Fraction Mr Research Articles

Ablative 5-Fraction CT vs. MR-Guided Pancreatic SBRT: Evaluation of Interfraction Anatomic Changes on Dosimetric Constraints.

CT-guided SBRT for locally advanced pancreatic cancer (LAPC) is usually non-ablative (BED < 100 Gy10) to minimize grade 3+ toxicity risks given the concern of interfraction anatomic changes (IACs) in GI anatomy and imaging quality associated with kV-CBCT. Emerging data demonstrate that MR guidance facilitates 5-fraction (fx) dose escalation due to superior soft tissue contrast, continuous intrafraction imaging, automatic beam gating, and on-table adaptive replanning capability. Treatment outcomes for ablative 5-fx CT- vs. MR-guided SBRT are not well characterized, nor are differences in predicted GI OAR doses when accounting for IACs. Weevaluated 40 plans (20 CT, 20 MR) for 20 LAPC patients (pts) previously treated in breath hold (BH) on a 0.35 T MR-Linac. Prescribed dose was 50 Gy (gross disease) and 33 Gy (elective) in 5 fx using a simultaneous integrated boost technique. CT plans were retrospectively created using 2-3 VMAT arcs with the same prescription dose, target volumes (assuming BH), and constraints (prioritizing OARs over target coverage) as the MR IMRT plans (∼20-40 fields). CT planners were blinded to MR plans. We compared predicted GI OAR dose of CT vs. MR plans across each of the 5 fx for all 20 patients to evaluate the dosimetric impact of IACs by coregistering CT plans to the anatomy of the day based on 0.35T MR scans acquired for GI OAR segmentation and treatment delivery. MedianV100% of the GTV, CTV, PTV50, and PTV33 across the original CT vs. MR plans were 97.5% vs. 91.3% (p = 0.017), 99.9% vs. 98.2% (p<0.01), 86.2% vs. 79.3% (p = 0.39), and 97.2% vs. 93.0% (p<0.01), respectively. GI OAR constraints were met for all original CT/MR plans although it was predicted that 1+ GI OAR constraint would be violated (most commonly duodenum) for 88/100 CT vs. 85/100 MR fractions. Across the 88 violated CT fractions, the median predicted GI OAR doses were duodenum V35: 3.3 cc (range: 0.16-18.0cc), duodenum V40: 1.2 cc (range: 0.01-11.9cc), small bowel V35: 1.2 cc (range: 0.4-10.9cc), small bowel V40: 0.2 cc (range: 0.04-7.0cc), stomach V35: 1.5 cc (range: 0.52-6.8cc), stomach V40: 0.3 cc (range: 0.05-2.8cc). GI OAR doses across the 85 violated MR fractions were similar. Median fxs per pt with 1+ predicted GI OAR violation was 5 (range: 1-5) for both CT and MR plans. This isthe first evaluation of IAC effects on predicted GI OAR dose for 5-fx CT- vs. MR-guided SBRT. Although VMAT arcs facilitated higher target coverage in the initial CT plans, GI OAR constraint violations were observed in 85-88% of CT/MR plans. Although on-table adaptive replanning is routine on MR-guided Linacs it is not commonly available on CT-guided Linacs. As such, ablative 5-fx SBRT delivered with CT guidance is expected to result in significant toxicity due to exceeding GI OAR constraints for most delivered fractions.

The relationship between mitral regurgitant severity, symptoms, and exercise capacity in primary degenerative mitral regurgitation.

