Non-invasive risk assessment of fetal sex chromosome aneuploidy through directed analysis and incorporation of fetal fraction.

OBJECTIVE: To assess the performance of a directed chromosomal analysis approach in the prenatal evaluation of fetal sex chromosome aneuploidy. METHODS: We analyzed 432 frozen maternal plasma samples obtained from patients prior to undergoing fetal diagnostic testing. The cohort included women greater than 18 years of age with a singleton pregnancy of greater than 10 weeks gestation. Samples were analyzed using a chromosome-selective approach (DANSR(TM) ) and a risk algorithm that incorporates fetal fraction (FORTE(TM) ). RESULTS: The cohort included 34 cases of sex chromosome aneuploidy. The assay correctly identified 26 of 27 (92.6%) cases of Monosomy X, one case of XXX, and all six cases of XXY. There were four false positive cases of sex chromosome aneuploidy among 380 euploid cases for an overall false positive rate of less than 1%. DISCUSSION: Analysis of the risk for sex chromosome aneuploidies can be accomplished with a targeted assay with high sensitivity.

Trisomy 13 detection in the first trimester of pregnancy using a chromosome-selective cell-free DNA analysis method.

OBJECTIVE: To assess the performance of chromosome-selective sequencing of maternal plasma cell-free DNA (cfDNA) in non-invasive prenatal testing for trisomy 13. METHODS: Two-phase case-control study on a single plasma sample per case. The first phase was used to optimize the trisomy 13 algorithm, which was then applied to a second dataset to determine the risk score for trisomy 13 by laboratory personnel who were blinded to the fetal karyotype. RESULTS: In the first phase, trisomy 13 risk scores were given for 11 cases of trisomy 13 and 145 euploid cases at 11-13 weeks' gestation. The test identified seven (63.6%) cases of trisomy 13 with no false positives. The trisomy 13 algorithm was subsequently modified and the trisomy 13 risk score was > 99% in all 11 cases of trisomy 13 and < 0.01% in all 145 euploid cases. In the second phase, the new algorithm was used to generate trisomy 13 risk scores for 10 cases of trisomy 13 and 1939 euploid cases. The trisomy 13 risk scores were > 99% in eight (80.0% (95% confidence interval (CI), 49.0-94.3%)) cases of trisomy 13. In the 1939 euploid cases the risk score for trisomy 13 was < 0.01% in 1937 (99.9%), 0.79% in one, and > 99% in one. Therefore, at the predefined risk cut-off of 1% for classifying a sample as high or low risk, the false-positive rate (FPR) was 0.05% (95% CI, 0.0-0.3%). CONCLUSIONS: Chromosome-selective sequencing of cfDNA can detect the majority of cases of trisomy 13 at an FPR of less than 0.1%.

Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris.

BACKGROUND AND PURPOSE: The prostamides (prostaglandin-ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX-2 from the respective endocannabinoids anandamide and 2-arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists. EXPERIMENTAL APPROACH: The isolated feline iris was used as a key preparation, where prostanoid FP receptors and prostamide activity co-exist. Activity at human recombinant FP and other prostanoid receptors was determined using stable transfectants. KEY RESULTS: In the feline iris, AGN 204396 produced a rightward shift of the dose-response curves for prostamide F2alpha and the prostamide F2alpha analog bimatoprost but did not block the effects of PGF2alpha and synthetic FP receptor agonists. Studies on human recombinant prostanoid receptors confirmed that AGN 204396 did not behave as a prostanoid FP receptor antagonist. AGN 204396 exhibited no antagonism at DP and EP1-4, but was a highly effective TP receptor antagonist. Contrary to expectation, the FP receptor antagonist AL-8810 efficaciously contracted the cat iris. AGN 204396 did not affect AL-8810 induced contractions, demonstrating that AL-8810 and AGN 204396 are pharmacologically distinct. Unlike AL-8810, the ethylamide derivate of AL-8810 was not an agonist. Al-8810 did not block prostamide F2alpha activity. Finally, AGN 204396 did not block PGE2-glyceryl ester activity. CONCLUSIONS AND IMPLICATIONS: The ability of AGN 204396 to selectively block prostamide responses suggests the existence of prostamide sensitive receptors as entities distinct from receptors recognizing PGF2alpha and PGE2-glyceryl ester.

