Pharmacokinetic profiles of the active metamizole metabolites in healthy horses.

Metamizole (MT) is an analgesic and antipyretic drug labelled for use in humans, horses, cattle, swine and dogs. MT is rapidly hydrolysed to the active primary metabolite 4-methylaminoantipyrine (MAA). MAA is formed in much larger amounts compared with other minor metabolites. Among the other secondary metabolites, 4-aminoantipyrine (AA) is also relatively active. The aim of this research was to evaluate the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.) and intramuscular (i.m.) routes in healthy horses. Six horses were randomly allocated to two equally sized treatment groups according to a 2 × 2 crossover study design. Blood was collected at predetermined times within 24 h, and plasma was analysed by a validated HPLC-UV method. No behavioural changes or alterations in health parameters were observed in the i.v. or i.m. groups of animals during or after (up to 7 days) drug administration. Plasma concentrations of MAA after i.v. and i.m. administrations of MT were detectable from 5 min to 10 h in all the horses. Plasma concentrations of AA were detectable in the same range of time, but in smaller amounts. Maximum concentration (Cmax ), time to maximum concentration (Tmax ) and AUMC0-last of MAA were statistically different between the i.v. and i.m. groups. The AUCIM /AUCIV ratio of MAA was 1.06. In contrast, AUC0-last of AA was statistically different between the groups (P < 0.05) with an AUCIM /AUCIV ratio of 0.54. This study suggested that the differences in the MAA and AA plasma concentrations found after i.m. and i.v. administrations of MT might have minor consequences on the pharmacodynamics of the drug.

Immunolocalisation of nitric oxide synthase isoforms in the ductuli efferentes and epididym.

The present research used immunohistochemistry to analyse the detection and localisation of nitric oxide synthase (NOS) isoforms in the ductuli efferentes and epididymis of prepubertal and adult alpaca. In the ductuli efferentes and epididymis of prepubertal and adult animals, nNOS and eNOS were similarly expressed in epithelial lining cells, conversely differences were observed in the immunopresence of iNOS. Our data provide evidence that NOS isoforms may have roles in reproductive functions and in the developmental processes of the excurrent duct system in the alpaca.

Pharmacokinetics of tramadol and its major metabolite after intramuscular administration in piglets.

Tramadol (T) is a centrally acting atypical opioid used for treatment of dogs. Piglets might experience pain following castration, tooth clipping and tail docking and experimental procedures. The aim of this study was to assess the pharmacokinetics of T and its active metabolite M1 in male piglets after a single intramuscular injection. Six healthy male piglets were administered T (5 mg/kg) intramuscularly. Blood was sampled at scheduled time intervals and drug plasma concentrations evaluated by a validated HPLC method. T plasma concentration was quantitatively detectable from 0.083 to 8 h. M1 was quantified over a shorter time period (0.083-6 h) with a Tmax at 0.821 h. The study demonstrated that piglets produce a larger amount of M1 compared with dogs, horses and goats. The human minimum effective concentration of M1 (40 ng/mL) was exceeded for over 3 h in piglets. If it is assumed to also apply to piglets, it could be speculated that the drug efficacy might exert its action over 3 h or longer. This assumption has to be confirmed by further specific pharmacokinetic/pharmacodynamic studies.

Expression of prostate glycoconjugates in the stallion and castrated horse.