Although it is assumed that more severe MR is associated with a greater burden of symptoms and lower exercise capacity, the relationship between symptoms, exercise capacity, and mitral regurgitant severity has not been well studied. We prospectively studied 67 (63 ± 11 years, 72% male) patients with at least mild degenerative MR and left ventricular ejection fraction ≥ 50% who underwent stress echocardiography, CMR, and evaluation with the Kansas City Cardiomyopathy questionnaire (KCCQ). Symptoms and exercise capacity were evaluated in the context of MR severity. Patients reporting dyspnea had lower KCCQ symptom scores (79 ± 23 vs. 96 ± 9, p = 0.01) and achieved lower percentage of age and gender predicted METs (114 ± 37 vs. 152 ± 43%, p < 0.001) compared to those without dyspnea. There was no significant difference in MR volume between those with vs. without dyspnea by CMR (43 ± 26 ml vs. 51 ± 28 ml, p = 0.3) or echocardiography (64 ± 28 vs. 73 ± 41ml, p = 0.4). Those with severe MR by CMR had similar KCCQ symptom scores (96 ± 10 vs. 89 ± 17, p = 0.04) and percentage of age and gender predicted METs (148 ± 42 vs. 133 ± 47%, p = 0.2) to those without severe MR. Those with severe MR by echocardiography had similar KCCQ symptom score (93 ± 15 vs. 89 ± 16, p = 0.3) and percentage of age and gender predicted METs (138 ± 43 vs. 153 ± 46%, p = 0.2) to those without severe MR. Patients with degenerative MR assessed by CMR and stress echocardiography, there was no relationship between MR severity and either symptoms or exercise capacity. These findings highlight the disconnect between symptoms and the severity of MR and challenge the assumption that correcting MR can be counted on to improve symptom status in patients with degenerative MR.

The Effect of Angiotensin Receptor and Neprilysin Inhibitors on Quality of Life in Patients with Heart Failure with Reduced Ejection Fraction and Functional Mitral Regurgitation

Aim. To compare the change in quality of life indicators and the main clinical and instrumental parameters in patients with chronic heart failure (CHF) and functional mitral regurgitation (FMR) under the influence of sacubitril/valsartan compared with valsartan in an outpatient practice.Material and Methods. The study included 90 patients with chronic FMR, who were observed for 12 months. They received sacubitril/valsartan or valsartan. Efficiency criteria were: the difference between groups in scores according to the Kansas questionnaire for patients with cardiomyopathy; MR degree parameters (change in effective regurgitation orifice area, vena contracta width, MR volume and MR fraction); indicators of the severity of myocardial remodeling (left ventricular EF; the level of N-terminal brain natriuretic propeptide), exercise tolerance based on a 6-minute walk test, a change in the functional class of heart failure according to NYHA.Results. In a treatment efficacy analysis, the Kansas City Cardiomyopathy Questionnaire–Overall Summary Score improved by 22.1 points in the sacubitril/valsartan group and by 4.5 points in the valsartan group (p&lt;0.001). EF, exercise tolerance, and the number of patients transitioning from NYHA functional class III to II increased in the sacubitril/valsartan group (p&lt;0.05). Also, in this group, the effective area of the regurgitation opening, the width of the vena contracta, the volume of regurgitation, the fraction of regurgitation, and the level of N-terminal brain natriuretic propeptide (p&lt;0.05) decreased more pronouncedly (p&lt;0.05).Conclusion. Compared with valsartan, treatment with sacubitril/valsartan leads to a significant improvement in the quality of life in patients with FMR and HF with reduced EF, which is largely associated with a change in NT-proBNP, echocardiographic characteristics of the severity of MR and the degree of myocardial remodeling.

Abstract 13337: Left Atrial Ejection Fraction by Cardiac Magnetic Resonance Imaging as a Predictor of Adverse Outcomes in Patients With Advanced Ischemic Cardiomyopathy

Background: The prognostic significance of cardiac magnetic resonance (CMR) based left atrial ejection fraction (LAEF) is not well defined in the ischemic cardiomyopathy (ICM) cohort.We assess the additive effect of LAEF in a model to predict outcomes in patients with ICM. Methods: Patients with ICM, who underwent CMR between April 2001 and March 2019 were retrospectively included. Clinical characteristics and CMR parameters were collected and analyzed and LAEF was calculated in addition to myocardial infarct size (MIS) using late gadolinium enhancement (LGE) and CMR based mitral regurgitant fraction (MRF). The primary clinical endpoint was a composite of all-cause mortality and cardiac transplant. A multiple-variable Cox proportional hazards regression model which included established predictors of outcome was constructed, followed by the addition of LAEF. Four pre-specified interactions of LAEF with left ventricular end systolic volume index (LVESVi), MIS and mitral regurgitant fraction (MRF) were tested at a significance level of 0.05. RESULTS: LA functional data was measured in 718 patients. There were 416 deaths and/or transplants, with a median duration of follow up of 1763 days (4.8 years) The mean LA EF was 36 [2 ,74]. In univariate analysis, lower LAEF and higher LAVI were significant predictors of worse outcome, HR= 0.12, 95% CI [0.06, 0.25], p&lt;0.001; HR 1.008, (1.003-1.014) p = 0.003. After addition to the multivariable model, a normal LAEF was highly predictive of reduced risk, HR 0.24, (0.12, 0.48), p&lt;0.001. In the interaction between LAEF and MIS with MR Fraction, the highest risk was observed in patients with an LAEF &lt; 20% and an MIS of &gt; 30%, with MR Fraction of &gt;35%, HR of 3.2 (1.73-5.93), p= 0.009. The lowest risk was in patients with an LAEF of &gt;50% with an MIS of &lt;15 and MR Fraction of &lt;35%, HR 1.07 (0.81-1.42), p=0.009. Figure 1. CONCLUSION: Reduced LAEF is an important predictor of increased mortality in patients with ICM and additive to MIS and MR Fraction.

