A review on inflammation and angiogenesis as key mechanisms involved in the pathogenesis of bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of reproductive failure in dairy cattle. The main aim of this review is to discuss some aspects related to inflammation and angiogenesis that seem to be involved in the development of follicular cysts in domestic animals, with special emphasis on the bovine species, in an attempt to elucidate the relationship between these two processes in the early stages of persistence and in the development of bovine COD. We describe the changes in the expression of cytokines and angiogenic factors that seem to generate disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. Results show that pro-inflammatory and anti-inflammatory cytokines behave as regulators of angiogenesis through direct and indirect effects, like overexpression of pro-angiogenic factors, particularly in bovine ovarian cells from follicular cysts and persistent follicles. We conclude that, in dairy cattle, an imbalance in the expression of cytokines and pro-angiogenic growth factors related to ovulation and the processes associated with it would contribute to follicular persistence and to the recurrent appearance of COD.

Contribution of the VEGF system to the follicular persistence associated with bovine cystic ovaries.

Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (p < 0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (p < 0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (p < 0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts.

Glargine blood biotransformation: in vitro appraisal with human insulin immunoassay.

AIM: Glargine, a long-acting insulin analogue, is metabolized in the bloodstream and in subcutaneous tissue. Glargine metabolism and its implications for diabetes therapy remain poorly understood. The aim of our study was to assess in vitro the glargine blood biotransformation and its inter-individual variability. METHODS: Formation of M1 glargine metabolite in vitro was studied with Elecsys Insulin immunoassay in pools of sera and sera from patients spiked with glargine. Elecsys Insulin assay is specific of human insulin, does not recognize glargine and its M2 metabolite but does recognize its M1 metabolite. RESULTS: Glargine incubation with serum resulted in M1 metabolite formation which was detected and characterized as an enzymatic process: metabolite kinetics were dependant on temperature, substrate concentration and serum proportion. Carboxypeptidase inhibitors and chelating agents partially inhibited the activity of the enzyme(s). Glargine biotransformation was decreased when blood was collected on EDTA tubes. After 30 min incubation of glargine (100 mU/l) in 69 sera at 37 degrees C, percentage of glargine converted into M1 ranged from 46% to 98% (mean 72%; S.D. 11%). CONCLUSION: Glargine blood biotransformation is an enzymatic process probably involving serum carboxypeptidase(s). Metabolite formation is rapid and non negligible. Inter-individual variability of glargine biotransformation is noteworthy and should be confronted to M1 metabolite bioactivity which has not been fully documented yet.

Purification and properties of methanol dehydrogenase from Methylophaga marina.

Purification of methanol dehydrogenase from Methylophaga marina, in order to avoid the instability observed in crude extracts, was achieved initially by a rapid procedure using mainly an aqueous two-phase partition system composed of polyethylene glycol 1000 (50%, v/v) and potassium phosphate (50%, w/v). The purified enzyme gave a single band of protein after SDS-polyacrylamide gel electrophoresis. Antiserum raised against purified methanol dehydrogenase was used to detect possible protein contaminants in the enzyme preparation. The enzyme is an NAD-independent dehydrogenase containing pyrrolquinoline quinone (PQQ) as a prosthetic group. It is made of two apparently identical subunits giving a total MW of 145,000 for the native enzyme. The isoelectric point is 6.4. In addition to methanol and formaldehyde, multicarbon primary alcohols and aldehydes as well as secondary alcohols can be used as substrates. Except for ammonium chloride, which is a necessary activator in vitro, no effector was found which could modify the rate of enzyme activity under standard conditions of the assay. Although the main properties of this methanol dehydrogenase are similar to those already described in the literature, it does not belong in any of the five categories described by Anthony.

Sequence and functional analysis of an Escherichia coli DNA fragment able to complement pqqE and pqqF mutants from Methylobacterium organophilum.