The metabolism and deamination of [14C]sulphamethazine in a germ-free pig: the influence of nitrate and nitrite.

Twenty-four hours after feeding nitrite in combination with [14C]sulphamethazine to a germ-free and a conventional control pig the level of [14C]desaminosulphamethazine in tissues from both pigs was high, accounting for 11 to 30% of the total tissue 14C. When another germ-free pig was fed [14C]sulphamethazine in combination with nitrate, a trace amount of [14C]desaminosulphamethazine was found by gas chromatography-mass spectroscopy in the skeletal muscle but not in other tissues. When a control pig was fed [14C]sulphamethazine and nitrate, [14C]desaminosulphamethazine was found in all tissues examined. The results from this study show that feeding pigs nitrite together with sulphamethazine increases the amount of desaminosulphamethazine in the tissues. Most of the desaminosulphamethazine found in the tissues of the control pig fed nitrate was presumably the secondary effect of bacterial reduction of nitrate to nitrite.

Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters.

Accumulation of acids in anaerobic digesters after organic overload can inhibit or stop CH4 production. Therefore, methods to reduce acid concentrations would be helpful. One potential method to improve recovery involves bioaugmentation, addition of specific microorganisms to improve performance. In this study, transiently overloaded digesters were bioaugmented with a propionate-degrading enrichment culture in an effort to decrease recovery time. Biomass samples from 14 different, full-scale anaerobic digesters were screened for specific methanogenic activity (SMA) against propionate; the microbial communities were also compared. SMA values spanned two orders of magnitude. Principal component analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns for a functional gene (mcrA) suggested an underlying community structure-activity relationship; the presence of hydrogenotrophic methanogens closely related to Methanospirillum hungatei and Methanobacterium beijingense was associated with high propionate SMA values. The biomass sample demonstrating the highest SMA was enriched for propionate degrading activity and then used to bioaugment overloaded digesters. Bioaugmented digesters recovered more rapidly following the organic overload, requiring approximately 25 days (2.5 solids retention times (SRTs)) less to recover compared to non-bioaugmented digesters. Benefits of bioaugmentation continued for more than 12 SRTs after organic overload. Bioaugmentation is a promising approach to decrease recovery time after organic overload.

In situ intestinal absorption of 2-chloro-N-isopropylacetanilide (propachlor) and non-biliary excretion of metabolites into the intestinal tract of rats, pigs and chickens.

1. Propachlor was absorbed from in situ intestinal loops of rats and pigs, with absorption half-times of 7.5 and 16.5 min, respectively. 2. Water-soluble 14C-labelled metabolites that accumulated in the intestinal loops accounted for 31%, 53%, and 25% of the starting 14C for rats, pigs and chickens, respectively. 3. Propachlor(S)cysteine was identified as the major metabolite in the pig intestinal lumen (43% of the water-soluble 14C). 4. It is concluded that intestinal metabolism and intestinal excretion of water-soluble metabolites of propachlor are important physiological processes that occur in a variety of animal species. These processes provide a route by which metabolites of xenobiotics may reach the intestinal lumen in animals which are poor biliary excretors.

Evidence for involvement of non-biliary excretion into the intestines in the formation of methylsulphonyl-containing metabolites of 2-chloro-N-isopropylacetanilide (propachlor) by swine and rats.

