Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP).

BACKGROUND: Recombinant factor VIIa (rFVIIa) is approved for use in controlling bleeding episodes in people with hemophilia who have developed inhibitors to replacement therapy. Due to its short half-life (t½), frequent injections are required, limiting its use as a prophylactic treatment. A novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) has been developed to extend the t(½) of rFVIIa. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic/pharmacodynamic characteristics of rVIIa-FP in preclinical animal species. METHODS: Pharmacokinetic (PK) parameters were derived after single intravenous dosing in hemophilia A mice, rats, rabbits and monkeys. PK analysis was based on human FVII plasma levels determined by measuring FVII antigen levels by ELISA in mice and rats, and FVIIa activity using STACLOT® VIIa-rTF in rabbits and monkeys. Induction of thrombin generation was investigated in mice, while hemostatic activity was assessed by thrombus formation in rabbits. RESULTS: Compared with rFVIIa, rVIIa-FP displayed a prolonged t(½), enhanced in vivo recovery and reduced clearance in all species investigated. In mice, 16 h after treatment with rVIIa-FP, thrombin levels were quantifiable, indicating prolonged efficacy, whereas values had approached baseline at this time after treatment with rFVIIa. After 12 h, hemostatic efficacy was negligible in rFVIIa-treated rabbits, but sustained in animals receiving rVIIa-FP. CONCLUSIONS: These studies indicate that the longer t(½) of rVIIa-FP compared with rFVIIa translates into extended activity. These findings suggest that rVIIa-FP has the potential to be administered less frequently than rFVIIa-containing concentrates in clinical use.

Resposta reprodutiva de vacas de corte associada a marcadores moleculares relacionados à fertilidade / Reproductive performance of beef cattle cows associated with molecular markers related to the fertility

O objetivo deste estudo foi buscar associação entre a taxa de prenhez após inseminação e natalidade com marcadores moleculares ligados aos genes do receptor para IGF-1, LHβ, Leptina e receptores do FSH e LH. Utilizaram-se 249 vacas adultas Aberdeen Angus, das quais 199 foram submetidas a protocolos distintos para a IATF, seguida pelo repasse com touros, e 50 vacas formaram o grupo controle representado pelo acasalamento com touros. Foram avaliados o escore de condição corporal (ECC) e o escore de condição ovariana (ECO) ao início da estação reprodutiva. O ECC influenciou a taxa de natalidade, respectivamente de 55,6%, 75,8% e 82,4% (P<0,05) para os animais com ECC menor que 2,5, entre 2,5 a 2,9, e maior ou igual a 3,0, por ocasião da estação reprodutiva. Os marcadores relacionados ao gene do receptor para o IGF-1 (AFZ-1 e HEL5) mostraram associação com a taxa de natalidade. Vacas homozigóticas para o marcador AFZ-1 apresentaram 84,4% de natalidade em comparação às heterozigóticas, com 71,5% (P<0,05). A presença do alelo*161 para o marcador HEL5 foi negativa sobre a natalidade, respectivamente de 33,3% e 76,5% para vacas com e sem esse alelo (P<0,05). Esses resultados demonstram uma importante associação entre os marcadores envolvidos com o receptor para o IGF-1 e desempenho reprodutivo de vacas Angus.

The association between the reproductive performance, expressed by pregnancy rate at fixed timed artificial insemination and birth rate in the subsequent season in beef cows, and molecular markers linked to genes for IGF-1 receptor, LHβ, leptin, and FSH and LH receptors were evaluated. Data from 249 Aberdeen Angus adult cows were used in this study. One hundred and ninety-nine cows were subjected to four different protocols for FTAI, followed by clean-up bulls and 50 cows formed the control group, matted only with bulls for 90 days during the mating season. Body condition score (BCS) and ovarian condition score (OCE) were evaluated at the beginning of the breeding season. The birth rate in the following year was 75.5%, with no treatments influence. The BCS has influenced the birth rate, respectively 55.6%, 75.8% and 82.4% (P<0.05) for animals with BCS less than 2.5; 2.5 to 2.9; and greater than or equal to 3.0, at the beginning of the breeding season. The markers related to IGF-1 receptor gene (AFZ-1 and HEL5) were associated with the birth rate in beef cows. Cows homozygous for AFZ-1 marker showed 84.4% of birth rate, while heterozygous cows showed 71.5% (P <0.05). The presence of allele *161 to the HEL5 marker was negative on birth rate. Cows with this allele had only 33.3% of birth rate, while cows without this allele had 76.5% of birth rate (P <0.05). These results demonstrate a significant association between the markers involved with the IGF-1 receptor and reproductive performance of Aberdeen Angus beef cows.

