, cup-to-disc (C/D) area ratio, and vertical C/D ratio, were computed based on both machine- and expert-defined margins and compared between the methods. RESULTS: All automated ONH measurements showed good correlation with the expert defined margins (Pearson r = 0.90, disc area; 0.56, rim area; 0.78, rim volume; 0.88, cup area; 0.93, cup volume; 0.69, C/D area ratio; and 0.67, vertical C/D ratio; all P or= 0.21). The mean or median of automatically defined disc and cup areas was significantly higher than the subjective assessment (disc area P = 0.0001, t-test; cup area P = 0.036, Wilcoxon signed ranks test), although they had high correlation coefficients. The software failed to detect the disc margin for all the disc photographs with peripapillary atrophy. CONCLUSIONS: The automated ONH analysis method provides an objective and quantitative ONH evaluation using widely available stereo disc photographs.

Optical coherence tomography scan circle location and mean retinal nerve fiber layer measurement variability.

PURPOSE: To investigate the effect on optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) thickness measurements of varying the standard 3.4-mm-diameter circle location. METHODS: The optic nerve head (ONH) region of 17 eyes of 17 healthy subjects was imaged with high-speed, ultrahigh-resolution OCT (hsUHR-OCT; 501 x 180 axial scans covering a 6 x 6-mm area; scan time, 3.84 seconds) for a comprehensive sampling. This method allows for systematic simulation of the variable circle placement effect. RNFL thickness was measured on this three-dimensional dataset by using a custom-designed software program. RNFL thickness was resampled along a 3.4-mm-diameter circle centered on the ONH, then along 3.4-mm circles shifted horizontally (x-shift), vertically (y-shift) and diagonally up to +/-500 microm (at 100-microm intervals). Linear mixed-effects models were used to determine RNFL thickness as a function of the scan circle shift. A model for the distance between the two thickest measurements along the RNFL thickness circular profile (peak distance) was also calculated. RESULTS: RNFL thickness tended to decrease with both positive and negative x- and y-shifts. The range of shifts that caused a decrease greater than the variability inherent to the commercial device was greater in both nasal and temporal quadrants than in the superior and inferior ones. The model for peak distance demonstrated that as the scan moves nasally, the RNFL peak distance increases, and as the circle moves temporally, the distance decreases. Vertical shifts had a minimal effect on peak distance. CONCLUSIONS: The location of the OCT scan circle affects RNFL thickness measurements. Accurate registration of OCT scans is essential for measurement reproducibility and longitudinal examination (ClinicalTrials.gov number, NCT00286637).

Repeated, noninvasive, high resolution spectral domain optical coherence tomography imaging of zebrafish embryos.

PURPOSE: To demonstrate a new imaging method for high resolution spectral domain optical coherence tomography (SD-OCT) for small animal developmental imaging. METHODS: Wildtype zebrafish that were 24, 48, 72, and 120 h post fertilization (hpf) and nok gene mutant (48 hpf) embryos were imaged in vivo. Three additional embryos were imaged twice, once at 72 hpf and again at 120 hpf. Images of the developing eye, brain, heart, whole body, proximal yolk sac, distal yolk sac, and tail were acquired. Three-dimensional OCT data sets (501 x 180 axial scans) were obtained as well as oversampled frames (8,100 axial scans) and repeated line scans (180 repeated frames). Scan volumes ranged from 750 x 750 microm to 3 x 3 mm, each 1.8 mm thick. Three-dimensional data sets allowed construction of C-mode slabs of the embryo. RESULTS: SD-OCT provided ultra-high resolution visualization of the eye, brain, heart, ear, and spine of the developing embryo as early as 24 hpf, and allowed development to be documented in each of these organ systems in consecutive sessions. Repeated line scanning with averaging optimized the visualization of static and dynamic structures contained in SD-OCT images. Structural defects caused by a mutation in the nok gene were readily observed as impeded ocular development, and enlarged pericardial cavities. CONCLUSIONS: SD-OCT allowed noninvasive, in vivo, ultra-high resolution, high-speed imaging of zebrafish embryos in their native state. The ability to measure structural and functional features repeatedly on the same specimen, without the need to sacrifice, promises to be a powerful tool in small animal developmental imaging.