1. Radiolabelled metabolites excreted in the urine of swine after an oral dose of 14C-propachlor were similar to those previously reported with rats and included methylsulphonyl (CH3SO2-)-containing metabolites. 2. A bile-duct-cannulated pig dosed orally with 14C-propachlor excreted 7.6% dose in the bile compared with approx. 75% dose with rats. Although enterohepatic circulation had been prevented with the bile-duct-cannulated pig. CH3SO3 metabolites of propachlor were excreted in the urine. Enterohepatic circulation had been reported to be necessary for formation of CH3SO2 metabolites of propachlor with rats. 3. Germ-free pigs dosed orally with 14-C-propachlor did not excrete urinary CH3SO2 metabolites, indicating involvement of the enteric flora in the production of these metabolites as shown previously with rats. 4. A g.l.c.-mass spectrometric specific ion monitoring technique indicated that trace amounts of CH3SO2 metabolites were present in the urine and bile of bile-duct-cannulated rats dosed orally with 14C-propachlor. This was confirmed by isolation and mass-spectral analyses. 5. It is concluded that after oral administration of propachlor to rats and swine, glutathione (GSH) conjugation and cysteine conjugate beta-lyase activity in the enteric flora are involved in formation of CH3SO2 metabolites. Presumably, the GSH conjugate is formed in mammalian tissue and must be returned to the lumen of the gut to be acted upon by the gut microflora. In rats this is accomplished primarily by biliary excretion, whereas in swine a non-biliary mechanism is much more important.

Spontaneous reversal of a cardiomyostimulator to asynchronous mode.

A patient who underwent dynamic cardiomyoplasty at our institute 10 months earlier was recently found to have asynchronous function of his cardiomyostimulator (model SP1005A, Medtronic, Inc.). This patient had been exposed to metal detection equipment 3 months earlier at an airport security gate, and this equipment was deemed responsible for this change in device programming. Despite the asynchronous function of the device, the patient did not suffer any functional impairment during this 3-month period. Moderate electromagnetic interference is capable of resetting the SP1005A cardiomyostimulator to the asynchronous mode of operation. Muscle stimulation, therefore, will not precisely occur during the systolic phase of the cardiac cycle and systolic augmentation may be lost. Despite asynchronous muscle stimulation, no impairment of this patient's functional Class was observed. This stability may be credited purely to the effect of the muscular wrap around the ventricles in which ventricular wall stress is decreased.

Catabolism of premercapturic acid pathway metabolites of naphthalene to naphthols and methylthio-containing metabolites in rats.

[14C]Naphthalene was given orally to rats with cannulated bile ducts and to germ-free rats. Bile and urine from the cannulated rats and urine from the germ-free rats contained no radioactive 1,2-dihydro-1-hydroxy-2-methylthionaphthalene and only trace amounts of radioactive naphthols or naphthol conjugates. Urine of control rats contained 4.6% of the 14C dose as naphthols and/or naphthol glucuronides. Appreciable quantities of 1- and 2-naphthol (7-20% of dose) and 1,2-dihydro-1-hydroxy-2-methylthionaphthalene (1-35% of dose) were in urine from rats dosed orally or intracecally with 1,2-dihydro-1-hydroxy-2-S-cysteinylnaphthalene and 1,2-dihydro-1-hydroxy-2-S-(N-acetyl)cysteinylnaphthalene. Apparently, in vivo, naphthols and methylthio-containing metabolites of naphthalene are formed during enterohepatic circulation of 1,2-dihydro-1-hydroxy-2-S-cysteinylnaphthalene and 1,2-dihydro-1-hydroxy-2-S-(N-acetyl)cysteinylnaphthalene in a process dependent upon intestinal microflora. A possible pathway for the formation of naphthols is aromatization of the precursor compounds by elimination of the appropriate substituent group from these metabolites. This discovery of the essential role of the intestinal microflora in the formation of naphthols from naphthalene indicates the existence of a novel pathway for hydroxylation of aromatic systems and challenges the current concept of the in vivo relevance of the in vitro production of naphthols from naphthalene 1,2-oxide.

Bacterial infection of cardiomyostimulator abdominal pocket following cardiomyoplasty procedure: an original approach to preserve synchronous muscle stimulation.