Quantification of Interfraction Gastric Motion by Daily Magnetic Resonance Imaging and Implications for Radiation Treatment Planning

Accurate radiation therapy targeting for stomach cancer is challenging given daily variations in the stomach's size and position. We evaluated daily MR-based imaging to quantify changes in stomach volume and characterized the impact of inter-fractional variation on dosimetry for gastric cancer radiation plans.We assessed inter-fraction changes in the volume and position of the stomach via MR-based imaging for 10 patients who previously underwent abdominal stereotactic body radiation therapy. All patients were simulated and treated over 5 fractions and were instructed to fast for 3 hours before each session. The stomach and regional organs-at-risk (OARs) were contoured on each fraction's MR scan according to standard RTOG guidelines. Inter-fraction variation in the size and position of the stomach was quantified by calculating the percentage volume change and dice similarity coefficient (DSC) as compared to the simulation scan. To determine the potential impact of inter-fraction stomach motion on treatment planning, we generated mock plans to recapitulate neoadjuvant or definitive stomach cancer treatments. For the purpose of this study, the stomach contour was symmetrically expanded by 1.7 cm to generate a planning treatment volume (PTV) and a prescription dose of 45 Gy in 1.8 Gy fractions was applied. Intensity-modulated radiation therapy plans were then generated to meet the following dosimetric goals: PTV V95% of 95%, liver V30 less than 30%, and bilateral kidney V20 less than 20%. Finally, the impact of gastric variation on these dose metrics was assessed.Despite standard daily preparation and set up procedures, we observed significant variability in the size and location of the stomach between fractions. The mean absolute daily change in gastric volume compared to the planning MRI was 19.1% (total range: -42.0% to 58.4%). The average stomach DSC was 0.69 (range: 0.40 to 0.86). Given this degree of variability, mock gastric cancer treatment plans displayed suboptimal dose metrics. The average PTV V45 was 81.5% (range: 58.8% to 99.2%. 95% CI: 78.8% to 84.2%). Average liver V30 values were 12.7% (range: 3.5% to 20.1%. 95% CI: 11.7% to 13.9%) and bilateral kidney V20 values were 12.3% (range: 0.5% to 31.3%. 95% CI: 9.2% to 15.3%). Target metrics were highly correlated with the percent change in stomach volume-a 10% increase in stomach volume was associated with a 2.4% decrease in the V45 (P < 0.001).We observed significant inter-fraction variability in the dimensions of the stomach using daily MRI-based imaging, leading to dosimetrically inferior mock gastric cancer treatment plans. Our study suggests that larger PTV margins may be necessary for conventional stomach plans, which would come at the expense of greater OAR exposure. Alternatively, MRI-based image guidance and daily adaptive treatments may improve the therapeutic window of radiation therapy for gastric tumors.

MR Guided Brachytherapy for Cervical Cancer: Are There Factors to Define Who Benefits Most From MR on Every Fraction?