Extending the pharmacokinetic half-life of coagulation factors by fusion to recombinant albumin.

The prophylactic treatment of haemophilia B and the management of haemophilia A or B with inhibitors demand frequent administrations of coagulation factors due to the suboptimal half-lives of the products commercially available and currently in use, e.g. recombinant factor IX (rFIX) and recombinant factor VIIa (rFVIIa), respectively. The extension of the half-lives of rFIX and rFVIIa could allow for longer intervals between infusions and could thereby improve adherence and clinical outcomes and may improve quality of life. Albumin fusion is one of a number of different techniques currently being examined to prolong the half-life of rFIX and rFVIIa. Results from a phase I clinical trial demonstrated that the recombinant fusion protein linking FIX to albumin (rIX-FP) has a five-times longer half-life than rFIX, and preclinical studies with the recombinant fusion protein linking FVIIa to albumin (rVIIa-FP) suggest that rVIIa-FP possesses a significantly extended half-life versus rFVIIa. In this review, we describe albumin fusion technology and examine the recent progress in the development of rIX-FP and rVIIa-FP.

Identification of the e allele at the Extension locus (MC1R) in Brazilian Creole sheep and its role in wool color variation.

The melanocortin 1 receptor (MC1R) gene has been described as responsible for the black color in some breeds of sheep, but little is known about its function in many colored breeds, particularly those with a wide range of pigmentation phenotypes. The Brazilian Creole is a local breed of sheep from southern Brazil that has a wide variety of wool colors. We examined the MC1R gene (Extension locus) to search for the e allele and determine its role in controlling wool color variation in this breed. One hundred and twenty-five animals, covering the most common Creole sheep phenotypes (black, brown, dark gray, light gray, and white), were sequenced to detect the mutations p.M73K and p.D121N. Besides these two mutations, three other synonymous sites (429, 600, and 725) were found. The dominant allele (E(D): p.73K, and p.121N) was found only in colored animals, whereas the recessive allele (E⁺: p.73M, and p.121D) was homozygous only in white individuals. We concluded that MC1R is involved in the control of wool color in Brazilian Creole sheep, particularly the dark phenotypes, although a second gene may be involved in the expression of the white phenotype in this breed.

Mitochondrial and nuclear DNA analyses reveal population differentiation in Brazilian Creole sheep.

Using ND5 sequences from mtDNA and 10 nuclear markers, we investigated the genetic differentiation of two South American Creole sheep phenotypes that historically have been bred in different biomes in southern Brazil. In total, 18 unique mtDNA haplotypes were detected, none of which was shared between the two phenotypes. Bayesian analysis also indicated two different groups (k = 2). Thus, these varieties are supported as being genotypically distinct. This situation could have resulted either from geographical isolation, associated with differences in the cultural habits of sheep farmers and in the way that flocks were managed, or more likely, from the introduction of different stocks four centuries ago.

Clinical pathology and parasitologic evaluation of free-living nestlings of the Hyacinth Macaw (Anodorhynchus hyacinthinus).