Ultrahigh-resolution spectral domain optical coherence tomography imaging of the lamina cribrosa.

Study of the structure of the lamina cribrosa is critical in glaucoma research. The purpose of this study is to determine the optimal spectral domain optical coherence tomography imaging protocol for the digital isolation and display of the lamina cribrosa. Three-dimensional datasets centered on the lamina cribrosa were obtained with 200 X 200 to 512 X 512 A-scan densities. The effect of scan density and c-mode slab thickness was subjectively compared. Increasing slab thickness reduced the sharpness of visible prelamina and lamina cribrosa structures. In retrolamina structures, thin slabs provided good visualization, but increased slab size increased the visibility of deeper structures. Scan times as short as 2.3 seconds (256 X 256 A-scans) degraded visualization of the shape of the optic nerve head. The optical scan protocol for lamina cribrosa imaging appears to be a 3 x 3 mm 200 X 200 A-scan volume with the lamina cribrosa positioned near direct current.

Spectral oximetry assessed with high-speed ultra-high-resolution optical coherence tomography.

We use Fourier domain optical coherence tomography (OCT) data to assess retinal blood oxygen saturation. Three-dimensional disk-centered retinal tissue volumes were assessed in 17 normal healthy subjects. After removing DC and low-frequency a-scan components, an OCT fundus image was created by integrating total reflectance into a single reflectance value. Thirty fringe patterns were sampled; 10 each from the edge of an artery, adjacent tissue, and the edge of a vein, respectively. A-scans were recalculated, zeroing the DC term in the power spectrum, and used for analysis. Optical density ratios (ODRs) were calculated as ODR(Art)=ln(Tissue(855)Art(855))ln(Tissue(805)Art(805)) and ODR(Vein)=ln(Tissue(855)Vein(855))ln(Tissue(805)Vein(805)) with Tissue, Art, and Vein representing total a-scan reflectance at the 805- or 855-nm centered bandwidth. Arterial and venous ODRs were compared by the Wilcoxon signed rank test. Arterial ODRs were significantly greater than venous ODRs (1.007+/-2.611 and -1.434+/-4.310, respectively; p=0.0217) (mean+/-standard deviation). A difference between arterial and venous blood saturation was detected. This suggests that retinal oximetry may possibly be added as a metabolic measurement in structural imaging devices.

Autocrine metabolism of vitamin D in normal and malignant breast tissue.

PURPOSE: Vitamin D seems to exert a protective effect against common cancers, although this does not correlate with circulating levels of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], indicating a more localized activation of vitamin D. The aim of this study was to investigate the significance of this in breast cancer. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR analysis of mRNA expression was carried out for the vitamin D-activating enzyme 1alpha-hydroxylase, the catabolic enzyme 24-hydroxylase, and the vitamin D receptor in 41 tumors and paired nonneoplastic tissue as well as breast cancer cell lines. Immunohistochemistry was used to assess 1alpha-hydroxylase protein expression, and enzyme assays were used to quantify vitamin D metabolism. RESULTS: Expression of mRNA for 1alpha-hydroxylase (27-fold; P < 5 x 10(-11)), vitamin D receptor (7-fold; P < 1.5 x 10(-8)), and 24-hydroxylase (4-fold; P < 0.02) was higher in breast tumors. 1alpha-Hydroxylase enzyme activity was also higher in tumors (44.3 +/- 11.4 versus 12.4 +/- 4.8 fmol/h/mg protein in nonneoplastic tissue; P < 0.05). However, production of inactive 1,24,25-trihydroxyvitamin D3 was also significantly higher in tumors (84.8 +/- 11.7 versus 33.6 +/- 8.5 fmol/h/mg protein; P < 0.01). Antisense inhibition of 24-hydroxylase in vitro increased antiproliferative responses to 1,25(OH)2D3. CONCLUSION: These data indicate that the vitamin D-activating enzyme 1alpha-hydroxylase is up-regulated in breast tumors. However, dysregulated expression of 24-hydroxylase seems to abrogate the effects of local 1,25(OH)2D3 production in tumors by catalyzing catabolism to less active vitamin D metabolites. The enzymes involved in autocrine metabolism of vitamin D in breast tissue may therefore provide important targets for both the prevention and treatment of breast cancer.