It has been previously reported that an MR on the first fraction of brachytherapy for cervical cancer may sufficiently define the target geometry for future fractions, and CT based normal tissue definition may allow for adequate customization of the plan to spare organs at risk (OAR), obviating the need for an MR on each fraction. The current report expands upon this with a larger cohort to determine if there are any subpopulations, such as large tumors, or those with residual parametrial extension that may benefit more from MR imaging on every brachytherapy fraction. As previously reported, women treated with tandem and ovoid or ring brachytherapy from 2011-2014 for cervical cancer were identified, in whom a planning MR was obtained on every fraction. The dose from this “MR optimized course” was captured for the HRCTV (D90 and D98), the IRCTV (D90), and OARs (bladder, rectum, sigmoid, and bowel, calculated to D2cc). Two simulated courses were then developed. The dwell positions from the first MR guided fraction were applied to the later fractions without any edits to define a “single plan course.” A “CT optimized course” was then created by developing new plans using only CT OAR anatomy from the latter fractions, with the HRCTV fused from the first fraction. If the applicator was changed, both simulations assumed that a new MR based plan would be developed with the new applicator. Doses were then calculated for the MR based HRCTVs and OARs, and both simulations were compared to the MR optimized course. Doses were converted to the equivalent dose at 2Gy per fraction (EQD2) via the BED equation (α/β = 10 for target, and 3 for normal tissue). Three patient factors at the time of the first fraction were then examined for impact on these findings: MR complete response (CR) vs partial response (PR), residual parametrial extension (PME), and the HRCTV volume. The Wilcoxon signed rank test was used for paired data, and the Mann-Whitney U test for unpaired, with a two tailed p-value of <0.05 considered significant. 19 women were identified; each undergoing 4-5 fractions of MR guided brachytherapy after 45Gy external beam with weekly cisplatin, 7 of whom had residual PME, and 7 with an MR CR. There was no systematic deviation in the D90 or D98 dose to HRCTV, or IRCTV D90 in either simulated course compared to the MR optimized fraction (p>0.05 for all comparisons). Fractional doses as a percent of the MR-optimized plan to the rectum were significantly lower with the CT-optimized plan (Rectal D2cc 94%,IQR 71-107%, p=0.024), higher to the bladder with the single plan course (Bladder D2cc 110%, IQR 89-124%, p=0.047), and higher to the small bowel with the single plan course (Bowel D2cc 105%, IQR 90-123%, p=0.011). There were no other significant systematic deviations in normal tissue dose. For those with greater than the median HRCTV volume (23.7cc), the doses to targets were no lower than those with smaller tumors. There was a modest significant decrease in D98 dose as a fraction of the MR optimized dose in those with residual PME, compared to those without (97% with PME, 102% without, p=0.018), and there were no other. The presence of MR CR or PR had no systematic effect on doses with either strategy. Women with residual parametrial extension at first brachytherapy had a slightly lower coverage of D98 with a CT-optimized plan compared to those without, however the absolute difference is likely clinically insignificant. Otherwise, neither tumor size, nor the lack of an MR complete response predicted for worse performance of either the single plan or CT-optimized plan. The CT-optimized plan had improved sparing of rectum, bladder, and small bowel compared to the single plan.

Significant Reduction In Mitral Regurgitation After Transcatheter Aortic Valve Implantation In a Patient With Severe Degenerative Aortic Valve Disease, Moderate to Severe Mitral Regurgitation and Normal Ejection Fraction

The best treatment modalities in severe aortic valve disease are surgery and, in selected patients, transcatheter aortic valve implantation (TAVI). At the present time, transcatheter aortic valve implantation performed in inoperable patients or in patients with high surgical risk not only treats the aortic valve stenosis but also treats the low ejection fraction and mitral regurgitation that result from it. An 83-year-old female patient presented with shortness of breath and class 3 angina. Transthoracic and transesophageal echocardiography revealed severe AS, moderate-to-severe MR, and normal ejection fraction. The patient was considered at high risk for aortic valve replacement (Logistic Euroscore 20, STS score 15). A dramatic decrease in the degree of mitral regurgitation was detected after the successful TAVI.