This study evaluated the health status and established hematologic and serum biochemistry parameters for free-living nestlings of the Hyacinth Macaw (Anodorhynchus hyacinthinus) from the Brazilian Pantanal (19 degrees 51'-19 degrees 58'S; 56 degrees 17'-56 degrees 24'W), for four consecutive years (from December 2003 through December 2006). Physical examinations indicated that all the birds were in good health. Endoparasites and blood parasites were not detected in any of the nestlings, and ectoparasites seemed to be limited to Philornis sp. (Diptera: Muscidae). Significantly higher levels of total white blood cells and heterophils, glucose, total protein, triglycerides, and phosphorus were observed in females. In females, higher cholesterol levels and packed cell volumes were observed in older birds, and total white blood cell and heterophil counts were higher in young animals. In males, uric acid levels were higher in older individuals. Wild Pantanal Hyacinth Macaws feed on only two species of palm nuts (Acrocomia totai and Scheelea phalerta). This limited food habit has a strong impact on population size and may alter the clinical pathology parameters of these birds. Therefore, knowledge of blood levels in normal individuals is essential to assess the physiologic and pathologic condition of wild macaws, to assess the effects of environmental changes on their health, and to contribute to conservation strategies of this endangered species.

Molecular diagnosis of Salmonella species in captive psittacine birds.

Cloacal swabs were collected from 280 captive psittacine birds belonging to 13 species. Samples of dna were tested by PCR using a pair of primers that amplify a 284 base pair fragment of the Salmonella genus invA gene, and the PCR-positive samples were tested by standard microbiological techniques. Thirteen per cent of the samples were positive by PCR, but negative by microbiological techniques. The infection rates were significantly different among the 13 species, the most commonly infected being Amazona amazonica (28 per cent) and Amazona pretrei (20 per cent). Specific tests for Salmonella Typhimurium Salmonella Enteritidis, Salmonella Pullorum and Salmonella Gallinarum did not produce positive results.

Effect of polymorphisms linked to LEP gene on its expression on adipose tissues in beef cattle.

In cattle, genetic markers at the leptin (LEP) gene and at those linked to the gene have been described as affecting calving interval (markers LEPSau3AI and IDVGA51), or daily weight gain (BMS1074 and BM1500). This work investigated the effect of these alleles on LEP mRNA levels in cattle subcutaneous and omental adipose tissues. A sample of 137 females of a Brangus-Ibage beef cattle herd was analysed to evaluate the distribution of the polymorphisms; then, animals having at least one of the IDVGA51*181 (allele 181 at marker IDVGA51; six animals), LEPSau3AI*2 (four), BMS1074*151 (13), BM1500*135 (six) alleles and a control group composed of animals without any of these alleles (four animals) were submitted to surgery to obtain omental and subcutaneous adipose tissues. Leptin mRNA expression was quantified by TaqMan RT-PCR, using 18S rRNA as internal control and adjusted for the effect of body condition score, through regression analysis. Omental fat had LEP gene expression 33% lower than the subcutaneous tissue. Carriers of IDVGA*181 and BMS1074*151 showed subcutaneous fat leptin mRNA levels higher than the controls. Leptin controls feed intake and coordinates reproduction; therefore, animals with higher LEP gene expression will probably have lower daily weight gain than others with similar forage offer and nutritional condition and probably will also have longer calving interval.

Purity of spiking agent affects partitioning of prions in plasma protein purification.

Prions are not detectable in the blood or plasma of persons afflicted with classical or variant Creutzfeldt-Jakob disease, and they have never been shown to be transmitted by blood or plasma products. Despite the uncertainty as to the presence and biophysical properties of prions in plasma, prion removal studies have been conducted using brain homogenate or microsomes prepared from prion-infected rodent brains as model prions. In this study, we compare the partitioning of different prion spiking agents, having different biophysical properties, in the processes used for plasma protein purification. We have found that membrane-bound prion spiking agents partition similarly, whereas purified, unbound pathogenic prion proteins can have significantly different partitioning properties depending on the conditions in the production process. We conclude that prion spiking studies for the evaluation of prion reduction in plasma protein purification should employ spiking agents with different biophysical properties to mimic partitioning of the theoretical prion contaminant. This will give greater assurance as to the prion safety margins of the life-saving plasma protein therapeutics and excipients.

The frequent Marburg I polymorphism impairs the pro-urokinase activating potency of the factor VII activating protease (FSAP).