Sampling for international normalized ratios in patients on hemodialysis with central venous catheters.

The primary objectives of this study were to evaluate the effect of varying heparin concentrations on International Normalized Ratio values and to assess the accuracy of these values deter mined through the arterial line of the dialysis circuit Twenty-two patients on hemodialysis with central venous catheters were studied After a peripheral venipuncture, timed samples from the arterial line of the hemodialysis circuit were obtained after dialysis was initiated and prior to initiating heparin. Assays for coagulation parameters were performed. There was no signifcant difference in any coagulation parameter measured from the arterial line samples compared to the venipuncture samples. Serial heparin dilutions in vitro demonstrated that residual amounts of heparin in the central venous catheter may falsely elevate International Normalized Ratio values. This study demonstrates that accurate International Normalized Ratio values in patients on hemodialysis with heparinized central venous catheters can be obtained ericiently and cost-effectively from the arterial line within 1 minute of dialysis initiation.

Biological actions of extra-renal 25-hydroxyvitamin D-1alpha-hydroxylase and implications for chemoprevention and treatment.

The Vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase) is now known to be expressed in a much wider range of tissues that previously thought, suggesting a role for 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), which is more in keeping with a cytokine than a hormone. In this capacity, the function of 1alpha-hydroxylase in tumors is far from clear. Studies from several groups including ours have shown altered expression of 1alpha-hydroxylase in different types of neoplasm including breast, prostate and colon cancers. However, functional analysis of Vitamin D metabolism in cancer is complicated by the heterogenous composition of tumors. Immunohistochemical analysis of breast tumors has shown that 1alpha-hydroxylase is expressed by both epithelial cells and by tumor-infiltrating macrophages, suggesting an immunomodulatory component to 1,25(OH)(2)D(3) production in some types of cancer. The demonstration of 1alpha-hydroxylase activity in tumors and their equivalent normal tissues has implications for both the treatment and prevention of cancers. For example, in tumors chemotherapy options may include the use of non-1alpha-hydroxylated Vitamin D analogs to increase local concentrations of active metabolites without systemic side-effects. The role of 1alpha-hydroxylase in protection against cancer is likely to be more complicated and may involve anti-tumor immune responses.

Evaluation and validation of a serum bactericidal antibody assay for Haemophilus influenzae type b and the threshold of protection.

Prior to routine immunisation, Haemophilus influenzae serotype b (Hib) was a major cause of serious bacterial infections, particularly in young children. In the United Kingdom, introduction of the Hib conjugate vaccine into the national childhood immunisation schedule has led to a sustained decline in invasive Hib disease across all age-groups. Evaluation of the immune response to Hib conjugate vaccines involves measurement of serum IgG antibodies against the capsular polyribosyl-ribitol-phosphate (PRP) polysaccharide by enzyme-linked immunosorbent assay (ELISA), with accepted short-term and long-term protective thresholds of ≥0.15µg/mL and ≥1.0µg/mL, respectively. These levels were derived by passive immunisation or immunisation with pure polysaccharide, and their relevance for protection following immunisation with conjugate vaccines remains unclear. This study aimed to modify and optimise a serum bactericidal antibody (SBA) assay to evaluate the functional activity of Hib antibodies generated following Hib conjugate vaccination. Validation of the Hib SBA assay was deemed acceptable for all assay parameters tested. A strong correlation between anti-PRP IgG concentrations and SBA titres was observed in vaccinated adults (r=0.81), as well as infants after primary immunisation at 2, 3, and 4 months (r=0.635) and after the 12-month booster (r=0.746). The assay identified some children with high anti-PRP IgG but low SBA activity and vice versa. The predictive protective SBA titre corresponding to a post-booster anti-PRP IgG of 1.0µg/mL was 8. Thus, the optimised Hib SBA assay was specific and reproducible and correlated with anti-PRP IgG. Such assays may have a role in evaluating immune responses to conjugate vaccines in addition to measuring capsular antibodies.