Isolation of antibacterial peptides from Saudi Arabian honeybees and investigating the antimicrobial properties of natural honey samples

As part of the ongoing search for novel anti microbial agents and their use in singular or combined drug therapy, a polypeptide fractions of Mr about 14.500 and 15.00 KDa were isolated from the lymph fluid of two groups of intact and immunized Saudi Arabian honeybees (Apis mellifera) obtained from Al-Qasim and Hail locations around Riyadh during the honey season 2008 following experimental infection with 1.1 x l0 6 viable Escherichia coli cells (ATCC 25922). The polypeptide was purified to homogeneity by Reversed Phase High Performance Liquid Chromatography. Antibacterial activity of the isolated polypeptide was evaluated in vitro by an agar well diffusion method for E. coli strain (ATCC 25922) and Klebsiella pneumoniae strain (ATCC 11678) the major Gram negative pathogens causing urinary tract infections, and Staphylococcus aureus (ATCC 6538) as Gram positive bacteria. A total of ten honey samples collected from different floral areas around Riyadh were also investigated for their antimicrobial activity against one yeast, Candida albicans (ATCC 10231) and four standards bacteria strains, E. coli (ATCC 25922), Pseudomonas aeruginosa(ATCC 27853), S. aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) using standard antimicrobial assays. The isolated polypeptide and the different honey samples revealed comparable marked variations in antimicrobial activities and their sensitivity might be depending on their floral source.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

HDAC1 Cooperates with C/EBPα in the Inhibition of Liver Proliferation in Old Mice

Epigenetic control of liver proliferation involves cooperation between transcription factors and chromatin-remodeling proteins. In this work, we found that the levels of HDAC1 (histone deacetylase 1) are increased in quiescent livers of old mice. The elevation of HDAC1 in liver is mediated by the RNA-binding protein CUGBP1. We found that the age-associated CUGBP1-eIF2 complex binds to the 5' region of HDAC1 mRNA and increases translation of HDAC1 in the liver. Further analyses showed that CUGBP1 also increases expression of HDAC1 in cultured cells, in the livers of CUGBP1 transgenic mice, and in the livers of mice injected with cyclin D3, which enhances the formation of the CUGBP1-eIF2 complex. In livers of old mice, HDAC1 interacts with the transcription factor C/EBPalpha and is recruited by this protein to E2F-dependent promoters as a component of high M(r) C/EBPalpha-Brm complexes. The recruitment of HDAC1 to c-Myc and FoxM1B promoters leads to deacetylation of histone H3 at Lys-9 on these E2F-dependent promoters. We show that HDAC1 is an important mediator of growth-inhibitory activity of C/EBPalpha and that small interfering RNA-mediated inhibition of HDAC1 reduces the ability of C/EBPalpha to inhibit cell proliferation. In addition, we have found that both elevation of HDAC1 and interaction of C/EBPalpha with HDAC1 are controlled by cyclin D3-dependent mechanisms. Treatment of old mice with growth hormone, which reduces cyclin D3 levels, leads to the reduction of the CUGBP1-eIF2 complex, normalization of HDAC1 levels, and inhibition of interactions of HDAC1 with C/EBPalpha-Brm complexes. Thus, our data demonstrate that translational elevation of HDAC1 in livers of old mice is involved in the assembly of high M(r) protein-protein complexes that inhibit liver proliferation.

Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths

QuestionHow does the size of the heparin moiety in the anti-thrombin (AT)–heparin complex influence its anticoagulant properties? ApproachOf 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT–heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 μM of that complex. ResultsThe Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e. the enzymes bind the better the longer the heparin. Thrombin (Kd=1.86±0.13 μM) binds 11 times tighter than factor Xa (Kd=20.2 ±1.5 μM). Factor Xa inactivation velocity is proportional to the concentration of pentasaccharide-bound AT if Mr<10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain). ConclusionThe data fit a model in which thrombin and factor Xa bind at a random site on the heparin chain and, via one-dimensional diffusion, reach the AT that is bound to its specific binding site on the heparin. Factor Xa, but not thrombin, can dissociate from heparin before reaching bound AT.

Immunomodulatory activity of acidic polysaccharides isolated from Tanacetum vulgare L.