A 7361 kb fragment of E coli chromosomal DNA able to complement pqqE and pqqF mutants of Methylobacterium organophilum has been sequenced. Five open reading frames (ORF) have been identified. Four ORFs (102, 103, 106 and 107), belong to a single transcription unit. They are separated by a transcription termination site from a fifth ORF (ORF109). Polypeptides of 28, 85 and 82 kDa encoded by ORFs 102, 103 and 106 respectively were visualised in maxi-cell experiments. Both ORF106 and ORF107 are required for complementations of pqqE and pqqF mutants from M organophilum. The polypeptides encoded by ORFs102, 103 and 107 have no homologies with the products of pqq genes previously sequenced from Acinetobacter calcoaceticus, Klebsiella pneumoniae, and Methylobacterium extorquens AM1. The polypeptide encoded by ORF106 shows homology with the pqqF gene product of K pneumoniae, and seems to belong to a family of zinc proteases. The sequence of ORF109 is identical to the sequence of the gadB gene of E coli encoding for a glutamate decarboxylase.

Protein kinase C inhibition of in vitro FSH-induced differentiation in pig granulosa cells.

In granulosa cells, growth factor IGF I plays a major role in both growth and differentiation, acting through an autocrine/paracrine mechanism, and its production is regulated by FSH, via cyclic AMP (cAMP). As protein kinase C is also involved in granulosa cell function, we investigated the possibility that its activation could balance the positive effects of FSH. Using pig granulosa cells cultured in vitro, we studied the effects of protein kinase C activation by tetradecanoylphorbol acetate (TPA) on IGF I mRNA level. We also checked morphological modifications, cAMP production and steroidogenesis at the P450 side chain cleavage mRNA and progesterone levels. Our data demonstrate that protein kinase C activation antagonizes the in vitro FSH-induced differentiation, particularly morphological modifications and accumulation of IGF I mRNA. These inhibitory effects on FSH responses suggest that there could be a balance between protein kinase A and protein kinase C pathways in regulating differentiation in pig granulosa cells.

Gonadotropins induce accumulation of insulin-like growth factor I mRNA in pig granulosa cells in vitro.

Pig granulosa cells have been shown to synthesize insulin-like growth factor (IGF) I peptide in vitro, and this expression is regulated by gonadotropins via the cAMP pathway. By hybridizing an IGF I cDNA probe with total RNA isolated from pig granulosa cells cultured in vitro, we show that these cells contain two IGF I transcripts of about 0.9 kb and 9 kb in size. Treatment of the cells with gonadotropins (follicle-stimulating hormone, luteinizing hormone) or cAMP agonists (dibutyryl-cAMP, forskolin) induces an accumulation of the transcripts which can be abolished by transcriptional inhibitors, but not by translational inhibitors. We thus provide new evidence that pig granulosa cells are a site of IGF I synthesis, and we conclude that (1) gonadotropins increase IGF I mRNA levels; (2) the accumulation of IGF I mRNA results from an increased transcription; (3) the stimulation of IGF I gene transcription does not require ongoing protein synthesis; (4) these effects of follicle-stimulating hormone can be mimicked by cAMP agonists.

Complementation of Methylobacterium organophilum mutants affected in pyrroloquinoline quinone biosynthesis genes pqqE and pqqF by cloned Escherichia coli chromosomal DNA.

The hybrid plasmid pBGT3, a derivative of pLA2917 containing a 7.8-kb fragment of Escherichia coli DNA, was found to complement pqqE and pqqF mutants of Methylobacterium organophilum, both impaired in PQQ biosynthesis. The cloned fragment of E. coli DNA did not hybridize with DNA fragments containing pqqE or pqqF previously cloned from M. organophilum. Yet, in M. organophilum mutants, expression of pqqE and pqqF genes from E. coli resulted in a PQQ production estimated at 9-16% of the production observed in M. organophilum wild-type. The growth rate in methanol medium of the complemented M. organophilum mutants was about 60% of that of the wild-type.

Identification and characterization of the pqqDGC gene cluster involved in pyrroloquinoline quinone production in an obligate methylotroph Methylobacillus flagellatum.

Pyrroloquinoline quinone is a prosthetic group of bacterial methanol dehydrogenases as well as some alcohol and glucose dehydrogenases. Genes involved in pyrroloquinoline quinone production have previously been cloned from the representatives of the alpha and gamma subdivisions of the Proteobacteria. We report identification and the sequence of the pqqDGC gene cluster in the obligate methylotroph, Methylobacillus flagellatum, which belongs to the beta subdivision. The deduced products of the pqq genes from M. flagellatum appear to be more similar to their counterparts from non-methylotrophic species of the gamma subdivision than to a facultative methylotroph of the alpha subdivision. A non-polar mutation in pqqG was constructed and resulted in a strain impaired in growth on methanol. This mutant accumulated a detectable amount of intracellular pyrroloquinoline quinone, but in contrast to the wild type, did not excrete pyrroloquinoline quinone into the culture medium. The possible role of PqqG is discussed.