This work was undertaken to determine the glycoconjugates secreted by the epithelium of the prostate in the intact stallion and castrated horse using lectin histochemical procedures in conjunction with enzymatic digestion and deglycosylation treatments. Additionally, anti-5 and 13-16-cytokeratin antibodies were used to localize epithelial basal cells. In the stallion, lectin histochemistry showed the following sugar residues in the Golgi zone of the glandular cells: α-Glu/Man, α-Fuc and ß-Gal included in both O- and N-linked oligosaccharides as well as ß-GalNAc, GlcNAc and α-Gal, which belonged to O-glycoproteins. ß-Gal and ß-GalNAc moieties were also noted subterminal to sialyl residues. Sialic acid specific lectins identified Neu-5Ac(α2,3-6)-ß-Gal or Neu5Ac(α2,6)-ß-GalNAc sequences in both N- and O-bound glycoproteins. The prostatic glandular cells of the castrated horse expressed some of the same sugar moieties found in the stallions, such as α-Glu/Man, α-Gal and GlcNAc, but significant differences were also noted. In particular, ß-D-GalNAc was only detected subterminal to sialic acid, ß-D-Gal-(1-3)-D-GalNAc was found in N-linked glycans, whereas ß-D-Gal-(1-4)-D-GlcNAc and Neu5Acα2,6Gal/GalNAc were noted only in O-glycoproteins. These results indicate that the lectin binding patterns in glandular cells may be modified by sex hormones. No specific lectin labelling of basal cells was found in either the stallion or the castrated horse even though they were immunostained with specific anti-cytokeratin antibodies. These cells stained more strongly in the castrated horse than in the intact stallion suggesting that they are androgen responsive. The glycomolecules detected in the equine prostate secretions may contribute to the remodelling of the sperm surface, which occurs during sperm transit through the male genital tract and also after ejaculation in the seminal plasma. These changes may be important in the understanding of the stallion fertility.

Constant rate infusion of tramadol in isoflurane-anesthetized pigs undergoing experimental surgery.

The purpose of this study was to investigate the effect of tramadol (TM) (2 mg/kg) administered intramuscularly (IM) followed by a constant rate infusion (CRI) of TM (2 mg/kg/h) in pigs. Sixteen pigs undergoing experimental surgery were premedicated IM with a combination of alfaxalone (5 mg/kg) and midazolam (0.5 mg/kg). Anaesthesia was induced with propofol (2 mg/kg) intravenously (IV) and maintained with isoflurane. Pigs were randomly assigned to one of the two following groups: Group 1 (n=8): received a loading dose of TM (2 mg/kg) followed by a CRI of TM (2 mg/kg/h); Group 2 (n=8): a loading dose of TM (2 mg/kg) followed by a CRI of lactated Ringer's solution (2 ml/kg/h). Heart rate (HR), respiratory rate (RR), rectal temperature (RT), haemoglobin oxygen saturation (SpO2), fraction of inspired oxygen (FIO2), end-tidal concentration of isoflurane (FEISO), end-tidal carbon dioxide concentration (FECO2), pH, arterial oxygen partial pressure (PaO2), arterial carbon dioxide partial pressure (PaCO2) and bicarbonate concentration (HCO3-) were recorded immediately after loss of righting reflex (T=0 min) and at 15-min intervals over a period of 60 min. Continuous data were analysed using a repeated- -measure analysis of variance (ANOVA) and a p-value ⟨0.05 was considered significant. HR, RR and FEISO were significantly lower (p⟨0.05) in Group 1 at T30 and T45, which corresponded to the time of the most intense surgical stimulation. The results suggest that the TM infusion minimizes the HR and RR response, slightly reducing isoflurane requirements and determining a superior perioperative analgesia.

Phylogeographic and genome-wide investigations of Vietnam ethnic groups reveal signatures of complex historical demographic movements.

The territory of present-day Vietnam was the cradle of one of the world's earliest civilizations, and one of the first world regions to develop agriculture. We analyzed the mitochondrial DNA (mtDNA) complete control region of six ethnic groups and the mitogenomes from Vietnamese in The 1000 Genomes Project (1000G). Genome-wide data from 1000G (~55k SNPs) were also investigated to explore different demographic scenarios. All Vietnamese carry South East Asian (SEA) haplotypes, which show a moderate geographic and ethnic stratification, with the Mong constituting the most distinctive group. Two new mtDNA clades (M7b1a1f1 and F1f1) point to historical gene flow between the Vietnamese and other neighboring countries. Bayesian-based inferences indicate a time-deep and continuous population growth of Vietnamese, although with some exceptions. The dramatic population decrease experienced by the Cham 700 years ago (ya) fits well with the Nam tien ("southern expansion") southwards from their original heartland in the Red River Delta. Autosomal SNPs consistently point to important historical gene flow within mainland SEA, and add support to a main admixture event occurring between Chinese and a southern Asian ancestral composite (mainly represented by the Malay). This admixture event occurred ~800 ya, again coinciding with the Nam tien.