Tanacetum vulgare L. (Tansy) has been extensively used in folk medicine for treatment of a variety of medical disorders. In the present study, we isolated and purified four acidic polysaccharide fractions (designated T-I to T-IV) from Tansy florets by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, anion-exchange, and size-exclusion chromatography. The average Mr of fractions T-I through T-IV was estimated to be 326, 151, 64 and 9 kDa, respectively, as determined by high performance size-exclusion chromatography analysis. Sugar composition analysis revealed that Tansy polysaccharides consisted primarily of galacturonic acid, galactose, arabinose, and rhamnose. Fractions T-II through T-IV contained an arabinogalactan type II structure, as determined by reaction with Yariv reagent. High Mr fractions T-I and T-II exhibited potent macrophage/monocyte-activating activity, enhancing production of reactive oxygen species (ROS), nitric oxide (NO), and tumor necrosis factor α (TNF-α) by J774.A1 murine macrophages, and activating nuclear factor κB (NF-κB) in THP-1 human monocytes. In addition, Tansy polysaccharides stimulated human neutrophil function by greatly enhancing neutrophil myeloperoxidase (MPO) release. Furthermore, the low Mr fraction T-IV had potent complement-fixing activity, which may also contribute to the anti-inflammatory and would-healing properties of Tansy extracts. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Tansy extracts, and support the concept of using Tansy polysaccharides as an immunotherapeutic adjuvant.

Pharmacodynamic Analysis of Changes in Reticulocyte Subtype Distribution in Phlebotomy-Induced Stress Erythropoiesis

Changes in the reticulocyte subtype distribution (high, medium and low reticulocytes count (HR, MR, LR)) measured by flow cytometry following phlebotomy-induced stress erythropoiesis (abruptly dropping hemoglobin to 3-4 g/dl over 4-5 hr) and the pharmacodynamic (PD) relationship to the stimulated erythropoietin (EPO) was investigated in sheep. A PD model was developed that describes the relationship between EPO and the reticulocyte maturity distribution fractions (r=0.95+/-0.02, mean +/- SD). The lag-time between EPO activation of erythroid progenitor cells and the subsequent increase in the least mature HR fraction in the peripheral circulation was 0.72 +/- 0.08 days. The mean transition times (in days) for all three reticulocyte fractions changed at baseline from, T(HR) : 0.09 +/- 0.06, T(MR) : 0.06 +/- 0.04, and T(LR) : 0.46 +/- 0.24 to T(HR) : 0.13 +/- 0.08, T(MR) : 0.29 +/- 0.15, and T(LR) : 2.3 +/- 0.24 under stress erythropoiesis. The total mean residence time for a reticulocyte in the peripheral circulation, T(total) (T(HR) + T(MR) + T(LR)), increased from 0.60 +/- 0.33 days under basal to 2.8 +/- 0.09 days during stress erythropoiesis. The statistically significant increase observed for T(LR) and T(total) supports the hypothesis that stress erythropoiesis perturbs the mean reticulocyte transition times. A correlation analysis between various new, proposed metrics involving the HR, MR and LR fractions and the total reticulocyte count, with the latter indicative of stress erythropoiesis at higher total counts, revealed a highly significant correlation indicating these new metrics may be a valuable adjunct to the reticulocyte maturation index (RMI) and the immature reticulocyte fractions index (IRF) previously used in assessing erythropoietic activity in response to anemia.

W01-P-002 Comparative study of inflammatory cells in human arteries with clinically significant atherosclerotic sequelae

Tanacetum vulgare L. (Tansy) has been extensively used in folk medicine for treatment of a variety of medical disorders. In the present study, we isolated and purified four acidic polysaccharide fractions (designated T-I to T-IV) from Tansy florets by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, anion-exchange, and size-exclusion chromatography. The average Mr of fractions T-I through T-IV was estimated to be 326, 151, 64 and 9 kDa, respectively, as determined by high performance size-exclusion chromatography analysis. Sugar composition analysis revealed that Tansy polysaccharides consisted primarily of galacturonic acid, galactose, arabinose, and rhamnose. Fractions T-II through T-IV contained an arabinogalactan type II structure, as determined by reaction with Yariv reagent. High Mr fractions T-I and T-II exhibited potent macrophage/monocyte-activating activity, enhancing production of reactive oxygen species (ROS), nitric oxide (NO), and tumor necrosis factor α (TNF-α) by J774.A1 murine macrophages, and activating nuclear factor κB (NF-κB) in THP-1 human monocytes. In addition, Tansy polysaccharides stimulated human neutrophil function by greatly enhancing neutrophil myeloperoxidase (MPO) release. Furthermore, the low Mr fraction T-IV had potent complement-fixing activity, which may also contribute to the anti-inflammatory and would-healing properties of Tansy extracts. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Tansy extracts, and support the concept of using Tansy polysaccharides as an immunotherapeutic adjuvant.