The recently reported plasmatic, Factor Seven Activating Protease (FSAP), has also been found to be a potent activator of pro-urokinase [single-chain plasminogen activator, urinary type (scuPA)]. An initial epidemiological study surprisingly showed that plasmas of 5-10% of healthy blood donors had an impaired potential to activate scuPA. Analysis of the respective genomic DNAs revealed one particular single nucleotide polymorphism of FSAP resulting in an identical amino acid exchange (G511E), which correlates with the reduced activities. The corresponding mutation was named FSAP Marburg I. Thrombelastographies of wild-type and mutant plasmas were performed, facilitating the auto-activation of the intrinsic FSAP pro-enzymes by addition of dextran sulfate (DXS) and accelerated clot lysis by addition of scuPA. On these conditions, tissue-factor-induced coagulation revealed that clot lysis was significantly delayed in the Marburg I mutant plasmas as compared with wild-type plasmas. Furthermore, in the presence of DXS and scuPA, a FSAP-deficient plasma revealed significantly prolonged plasma clot lysis times, whereas the addition of purified FSAP pro-enzyme plus scuPA reversed this effect. These results support the hypothesis that FSAP contributes to the scuPA-dependent plasma fibrinolytic potential, which can be impaired in plasmas containing the FSAP Marburg I polymorphism, for instance.

Alu insertions versus blood group plus protein genetic variability in four Amerindian populations.

BACKGROUND: Do the population relationships obtained using DNA or blood group plus protein markers remain the same or do they reveal different patterns, indicating that the factors which influence genetic variation at these two levels of analysis are diverse? Can these markers shed light on the biological classification of the Aché, a Paraguayan tribe which only recently established more permanent contacts with non-Indians? SUBJECTS AND METHODS: To consider these questions we typed 193 individuals from four Amerindian tribes in relation to 12 Alu polymorphisms (five of them never studied in these populations), while 22 blood group plus protein systems were studied among the Aché. These data were then integrated with those previously available (blood groups plus proteins) for the three other populations. DNA extraction and amplification, as well as the other laboratory procedures, were performed using standard methods currently in use in our laboratory. The genetic relationships were obtained using the D(A) distance, and the trees were constructed by the neighbour-joining method, both developed by M. Nei and collaborators. Reliability of the trees was tested by bootstrap replications. Other population variability values were also determined using Nei's methods. RESULTS: Alu polymorphism was observed in all populations and for most of the loci; in the seven systems from which we could compare our results with those of other Amerindian groups agreement was satisfactory. Unusual findings on the blood group plus protein systems of the Aché were a very low (5%) HP*1 frequency and the presence of the C(W) phenotype in the Rh blood group. The intertribal patterns of relationship and other aspects of their variation were remarkably congruent in the two sets (Alu; blood group plus protein) of systems. CONCLUSIONS: The answer to the first question posed above is affirmative. However, the problem of whether the Aché derived from a Gê group that preceded the Guarani colonization of Paraguay, or are just a differentiated Guarani group, could not be answered with the genetic information available; the second hypothesis seems more likely at present, but the point to be emphasized is the striking genetic distinctiveness of the Aché as compared to other Amerindians.

New genetic data on Amerindians from the Paraguayan Chaco.

New data on 17 blood group and protein genetic systems obtained among the Ayoreo and Lengua Indians of Paraguay are presented. They include the first report on the red cell band-3 protein investigated among South American Indians. This information was integrated with previous results available for these two and four other groups. Five of the six populations reside in the Chaco area, while the sixth was included as an outgroup living elsewhere in Paraguay. Four of the five Chaco tribes exhibit good genetic homogeneity, but the Ayoreo are somewhat different. The results confirm the Chaco as a distinct biological (as well as cultural and economic) region, which should be considered in evaluations of genetic variability among South American Indians.

Genetic relationships between Amerindian populations of Argentina.