Admixture and genetic relationships of Mexican Mestizos regarding Latin American and Caribbean populations based on 13 CODIS-STRs.

Short tandem repeats (STRs) of the combined DNA index system (CODIS) are probably the most employed markers for human identification purposes. STR databases generated to interpret DNA profiles are also helpful for anthropological purposes. In this work, we report admixture, population structure, and genetic relationships of Mexican Mestizos with respect to Latin American and Caribbean populations based on 13 CODIS-STRs. In addition, new STR population data were included from Tijuana, Baja California (Northwest, Mexico), which represents an interesting case of elevated genetic flow as a bordering city with the USA. Inter-population analyses included CODIS-STR data from 11 Mexican Mestizo, 12 Latin American and four Caribbean populations, in addition to European, Amerindian, and African genetic pools as ancestral references. We report allele frequencies and statistical parameters of forensic interest (PD, PE, Het, PIC, typical PI), for 15 STRs in Tijuana, Baja California. This Mexican border city was peculiar by the increase of African ancestry, and by presenting three STRs in Hardy-Weinberg disequilibrium, probably explained by recurrent gene flow. The Amerindian ancestry in Central and Southeast of Mexico was the greatest in Latin America (50.9-68.6%), only comparable with the North of Central America and Ecuador (48.8-56.4%), whereas the European ancestry was prevalent in South America (66.7-75%). The African ancestry in Mexico was the smallest (2.2-6.3%) in Latin America (≥ 2.6%), particularly regarding Brazil (21%), Honduras (62%), and the Caribbean (43.2-65.2%). CODIS-STRs allowed detecting significant population structure in Latin America based on greater presence of European, Amerindian, and African ancestries in Central/South America, Mexican Mestizos, and the Caribbean, respectively.

Low red blood cell glutathione reductase and pyridoxine phosphate oxidase activities not related to dietary riboflavin: selection by malaria?

This study was designed to confirm that low dietary riboflavin does not contribute to the flavin-deficient red blood cells commonly found in subjects in Ferrara Province, northern Italy. In this area it is primarily an inherited characteristic believed to have been selected for by malaria, which was endemic from the 12th century. In parallel with assessment of daily riboflavin intake (DRI), flavin adenine dinucleotide-dependent glutathione reductase (EGR) and flavin mononucleotide-dependent pyridoxine phosphate oxidase (PPO) were measured in beta-thalassemic heterozygotes, their normal relatives, and normal spouses (representative of the normal population). In all of these groups there is a high incidence of deficiency of these flavin enzymes. We found that the majority had an adequate riboflavin intake and there was no significant correlation of EGR and PPO activities with DRI. Thus, interpretation of low EGR activity is discussed with reference to studies of EGR done to detect nutritional riboflavin deficiency in countries where there is malnutrition and endemic malaria.

HLA polymorphism in a Mataco South American Indian tribe: serology of class I and II antigens. Molecular analysis of class II polymorphic variants.