The inhibition of blood coagulation by heparins of different molecular weight is caused by a common functional motif—the C-domain

Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties. Find a common basis for their anticoagulant action. In 50 fractions of virtually single molecular weight (Mr), prepared from unfractionated heparin (UFH) and four low-molecular-weight heparins (LMWH), we determined: (i) the molar concentration of material (HAM) containing the antithrombin binding pentasaccharide (A-domain); (ii) the specific catalytic activity in thrombin and factor Xa inactivation; (iii) the capacity to inhibit thrombin generation (TG) and prolong the activated partial thromboplastin time (APTT). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries antithrombin activity (C-domain). The antithrombin activity and the effects on TG and APTT are primarily determined by the concentration of C-domain and independent of the source material (UFH or LMWH) or Mr. High Mr fractions (>15 000) are less active, probably through interaction with non-antithrombin plasma proteins. Anti-factor Xa activity is proportional to the concentration of A-domain, it is Ca2+- and Mr-dependent and does not determine the effect on TG and APTT. For any type of heparin, the capacity to inhibit the coagulation process in plasma is primarily determined by the concentration of C-domain, i.e. the AT-binding pentasaccharide with 12 or more sugar units at its non-reducing end.

Group B Streptococcus capsular polysaccharide-cholera toxin B subunit conjugate vaccines prepared by different methods for intranasal immunization.

Group B Streptococcus (GBS) type III capsular polysaccharide (CPS III) was conjugated to recombinant cholera toxin B subunit (rCTB) using three different methods which employed (i) cystamine and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), (ii) carbodiimide with adipic acid dihydrazide (ADH) as a spacer, or (iii) reductive amination (RA). The CPS III-rCTB conjugates were divided into large- and small-molecular-weight (M(r)) fractions, and the immunogenicities of the different preparations after intranasal (i.n.) immunization were studied in mice. Both large- and small-M(r) conjugates of CPS III-rCTB(RA) or CPS III-rCTB(ADH) induced high, almost comparable levels of CPS-specific immunoglobulin G (IgG) in serum, lungs, and vagina that were generally superior to those obtained with CPS III-rCTB(SPDP) conjugates or a CPS III and rCTB mixture. However, the smaller-M(r) conjugates of CPS III-rCTB(RA) or CPS III-rCTB(ADH) in most cases elicited a lower anti-CPS IgA immune response than the large-M(r) conjugates, and the highest anti-CPS IgA titers in both tissues and serum were obtained with the large-M(r) CPS III-rCTB(RA) conjugate. Serum IgG anti-CPS titers induced by the CPS III-rCTB(RA) conjugate had high levels of specific IgG1, IgG2a, IgG2b, and IgG3 antibodies. Based on the effectiveness of RA for coupling CPS III to rCTB, RA was also tested for conjugating GBS CPS Ia with rCTB. As for the CPS III-rCTB conjugates, the immunogenicity of CPS Ia was greatly increased by conjugation to rCTB. Intranasal immunization with a combination of CPS Ia-rCTB and CPS III-rCTB conjugates was shown to induce anti-CPS Ia and III immune responses in serum and lungs that were fully comparable with the responses to immunization with the monovalent CPS Ia-rCTB or CPS III-rCTB conjugates. These results suggest that the GBS CPS III-rCTB and CPS Ia-rCTB conjugates prepared by the RA method may be used in bivalent and possibly also in multivalent mucosal GBS conjugate vaccines.