New unified nomenclature for genes involved in the oxidation of methanol in gram-negative bacteria.

The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox, for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups.

Evaluation of Elecsys free triiodothyronine assay: relevance of age-related reference ranges.

OBJECTIVE: This study aimed at evaluating Elecsys free triiodothyronine (FT3) assay performed on an Elecsys 2010 system, while paying special attention to age relationship in euthyroid subjects. DESIGN AND METHODS: FT3 levels were measured in 149 euthyroid control subjects aged between 2 and 92 years old, 33 hyperthyroid and particular euthyroid patients: female in the last 3 months of pregnancy (n = 30), nonthyroidal ill hospitalized in medical (NTlm, n = 31), or intensive care units (NTlc, n = 31) and amiodarone-treated (n = 27). RESULTS: FT3 was inversely related to age in controls (r = -0.67). Three reference ranges were used: below 20 years 4.5-9.0 pmol/L, between 20 and 60 years 3.9-7.2, and over 60 years 2.4-6.5. Compared to age-matched controls, FT3 decreased in pregnancy, NTlm, NTlc, and amiodarone groups. Use of age-related reference ranges improved the specificity markedly in amiodarone patients and to a lesser extent in NTlm and TClc patients. CONCLUSIONS: The reliability of the Elecsys FT3 assay was found to be satisfactory for clinical use, when the age of patients was taken into account.

Determination of free triiodothyronine by six different methods in patients with non-thyroidal illness and in patients treated with amiodarone.

We performed a methodological comparison of free triiodothyronine (FT3) estimates in patients with liver cirrhosis and renal failure. Patients were classified in terms of severity of illness on the basis of their total triiodothyronine, total thyroxine and reverse triiodothyronine profiles. FT3 levels, measured in direct dialysis, microchromatography, labelled analogue and two-step immunoextraction assays were significantly (P < 0.01) lower than the control group in all patient categories. However, FT3 measured by a labelled antibody radioimmunoassay was significantly reduced only in the most severely ill sub-group of patients. In a further group of patients on long-term amiodarone therapy for cardiac disease all FT3 methods, with the exception of the labelled antibody radioimmunoassay and an analogue method, yielded significantly (P < 0.01) reduced levels. A significant negative association between FT3 and subject age was demonstrated for all methods except the labelled antibody radioimmunoassay, and a weak but significant negative correlation between log thyrotropin and FT3 was only seen with this assay. Three methods demonstrated a correlation (P < 0.02) with albumin levels in patients with the 'low T3 syndrome'. In this group, albumin had a predictive value (P < or = 0.02) for four out of six assays as determined by stepwise variable selection. Our findings suggest that users of FT3 assays should exercise caution in interpreting results in non-thyroidal illness and amiodarone treated patients, as there are method-related differences in the profiles obtained.

[Ultra-sensitive TSH levels: an aid in the screening for amiodarone-induced thyroid dysfunction]. / Le dosage de TSH ultra-sensible: une aide au dépistage des dysthyroïdies induites par l'amiodarone.

Amiodarone modifies thyroid hormone secretion and hypothyroidism occurs in some cases. The latter diagnosis is often difficult and is of particular importance in these patients as it may have serious consequences for the heart. Early diagnosis is therefore essential but difficult because of the induced hyperthyroxinemia with maintenance of euthyroidism and a hypotriiodothyronemia. The diagnostic performance of an ultrasensitive method of measuring TSH (TSH-U), capable of distinguishing hyper and euthyroidism were compared with standard thyroid function tests and TSH stimulation with TRH in 50 patients treated with amiodarone. Only 6 of the 14 patients with hyperthyroxinaemia had TSH-U values in the hyperthyroid range: only one of these patients had an increased triiodothyronine. In 2 cases the THS-U was low but the T4L was normal. In 4 patients, increased TSH-U allowed diagnosis of latent or patent hypothyroidism. There was a close correlation between results of the TRH stimulation test and those of the TSH-U in all cases. This test may therefore be used as an initial screening test for thyroid dysfunction in patients on amiodarone and is simple, reliable and relatively cheap to perform. It makes it unnecessary to measure all thyroid hormonal parameters and the TRH test simultaneously.