In the present study, HLA-A, B, C, DR, DQ, and DP loci were analyzed in a group of Mataco Amerindians of Argentina. Using reagents from the 11th International Histocompatibility Workshop (11th IHW), class I specifities such as Bw70, Bw75, and Bw48 were found in this population, other than the HLA determinants commonly described in South American Indians. The class II antigens found were DR4, DRw14, and DRw8 at the DR locus, and DQw4 and DQw7 at the DQ locus. The analysis of DRB1-DR4 related alleles, performed by PCR amplification and oligonucleotide probe hybridization, showed the presence of DRB1*0403, *0404, *0405, and *0411 in individuals from this ethnic group. By the analysis of DRB1-DRw14 related alleles, two variants were found: DRB1*1402 and DRB1*1406, the latter provisionally called DRB1 14.6 in 11th IHW. The DRw8-related allele present was DRB1*0802. The analysis of DRB3 gene revealed only the presence of DRB3*0101 allele in DRw14 individuals. DPB1 locus was also analyzed in unrelated individuals of the same population. Only five DPB1 alleles were found: DPB1*0201, *0301, *0402, *0501, and *1301 over the 19 previously described in the literature. These findings emphasize the restricted HLA class I and II variation observed in this ethnic group as it has been previously shown in other American groups. Some particular haplotypes in this Mataco tribe are described in this work.

HLA class I diversity in Kolla Amerindians.

Human leukocyte antigen (HLA) class I polymorphism was studied within a population of 70 unrelated Kolla Amerindians from the far northwest of Argentina close to the Bolivian border. The results indicate that the HLA-A, -B, and -C alleles typical of other Amerindian populations also predominate in the Kolla. These alleles belong to the following allele groups: HLA-A*02, *68, *31, *24, HLA-B*35, *15, *51, *39, *40, *48, and Cw*01, *03, *04, *07, *08, and *15. For the HLA-A locus, heterogeneity was seen for HLA-A*02 with A*0201, *0211, and *0222; and for A*68 with *68012 and *6817, the latter being a novel allele identified in this population. Analysis of HLA-B identified heterogeneity for all Amerindian allele groups except HLA-B*48, including the identification of the novel B*5113 allele. For HLA-C heterogeneity was identified within the Cw*07, *04, and *08 groups with Cw*0701/06, *0702, *04011, *0404, *0803, and *0809 identified. The most frequent "probable" haplotype found in this population was B*3505-Cw*04011. This study supports previous studies, which demonstrate increased diversity at HLA-B compared with HLA-A and -C. The polymorphism identified within the Kolla HLA-A, -B, and -C alleles supports the hypothesis that HLA evolution is subject to positive selection for diversity within the peptide binding site.

Assessment of prospective genetic counseling in the Ferrara area.

A group of 613 heterozygotes for beta thalassemia (267 married, 346 unmarried) who were screened mostly at elementary school age, were identified and interviewed at the average age of 23.7 years to assess their knowledge of the heterozygous state and its implications. It was found that 83% recalled some information about their heterozygous state; only 60% had some information about the meaning of being heterozygous, and only 26% said they knew of some relationship between Cooley's anemia and the heterozygous state for beta thalassemia. In the married group, the proportion of those having married another carrier was in agreement with random mating expectations. It was concluded that there is ample room for improvement in the procedures of delivery of prospective genetic counseling at the population level in this area.

Reproductive behavior of thalassemic couples segregating for Cooley anemia.

The reproductive behavior in 1984 of families segregating for Cooley anemia in Ferrara was compared with that of a control group of families, matched for some biological variables which affect fertility. At the resolution power of the sample, it was found that there is no significant difference in these variables due to segregation for Cooley anemia, and it appears that there is no longer significant reproductive compensation in thalassemic couples, although a tendency to compensate does still exist. The increased life span of children affected by Cooley anemia, due to improvements in treatment in the past decade, is probably the main reason why the compensatory reproductive behaviour of the past has almost disappeared.

Rheumatoid arthritis and its association with HLA-DR antigens. II. Antibodies to native connective tissue antigens detected by enzyme linked immunosorbent assay.

The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

New developments in stem cell transplantation with special reference to the first in utero transplants in humans.