A total of 495 individuals from five different Argentinian tribes was examined for variation in 23 blood group and protein genetic systems, and the results were integrated with previous data on some of these systems. These tribes generally present RH * R1, PGM1 * 1, and ACP * A frequencies lower and RH * R2, ESD * 1, and GLO * 1 prevalences higher than those observed in other South American Indian groups. Earlier studies with mitochondrial DNA showed that haplogroup A was present in low frequencies in these tribes, but haplogroup B showed a high prevalence among the Mataco. Average heterozygosities are very similar in the five tribes, while estimates of non-Indian ancestry are generally low. Both the blood group and protein, as well as the mtDNA data sets, divide the five tribes into two groups, and the relationships obtained with the blood group and protein systems are exactly those expected on the basis of geography and language. However, the topology obtained with the mtDNA results was different, possibly due to sampling effects or diverse patterns of exchange between the groups related to sex.

TP53 polymorphisms and haplotypes in South Amerindians and neo-Brazilians.

To evaluate the genetic diversity of Brazilian populations and contribute to the knowledge of their evolutionary history this study investigated three TP53 polymorphisms (BstUI and MspI RFLPs in exon 4 and intron 6, respectively, and a 16 bp duplication in intron 3). The populations studied were: 114 Amerindians from five Brazilian Indian tribes (Gavião, Surui, Zoró, Wai-Wai and Xavante), 95 Euro-Brazilians and 70 Afro-Brazilians. The polymorphisms were all analysed using PCR amplifications. Gene frequencies and haplotype prevalences were calculated using the ARLEQUIN software. The genetic affinities of these groups with other world populations were estimated by the D(A) distance and neighbour joining method, using the NJBAFD computer program. Neo-Brazilians (immigrants from Europe and Africa) generally presented more variability than Amerindians, Afro-Brazilians being the most variable population. Among Amerindians, Gavião is the only group polymorphic for the three markers. Wai-Wai showed variability in BstUI and MspI RFLPs, while the other tribes were monomorphic for the 16 bp A1 and MspI A2 alleles. A rare haplotype (1-2-1) was verified among the Wai-Wai. This haplotype was previously described in a Chinese sample only, but with low frequency. Therefore, either this combination was lost in the other tribes by genetic drift, recombination, or other factor, or it occurs in the Wai-Wai and Chinese by independent events. The Gavião also presented a haplotype (2-1-1) not observed in the other Amerindians; but since it is present in Euro- and Afro-Brazilians. its occurrence there is probably due to interethnic admixture. The relationships of several world populations obtained using TP53 indicates that this marker is very efficient in clustering populations of the same ethnic group.

High-titer screening PCR: a successful strategy for reducing the parvovirus B19 load in plasma pools for fractionation.

BACKGROUND: Human parvovirus B19 (B19) is regarded as a potential risk factor for certain patient populations receiving plasma components. STUDY DESIGN AND METHODS: The prevalence of B19 was determined in a limited plasma donor population. Conditions for high-titer screening PCR were designed to allow the removal of plasma donations in the acute phase of infection with virus loads >or=10(7) genome equivalents per milliliter before manufacturing. Antithrombin III lots originating from screened plasma were compared to lots originating from untested plasma with respect to their B19 DNA load by a sensitive PCR assay. RESULTS: B19 was shown to have a prevalence of about 1 per 800 plasma donations. Only a minority (1/8000) of occurrences were in the acute phase of infection. Removing plasma units with high virus load as determined by high-titer screening PCR significantly decreased peak virus loads of plasma pools for fractionation. Together with a virus-removal capacity of 10.4 log(10) of the manufacturing process, this screening resulted in a final antithrombin III product that was nonreactive for B19 on PCR. CONCLUSION: Combining the strategy of high-titer screening PCR with the virus reduction capacity of the manufacturing process considerably increased the margin of B19 virus safety of antithrombin III. This strategy should have positive impact on other plasma components as well.

Influence of beta-naphthoflavone on 7,12-dimethylbenz(a)anthracene metabolism, DNA adduction, and tumorigenicity in rainbow trout.