[Macroprolactin detection: a new approach]. / Détection de la macroprolactine: une nouvelle approche.

Macroprolactin is a complex of prolactin with immunoglobulins (IgG) that has limited or no biological activity in vivo. Immunoassays for prolactin have variable reactivity with macroprolactin. Therefore the presence of macroprolactin should be considered in the differential diagnosis of hyperprolactinemia. We compared a valid screening test for macroprolactin, polyethyleneglycol (PEG) precipitation, with the determination of the ratio of the results of two prolactin assays: Elecsys with high cross-reactivity with macroprolactin and Centaur with low cross-reactivity. In 59 negative samples subjected to the PEG test (precipitation < 50%), the Elecsys/Centaur ratio ranged between 1.11 and 1.45. Among 35 positive samples (precipitation > 60%), 33 had, as expected, an increased ratio (over 1.45), 1 a normal ratio and 1 a decreased ratio (1.07). This decreased ratio could be due to a particular form of macroprolactin poorly recognised by the Elecsys assay. Among 5 samples in the grey zone (precipitation between 50 and 60%), the ratio was increased in 2, normal in 1 and decreased in 2. Apart from one false negative case (normal ratio with positive PEG test), the results of the Elecsys/Centaur ratio method were in good agreement with those of the PEG test. The ratio method could be helpful for samples with PEG test results in the grey zone, before undertaking a complete analysis of circulating molecular forms by gel filtration chromatography. Out of the 5 five samples in the grey zone, the ratio was 4 times out of the reference range: 2 increased, 2 decreased. Our results also underline the necessity of reevaluating the Centaur prolactin reference range from samples without macroprolactin.

[Free triiodothyronine assays using labeled antibody: evaluation and comparison with an analog labeled assay]. / Dosages de la triiodothyronine libre utilisant un anticorps marqué: évaluation et comparaison à un dosage avec traceur analogue.

We evaluated analytically and clinically two new one-step labelled antibody assays for measuring free triiodothyronine (FT3): the first, radiolabelled with 125I, Amerlex-MAB (MAB) from Kodak Diagnostic, and the second, labelled with peroxidase, Enzymun-test FT3 (BM) from Boehringer Mannheim adapted for the Boehringer ES 600 analyser. The clinical results were compared with those obtained with a radiolabelled analog tracer kit, Amerlex-M (M) from Kodak Diagnostic. The latter kit is known to give low FT3 results in sera with low albumin concentrations. Analytical performances of the automated method (BM) were better than those obtained with the manual method (MAB): intra-assay reproducibility (CV < 3% vs CV about 5%), inter-assay reproducibility (CV < 4% vs CV between 4 and 8%) and mean drift (+1.25% vs -4.3%). The detection limit was low for both kits (< 1 pmol/l). In the euthyroid reference group (n = 98) we observed a significant difference between outpatient and hospitalized patient FT3 concentrations as measured with the M kit only. The reference range was 3.2-6.5 pmol/l for the MAB kit vs 5.4-9.2 pmol/l for the BM kit. This result underlines the problem of standardisation with the FT3 assays. Clinical sensitivity for hyperthyroidism (n = 38) was better for the MAB (92%) than for the BM kit (76%). Specificity in euthyroid L-thyroxine (T4) treated patients (n = 26) was good for both kits (MAB: 92%; BM: 88%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Proposed reference values for nine methods for measurement of free thyroxin]. / Proposition de valeurs de référence pour neuf techniques de dosage de la thyroxine libre dans le sérum.

In connection with a comparative study of nine kits for the measurement of free thyroxin, we determined reference values in a adult control group of 81 women and 73 men. The correlations observed between the kits are associated with very large differences in the results obtained. The reference ranges are more or less broad according to the kits, but narrower than those offered by the manufacturers.

[Two-center evaluation of eight kits for antithyroid peroxidase autoantibodies determinations]. / Evaluation bicentrique de huit kits utilisés pour la recherche des anticorps antithyroperoxydase.