Based on the experience acquired in post-natal liver transplantation since 1974, we recently initiated pre-natal, in utero stem cell transplantation from the human fetal liver. The first two fetuses that we treated had immunodeficiencies, the third one had thalassemia major. Donors and recipients were not matched. The fetal cells were infused in the umbilical vein of the first two patients and injected intraperitoneally into the third one, under ultrasonic visualization. The first patient, born in 1988, has both engraftment of donor cells and reconstitution of cell-mediated immunity. This child, who had bare lymphocyte syndrome, has no clinical manifestation of the disease and he lives normally at home. The second child, born in 1989, has not yet developed a significant reconstitution of immunity although donor cell engraftment has been proven (Y chromosome in this female patient). The third patient has also evidence of donor cell take (Y chromosome in a female patient) but the effect on thalassemia has not yet been fully analyzed (donor hemoglobin present in small quantity). In all 3 cases, no side-effect of any kind developed in the mother nor in the fetus. Several advantages appear to be associated with in utero FLT: increased probability of graft take, ideal isolation of patient (in the uterus), optimal environment for fetal cell development (in the fetal host).

Pharmacokinetics of sex steroids in patients with beta thalassaemia major.

AIMS: To assess the pharmacokinetics of oral, intramuscular, or transdermal hormone replacement in patients with beta thalassaemia major. METHODS: Oral (testosterone undecanoate 40 mg) and intramuscular (testosterone propionate 15 mg, phenylpropionate 30 mg, isocaproate 30 mg and decanoate 50 mg) testosterone and transdermal (17 beta oestradiol 25 micrograms and 50 micrograms) oestradiol were evaluated in 21 male (16-29 years) and 11 female (19-26 years) patients with beta thalassaemia major and various forms of hypogonadism. RESULTS: In male patients given oral testosterone, peak testosterone concentrations were observed either two to four hours or seven hours after administration; intramuscular testosterone produced peak values seven days after injection. Transdermal 17 beta oestradiol given to female patients produced a biphasic pattern with an initial peak concentration occurring at 36 hours and a secondary rise at 84 hours. CONCLUSIONS: The results indicate that oral androgens should be given twice daily in cases of hypogonadism, and where growth is incomplete, lower than recommended doses. If intramuscular testosterone is used, smaller doses of 10-25 mg should be given every one to two weeks. Transdermal administration of 25-50 micrograms 17 beta oestradiol generally produces a plasma E2 value in the early to mid-follicular phase range (100-300 pmol/l). This is appropriate in adults but excessive for prepubertal girls. Diffuse iron infiltration of tissues does not seem to interfere with the absorption of androgens and oestrogens from the gut, muscle, or skin.

Gonadal function in patients with beta thalassaemia major.

Endocrine studies were made on 23 female patients aged 13 to 29 years, with delayed puberty or primary amenorrhoea and beta thalassaemia major, and 12 healthy controls, of whom six were prepubertal and six were in Tanner's stage 3-4. Each patient and control received a single intravenous dose of 100 micrograms gonadotrophin releasing hormone (GnRH), and one week later, 10 U/kg body weight of human menopausal gonadotrophin (hMG) to stimulate ovarian function. The patients had decreased gonadotrophin reserves when compared with those of normal controls, only one of 23 patients had an intact luteinising hormone and follicle stimulating hormone response. Most of the thalassaemic patients with delayed puberty showed normal gonad response to human menopausal gonadotrophin (hMG), but three had very low responses, when compared with that of controls. The gonadal failure was even more severe in four of six patients with primary amenorrhoea. It is important to assess hypothalamic-pituitary-gonadal function in young women with beta thalassaemia major, so that those with glandular dysfunction may be started on replacement therapy.

A multi-center safety trial of the oral iron chelator deferiprone.