Metabolism, DNA adduction, and tumor induction by 7, 12-dimethylbenz(a)anthracene (DMBA) were examined in cultured trout liver cells and in vivo in trout. Modulating CYP1A1 activity indicated this enzyme plays a significant role in metabolizing DMBA to water-soluble compounds in isolated trout liver cells. The major DMBA metabolites identified in trout liver cells were 10-, 11-, 8,9-, and 5,6-DMBA dihydrodiols, and DMBA, 2- or 3- or 4-phenol; 7-OH-methyl-12-methyl-benz(a)anthracene and 12-OH-methyl-7-methyl-benz(a)anthracene were minor metabolites. A very small amount of DMBA-3,4-dihydrodiol was detected, and polar metabolites, which did not migrate with any DMBA metabolite standards, were observed. Incubating trout hepatocytes with DMBA-3, 4-dihydrodiol produced three prominent, nonpolar adducts indistinguishable from those in mouse embryo cells. However, DMBA-DNA adducts, formed in trout in vivo or in trout liver cells exposed to DMBA, were predominantly more polar than those formed in mouse embryo fibroblasts, and levels of DMBA-DNA adducts formed in trout liver cells were not significantly altered by modulating CYP1A1 activity. No significant repair of DMBA-DNA adducts was detected in cultured trout liver cells over a 48-h period, supporting previous studies indicating that fish are less efficient than mammals in repairing polyaromatic hydrocarbon DNA adducts. Compared to animals receiving DMBA alone, beta-naphthoflavone pretreatment in vivo did not affect hepatic CYP1A1, DMBA-DNA adducts, nor hepatic tumor response; but did significantly reduce tumor response in two other target organs. These results collectively indicate that DMBA bioactivation to DNA-binding metabolites in trout liver cells and mouse embryo cells predominantly involve different metabolic pathways to form the DNA-binding intermediates.

Increased safety level of serotype 3 Sabin oral poliomyelitis vaccine lots by improved seed virus, and tissue culture and virus infection conditions.

The content of 472U to 472C revertant virus in serotype 3 oral poliomyelitis monovalent bulk vaccines can be quantified by MAPREC (Mutant Analysis by PCR and Restriction Enzyme Cleavage). Besides other wildtype reversions identified in propagated type 3 Sabin strain populations, the 472U to 472C reversion correlates most prominently with neurovirulence in the monkey neurovirulence test. Therefore, the results can be used for the discrimination of 'good' and 'bad' vaccines on the molecular level. In international collaborative studies it has been well established that vaccine lots containing revertant genomes below a critical threshold pass the in vivo monkey neurovirulence test (MNVT), while vaccine lots containing more revertants fail the MNVT. In this communication we show that the MAPREC test is a sensitive tool for quality control and the demonstration of consistency in large scale production. Furthermore, MAPREC offers a possibility to assess the effect of changed production conditions on the rate of reversion and to find conditions for consistent production with low reversion rates.

Cytochrome P4501A1 polymorphisms in South American Indians.

A total of 131 individuals from five Brazilian Indian tribes were studied for two CYP1A1 gene polymorphisms. The presence of the *val allele at codon 462 varied from 54% in the Surui to 97% in the Xavante, while the presence of the MspI restriction site (*m2 allele) at position T6235C ranged from 72% in the Gavião to 95% in the Xavante. The haplotypes derived from these two sites showed a highly heterogeneous distribution among the five populations. The most common haplotype in South Amerindians was *val/*m2 (54% to 94%). This prevalence is the highest that has been observed in any world population.

Diversity of two short tandem repeat loci (CD4 and F13A1) in three Brazilian ethnic groups.

Two microsatellites (CD4 and F13A1) were investigated in seven Brazilian populations: one group each of European- and African-derived subjects from Porto Alegre, southern Brazil, and five Amerindian tribes (three Tupi-Mondé speaking [Gavião, Surui, and Zoró], one Macro-Gê [Xavante], and one Carib [Wai-Wai]). For both markers, neo-Brazilians presented with a high diversity, but Amerindians showed a low level of variability. Genotype frequency distributions were heterogeneous among populations, the only exception being similar CD4 frequencies in Afro- and Euro-Brazilians. Gene diversity analysis revealed that most of the total variation is due to intrapopulational diversity in all populations. Because of the high information content of these markers in Afro- and Euro-Brazilians, these systems are most appropriate for forensic analyses. The comparison among Brazilian and other world populations revealed high similarity among populations of the same ethnic group, indicating a high discriminative power for these markers.