Dynamic cardiomyoplasty (DC) represents a new technique in therapy for refractory heart failure. So far, DC has been applied to more than 500 cases worldwide but reports on postoperative complications and related management are still lacking. We present the case of a patient suffering from refractory chronic heart failure for which the DC procedure was applied also accompanied by the complication of an infection process at the cardiomyostimulator pocket that began 2 weeks postoperatively. Following trials with several unsuccessful conservative approaches, an original procedure was developed to temporarily retain the implanted stimulation system, while at the same time maintain the synchronous contractions of the wrapped muscle. Finally, reimplantation of the pacing system was achieved with a low-risk procedure, effective cardiac assistance was preserved, and the infection process was arrested 3 years following DC.

Metabolism of the mercapturic acid of 2,4',5-trichlorobiphenyl in rats and mice.

2,4',5-Trichloro[14C]biphenyl mercapturic acids (triCB-MA) were metabolized in part to methylsulphide, methylsulphoxide and methylsulphone metabolites after i.p., i.v. (hepatic portal vein) and p.o. administration to rats. Methylthio-triCB and triCB-MA were present in faeces and 3- and 4- methylsulphonyl-triCB (triCB-SO2CH3) were present in carcasses of all rats. Only triCB-SO2CH3 was present in lung tissue. As shown by autoradiography, radioactivity accumulated in the tracheo-bronchial mucosa of mice given either intracaecal or tail-vein injections of triCB-MA. Radioactivity extracted from the lungs of mice was shown to be present as triCB-SO2CH3.

Management of pacing system related complications in patients undergoing dynamic cardiomyoplasty.

Integrity of the electrical circuit is a necessary requirement for appropriate heart/wrapped skeletal muscle interaction to be achieved in cardiomyoplasty. This article describes the management of two different complications after a cardiomyoplasty procedure involving the electrical system (infection of the abdominal cardiomyostimulator pocket and intramuscular lead fracture). Minimal approaches were carried out, which ensured the successful treatment of the infective and of the mechanical insult, and represent useful strategy for solving such uncommon problems.

Metabolism of 2,6-dichlorobenzonitrile, 2,6-dichlorothiobenzamide in rodents and goats.

1. Twelve 14C-labelled metabolites were isolated from either urine or bile from either rats (11 metabolites) or goats (7 metabolites) given single oral doses of 2,6-dichlorobenzo[14C]nitrile (DCBN). Five of these metabolites were also excreted in urine from rats dosed orally with 2,6-dichlorothiobenz[14C]-amide (DCTBA). 2. All metabolites from either DCBN or DCTBA were benzonitriles with the following ring substituents: Cl2, OH (three isomers); Cl2, (OH)2; Cl, (OH)2; Cl, OH, SH; Cl, OH, SCH3; SCH3, SOCH3, OH; Cl2, S-(N-acetyl)cysteine; Cl, S-(N-acetyl)cysteine; Cl, OH, S-(N-acetyl)cysteine. 3. The thiobenzamide moiety of DCTBA was converted to the nitrile in all the excreted urinary metabolites. No hydrolysis of the nitrile in DCBN to either an amide or an acid was detected. 4. Urine was the major route for excretion; however, enterohepatic circulation occurred. 5. Whole-body autoradiography of 14C-DCBN and 14C-DCTBA in mice showed the presence of bound residues in the mucosa of the nasal cavity, trachea, tongue, oesophagus, the kidney, liver and the intestinal contents.

Tissue distribution and complex formation with IgE of an anti-IgE antibody after intravenous administration in cynomolgus monkeys.