Accuracy of fast spin echo magnetic resonance imaging in the diagnosis of vestibular schwannoma

Purpose: An ideal screening test is noninvasive, inexpensive, and has a high specificity and sensitivity. Auditory brain-stem response testing has been the usual screening test for the diagnosis of vestibular schwannoma, although its accuracy in diagnosing small vestibular schwannomas has caused its effectiveness as a screening test to be questioned. Magnetic resonance imaging (MRI) with gadolinium has a high sensitivity and specificity for the diagnosis of vestibular schwannoma. Its use as a screening test for vestibular schwannoma has been limited due to its high cost. Fast spin echo MRI is a technique that provides T2-weighted images with excellent contrast between fluid and neural structures, and its cost is a fraction of a gadolinium MR[ scan. This study compares the accuracy of fast spin echo MRI to gadolinium MRI in the diagnosis of vestibular schwannoma. Materials & Methods: Twenty-five patients (50 ears) in whom there was a clinical suspicion of vestibular schwannoma were scanned with both modalities. All studies were read independently and scored as positive, negative, or indeterminate. Results: There were 11 true positives and 39 true negatives. There were no false positives or negatives, resulting in a sensitivity of 100% and a specificity of 100%. Conclusion: Fast spin echo MRI appears to be an excellent choice as a screening test for vestibular schwannoma due to its low cost, noninvasiveness, and high sensitivity and specificity.

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l−1 exopolysaccharides with glucose and 30 mg l−1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (Mr) of 1.7 × 106 and 4 × 104 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low Mr of 4 × 104. The high-Mr fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-Mr weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-Mr fractions appeared to be dependent on the carbohydrate source, whereas the low-Mr fractions were produced more continuously.

STUDIES ON THE MECHANISM OF ACTION OF COPPER FINING AGENTS (κ CARRAGEENAN)

Carrageenan mediated clarification of brewers' wort comprises two reactions: interaction between carrageenan and soluble wort polypeptides and flocculation of particulate material. The dose/response characteristics of interaction between carrageenan and soluble polypeptide material suggested that carrageenan exhibits selectivity with respect to specific polypeptide fractions. Size exclusion chromatography of worts demonstrated that carrageenan treatment resulted in reductions in fractions of relative molecular mass Mr 70 000, 40 000 and 12 000. Fining trials demonstrated that the reactivity towards soluble polypeptides is independent of the presence of insoluble particulate material. Furthermore, both interaction with soluble polypeptides and flocculation of particulate material occurred in systems fined under high (80°C) and low (20–25°C) initial temperatures and this suggested that the presence of carrageenan in a helical conformation is essential to fining action rather than conformational transitions in polysaccharide structure. Additionally, carrageenan has no effect on the levels of low molecular mass wort solutes such as nucleic acid derivatives, aromatic and heterocyclic amino acids.

Purification of Inositol Triphosphate Kinase from Bovine Brain

Inositol 1,4,5-triphosphate() is a second messenger for mobilizing intracellular . It can be dephosphorylated by soluble and particulate forms on 5-phosphatase, or phosphorylated to produce inositol 1,3,4,5-tetrakisphosphate() by 3-kinase. These enzymes represent possible targets for the regulation of the signal. 3-kinase which catalyses th ATP-dependent phosphorylation of was purified from bovine brain tissue. All operation were carried out at . Fresh tissure was homogenized and centrifuged. The supernatant was pooled. Proteins were precipitated from 10% polyethylene glycol, and suspended solution was applied to DEAE cellulose column for chromatography. As the result of above procedure, two isozymes of 3-kinase, I and II were obtained. Each isozyme was applied to Matriz green gel, Calmodulin-Affigel 15 column and subsequent phenyl-TSK HPLC column. Specific activites(SA) and fold of puriety were observed at each purification step of chromatography. At DEAE cellulose chromatography, SA were I, 0.6 and II, 4.8 nM/min/mg, and folds were I, 17.2 and II, 16.6. At Matrix green gel chromatography, SA were I, 18 and II, 11 nM/min/mg, folds were I, 62.1 and II, 38.0. At calmodulin-Affigel 15 column chromatography, SA were I, 19 and II, 13 nM/min/mg, folds were I, 65.5 and II, 44.8. Finally kinase I and II were purified 3,103-fold and 2,310-fold, and SA were I, 900 and II, 670 nM/min/mg, respectively. SDS-polyacrylamide gel electrophoresis elucidated 3 distinct fractions of Mr of 145,000, 85,000 and 69,500 from isozyme I, and 2 distinct fractions of Mr of 79,000 and 57,000 from isozyme II.