Deferiprone, also known as L1, is an orally active iron chelator that has been studied extensively in clinical trials. The sporadic occurrence of agranulocytosis in association with deferiprone and the highly variable frequency of other possible side effects such as arthralgia have created uncertainty about the true incidence of deferiprone-related complications. A multi-center, 1-year trial was initiated to determine the safety profile of deferiprone. Using the Apotex formulation of deferiprone, 187 patients with thalassemia who were unable or unwilling to use deferoxamine were enrolled in four centers; 162 patients completed one year of therapy. Agranulocytosis (ANC < 500/mm3) occurred in one patient after 15 weeks of treatment, was not accompanied by infection and resolved following treatment with G-CSF. Nine other subjects developed less severe neutropenia (ANC 500-1500/mm3) with the lowest absolute neutrophil count reaching 500-1250/mm3. The neutropenia in these patients developed after 1-50 weeks of therapy, frequently accompanied febrile illnesses, and occurred predominantly in non-splenectomized patients. Reasons other than neutropenia for discontinuing use of deferiprone included nausea (4), voluntary withdrawal (3), high ALT (2), platelet count < 100,000/mm3 (2), low but unconfirmed ANC (1), protocol violation (1) fatigue (1), and depression (1). Mean ALT levels rose within three months of therapy and stabilized thereafter. Arthralgia and nausea and/or vomiting occurred in 6% and 24% of subjects, respectively. In this multi-center trial with weekly monitoring of blood counts, the incidence of agranulocytosis was 0.58 per 100 patient-years, and the frequency of agranulocytosis after one year was 0.5%. These findings support the safety of this formulation of deferiprone, using the careful monitoring system employed in this trial.

Psychosocial integration of adolescents and young adults with thalassemia major.

UNLABELLED: The aim of this study was to explore the effect of thalassemia major on the psychosocial adjustment of adolescents and young adults. DESIGN: unmarried adolescent and young adult patients were asked to fill in an ad hoc questionnaire; a semi-structured interview exploring marriage and family life was done with married patients. SAMPLE: group A: 90 unmarried patients, 50% females and 50% males, aged 14-22 years. The control group was formed by 100 matched subjects; group B: 19 thalassemic married subjects, 6 males and 13 females, aged 28-45 years, 7 patients had children and 12 did not. RESULTS: group A: subjects with thalassemia had normal psychological and social development and scored better than their normal peers in tests investigating social adjustment, self-esteem and self-description. Moreover the family relationships of adolescents with thalassemia appeared to be stronger than those reported by normal controls; group B: the behavior of thalassemic couples did not differ from the one observed in non-thalassemic couples in the course of previous investigations. CONCLUSIONS: Our data shows that neither thalassemic adolescents nor thalassemic married, well-treated, young adults differ significantly from the healthy young people in their ability to cope with life's difficulties both in adolescence and marital life.

Hypothalamic-pituitary-gonadal axis in thalassemic patients with secondary amenorrhea.

Eight thalassemic patients, aged 24-35 years, who developed amenorrhea 2-15 years after menarche, were studied. Mean basal serum LH and FSH levels and the peak levels after gonadotropin-releasing hormone were significantly less than corresponding values in normal controls. All patients showed low basal serum levels of estradiol and six had a poor or absent response to human menopausal gonadotropin. One subject had intact pituitary-gonadal function and one patient had an impaired LH and FSH response to gonadotropin-releasing hormone in the presence of a significant increase of estradiol after human menopausal gonadotropin stimulation. The findings regarding pituitary hormones other than gonadotropins suggest that iron overload damages tropic cells unequally and inconsistently. We conclude that both pituitary and gonadal damage may be responsible for the secondary amenorrhea in thalassemic patients.

Induction of spermatogenesis in thalassaemia.

In an attempt to induce or to augment pubertal development and to achieve spermatogenesis, 10 gonadotropin-deficient thalassemic patients 15 to 23 years of age (mean 18.9 years) were treated with exogenous gonadotropins for 1 to 4 years (mean 2.1 years). Seven patients produced sperm during human chorionic gonadotropin (hCG) treatment given for 6 to 14 months. However, full spermatogenesis was achieved only when human menopausal gonadotropin was added to hCG regimen. In one patient, despite cessation of gonadotropin treatment, sexual potency, libido, and spermatogenetic capacity were maintained during the past 2 1/2 years. Our study indicates that it is possible to induce or to restore spermatogenesis in the majority of thalassemic patients and that gonadotrope cells may not be irreversibly damaged by iron deposition.