A humanized antihuman IgE antibody, rhuMAb-E25, was designed to form complexes with free IgE, blocking its interaction with mast cells and basophils and thereby preventing the initiation of the allergic cascade. To characterize the rhuMAb-E25: IgE complexes formed in vivo and to examine the disposition of the antibody in a relevant animal model, 125I-rhuMAb-E25 was administered as an intravenous bolus dose to cynomolgus monkeys that have high levels of IgE. The pharmacokinetic values of unlabeled and radiolabeled antibody were similar, which indicated that the disposition of 125I-rhuMAb-E25 reflected that of rhuMAb-E25. Size-exclusion chromatography of serum samples showed that the rhuMAb-E25:IgE complexes were of limited size and were similar to the small complexes formed in vitro with human IgE. Pharmacokinetic analysis revealed that both rhuMAb-E25 and rhuMAb-E25:IgE complexes cleared the serum compartment, albeit slowly. No specific uptake of radioactivity was seen in any of the tissues collected from the cynomolgus monkeys at 1 hr and 96 hr postadministration; no association was observed between 125I-rhuMAb-E25, or the complexes, and blood cells. Urinary excretion was the primary route of elimination of radioactivity; > 90% of the radioactivity found in urine was not associated with protein. The lack of specific tissue uptake and blood cell association and the slow clearance of rhuMAb-E25:IgE complexes were consistent with low-avidity interaction of small complexes with Fc gamma receptors of leukocytes and the reticuloendothelial system.

The isolation and identification of 14C-sulfamethazine (4-amino-n-(4,6-dimethyl-2-pyrimidinyl)[14C]benzenesulfonamide) metabolites in the tissues and excreta of swine.

Pigs were given a single oral dose of 14C-sulfamethazine (4-amino-N(I4,6-dimethyl-2-pyrimidinyl)[14C]benzenesulfonamide). Approximately 78% of the 14C was eliminated in the urine and 18% was eliminated in the feces within 192 hr after dosing. The percentage of the 14C remaining in the animals after dosing was as follows: 6 hr, 88%; 24 hr, 49%; 48 hr, 14%; 192 hr, less than 1%. The 14C-labeled compounds in the tissues and excreta were isolated by solvent extraction and by conventional and high-pressure liquid chromatography, and then derivatized and characterized by infrared and mass-spectral analysis. Chemical structures were confirmed by synthesis. The major 14C-labeled compounds in the skeletal muscle, liver and kidney were identified as sulfamethazine, N4-acetylsulfamethazine, the N4-glucose conjugate of sulfamethazine, and N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide (desaminosulfamethazine). The major 14C-labeled compounds in the urine and feces were identified as sulfamethazine and N4-acetylsulfamethazine.

Biomechanical characteristics of unconditioned and conditioned latissimus dorsi muscles used for cardiocirculatory assistance.

An understanding of the biomechanical characteristics of striated skeletal muscles involved in cardiocirculatory assistance is a prerequisite to assess their efficacy and to evaluate their haemodynamic benefits. Six goats had their latissimus dorsi muscles evaluated by isometric strain gauge testing. Total tension, and both active and passive force development at different preloads were measured. The relationship between muscle impedance and starting length was also studied. Four additional muscles were submitted to isometric and isotonic strain gauge testing after 3 months of chronic electrical stimulation (Broussais Hospital protocol) with the contralateral muscle serving as a control. In isometric testing, both conditioned and unconditioned goat latissimus dorsi displayed a Frank-Starling length-tension curve, and a linear relationship between muscle impedance and starting length was found. Chronic stimulation preserved muscle mass and isometric force. Transformed muscles showed a mean 59% reduction of maximal shortening velocity; means (s.d.) residual shortening velocity at maximal work and power output was 0.17(0.07) m/s. The work and power output were both reduced 65% after stimulation, and the residual maximal power at optimal preload varied from approximately 7.7 and 9.6 W/kg. It is concluded that, following the Broussais protocol, the goat latissimus dorsi muscle retained mass and most of its isometric force-generating capacity, but lost significant work and power potential. The residual power output did not, however, preclude the possibility of a significant cardiocirculatory contribution, providing that the conditions for optimal energy transduction are adequately delineated.