We compared eight antithyroid peroxidase antibody assay kits in two centres, by use of panel sera from 269 patients: controls (n = 100), patients with autoimmune thyroid diseases (n = 77 Graves' disease, Hashimoto's thyroiditis), with non autoimmune thyroid diseases (n = 69 nodular goiter, differentiated thyroid carcinoma), and with autoimmune disease without thyroid pathology (n = 23 diabetic subjects, rheumatoid polyarthritis). On the controls sera we observed different distributions of values. The cut-off values of each kit was, in most cases, similar to the value noted in the manufacturer's instructions. In the clinical study, we observed few differences. The majority of assays demonstrated high diagnostic performance. Some false positive results and the non assessment of some sera by competitive immunoassay were observed.

[Assay of free thyroxine by immunoenzyme method in general diseases]. / Dosage de la thyroxine libre (T4 L) par méthode immunoenzymatique dans les maladies générales.

In non thyroid illnesses the degree of serum FT4 levels perturbation may be variable according to the assay used. The performance of a new FT4 immunoenzymatic assay (Enzymmum test, BMF) was evaluated in 78 euthyroid controls and in 99 sick patients with renal insufficiency, severe diabetes, hypoalbuminemia, severe general disease or under heparinate treatment. Results were compared to two RIA assays: by immunoextraction (CA2), known to be little disturbed by hypoalbuminemia and considered as a reference method, and by a labelled antibody (MAB, Amerlex) easier to use. In controls mean values and T4 L confidence intervalls were comparable with the three assays. In patients FT4 levels were diminished in case of renal insufficiency but the mean values obtained by BMF and CA2 did not differ. FT4 levels of heparinate treated patients were elevated with BMF and CA2. Generally patients are better classified as euthyroid with the CA2 assay (5.6% misclassification) than with BMF (11.2%) or MAB (12.4%), particularly in case of serious disease. A good linear correlation was found between the three methods. With the new BMF assay results were grossly comparable to those obtained by other assays. However the determination of FT4 levels alone appeared no sufficient to characterize the patients thyroid status.

[Effects of amiodarone on thyroid hormonal profile. Updating based on new assay methods]. / Effets de l'amiodarone sur le profil hormonal thyroïdien. Actualisation à propos de nouvelles méthodes de dosage.

Amiodarone (A) treatment alters the levels of thyroid hormones. We investigated whether new hormonal assays are also altered by this drug. Thyroid function was determined in 21 patients chronically treated with A and in 30 controls. TSH was determined with a third generation assay. Free T3 and Free T4 levels were determined by 5 different immunoassays. Equilibrium dialysis (E.D.) was considered as the reference assay for FT3 and FT4. With this method FT3 is diminished, reverse T3 ans FT4 are increased whereas TSH remained normal. Only FT3 determined by an assay using a labelled monoclonal antibody (MAB) appears not modified by A. Other methods (chromatography, chemiluminescence and radioisotopic) give results in agreement with E.D. Some of these alterations may be explained by a marked increase in plasma levels of non esterified fatty acids. Hormonal changes induced by A are typically with all but one assays. Whatever the method used the determination of TSH remains necessary to avoid misinterpretation of the thyroid function tests in A-treated patients.

[Evaluation of thyroid function after myocardial infarction]. / Evaluation de la fonction thyroïdienne après infarctus du myocarde.

The myocardial infarction (M.I.) constitutes an exemplary acute severe affection able to modifie hormonal concentrations. The total and unbound thyroid hormones, reverse T3 (rt3), TSH, and cortisolemia were determined in 24 patients during a period of 21 days in order to compare them to different markers of severity of MI. The initial phase of the disease is characterized by low concentrations of total and free T3 and high concentrations of rT3 combined with more often than not normal total and free T4 and TSH values contrasting with an increase in cortisol levels. The abnormalities were more pronounced the day after admission and then progressively amend. There are several statistic relationship between the marker of severity of MI and thyroid hormones. In the same way total T3 is all the more decreased especially since myoglobin, CPK-MB, ST amplitude and ventricle ejection fraction are more disturbed. Severe forms of MI induces a pseudo central thyroid insufficiency with low T3, low T4 and a tendency to TSH decrease. Total T3 blood levels may usefully contribute to the elaboration of an MI severity index.