Effect of supplementing an α-amylase enzyme or a blend of essential oil components on the performance, nutrient digestibility, and nitrogen balance of dairy cows.

Lowering dietary protein content is a promising strategy to reduce N excretions in cattle but it requires improved N utilization by the animal. Feed enzymes (e.g., exogenous α-amylase) and plant extracts (e.g., essential oils [EO]) are 2 additives that may enhance rumen function and possibly also microbial protein yield. This may increase fat- and protein-corrected milk yield (MY) and milk nitrogen efficiency and thus lower N losses from dairy cows. Both types of additives were studied in an experiment including 39 Holstein cows that had (average ± SD) 40.7 ± 7.95 kg/d MY, 89 ± 43 DIM, 2.7 ± 1.5 lactations, and 677 ± 68.6 kg of BW, consisting of a covariate (4 wk) and treatment period (5 wk). During the whole experiment cows were fed a typical Benelux diet (CTRL), supplemented with concentrates to meet individual requirements for energy and MP, which were fulfilled for 100% and 101%, respectively. The total diet was low in CP (15.5%) and relatively high in starch (22.6% and 6.6% rumen bypass starch). Cows were balanced for parity, DIM, MY, and roughage intake and randomly assigned to one of 3 groups, which received the following treatments in the treatment period: (1) CTRL (n = 13); (2) CTRL + 14 g/cow per day Ronozyme RumiStar α-amylase enzyme (AMEZ, n = 13; DSM); and (3) CTRL + 2.5 g/cow per day Crina Protect, a blend of EO components (ESOL, n = 13; DSM). Animal performance, ruminal pH, and enteric gas emissions were monitored throughout the experiment. During the last week of the covariate and treatment periods, nitrogen balances were conducted, total-tract nutrient digestibility was determined, and urinary allantoin and uric acid were determined as indicators for microbial N production. The statistical model applied to these variables contained group and DIM during treatment period as fixed effects and the values from the covariate period as covariate. Post hoc Dunnet-corrected comparisons between each treatment group and the control group were explored. The α-amylase enzyme tended to increase apparent total-tract starch digestibility and increased milk lactose concentration. The EO blend tended to increase MY and increased milk N output, milk nitrogen efficiency, and feed efficiency. Therefore, when feeding reduced dietary protein levels, EO have potential to improve the N-use efficiency in cattle, whereas the α-amylase enzyme might increase starch digestibility and milk lactose. However, additional research is necessary to substantiate our findings.

The effect of maternal supply of rumen-protected protein to Holstein Friesian cows during the dry period on the transfer of passive immunity and colostral microbial composition.

The objective of this study was to analyze if maternal supply of rumen-protected protein during the dry period can affect the IgG concentration and microbial composition of colostrum and the IgG absorption and fecal microbial composition in the calf. Seventy-four multiparous Holstein Friesian (HF) dairy cows were stratified per parity and randomly assigned to one of 2 different dry period diets, a diet with a low crude protein (CP) level (LP) and a diet with a high CP level (HP) by addition of rumen-undegraded protein (RUP; formaldehyde-treated soybean meal, Mervobest, Nuscience, Drongen, Belgium). Colostrum was collected within 1 h after calving and IgG concentration was quantified by radial immunodiffusion analysis. Forty-nine calves (23 female and 26 male) were enrolled in the trial with a 2 × 2 factorial design, with prenatal and postnatal treatment as the 2 independent variables. This led to 4 experimental groups: LPLP, LPHP, HPLP, and HPHP, in which the first 2 letters refer to the prenatal treatment (diet of the dam) and the last 2 refer to the postnatal treatment (diet of the colostrum-producing cow). Calves received 3× 2 L of colostrum within 2, 6, and 24 h after birth. Meconium and feces were collected solely from female calves (n = 18) by digital palpation of the rectum, immediately after birth and before colostrum administration and at d 3 of age. Microbial DNA was extracted from meconium (n = 9), feces (n = 15), and colostrum (n = 49). Amplicon sequencing of the bacterial V3-V4 region of the 16S rRNA gene was performed for characterization of the bacterial communities. Colostrum IgG concentration was higher in cows that were supplemented with RUP, especially in cows entering their second lactation (LSM ± SEM 61.3 ± 2.3 vs. 55.2 ± 2.8 g of IgG/L). Calves born out of LP cows that received colostrum from HP cows (LPHP) had a lower serum IgG level compared with HPHP and LPLP calves (LSM ± SEM 14.2 ± 1.3 vs. 18.8 ± 1.2 and 20.9 ± 1.3 g of IgG/L in HPHP and LPLP, respectively). The most abundant phyla in colostrum were Proteobacteria (48.2%), Firmicutes (24.8%), Bacteroidetes (9.5%), and Actinobacteria (5.0%). The most abundant phyla in calf meconium and feces were Firmicutes (42.5 and 47.5%), Proteobacteria (21.7% and 33.7%), Bacteroidetes (16.8% and 15.7%), and Actinobacteria (2.9% and 3.1%). There was no difference in the overall microbial communities between colostrum from HP and LP cows. However, 2 genera (both members of the family Lachnospiraceae) were more abundant in colostrum from HP cows compared with LP cows. The microbial composition of meconium, feces and colostrum differed from each other. Fecal samples were more similar to each other and are characterized by a lower intersample diversity compared with colostrum and meconium samples. To conclude, increasing the CP level by addition of RUP in the dry period diet affected the colostrum IgG concentration and the transfer of passive immunity, but did not change the overall microbial composition of colostrum nor of meconium and feces in the calf.

Effect of supplementing rumen-protected methionine, lysine, and histidine to low-protein diets on the performance and nitrogen balance of dairy cows.

Lowering the dietary protein content can reduce N excretions and NH3 emissions from manure and increase milk N efficiency of dairy cows. However, milk yield (MY) and composition can be compromised due to AA deficiency. Methionine and Lys are known as first limiting EAA for dairy cows, and recently His is also mentioned as limiting, especially in grass-based or low-protein diets. To examine this, a trial was conducted with a 3-wk pre-experimental adaptation period (diet 16.5% crude protein), followed by a depletion period of 4 wk, in which 39 cows (average ± standard deviation: 116 ± 29.3 d in milk, 1.8 ± 1.2 lactations, 638 ± 73.2 kg of body weight, and 32.7 ± 5.75 kg MY/d) received a low-protein diet (CTRL) (14.5% crude protein). Then, taking into account parity, His plasma concentration, and MY, cows were randomly assigned to 1 of 3 treatment groups during the rumen-protected (RP) AA period of 7 wk; (1) CTRL; (2) CTRL + RP-Met + RP-Lys (MetLys); (3) CTRL + RP-Met + RP-Lys + RP-His (MetLysHis). Products were dosed, assuming requirements for digestible (d) Met, dLys, and dHis being, respectively, 2.4%, 7.0%, and 2.4% of intestinal digestible protein. In the cross-back period of 5 wk, all cows received the CTRL diet. During the last week of each period, a N balance was conducted by collecting total urine and spot samples of feces. Total feces production was calculated using the inert marker TiO2. Statistical analysis was performed with a linear mixed model with cow as random effect and data of the last week of the pre-experimental period used as covariate for the animal performance variables. No effect of supplementing RP-Met and RP-Lys nor RP-Met, RP-Lys, and RP-His on feed intake, milk performance, or milk N efficiency was observed. However, the plasma AA profile indicated additional supply of dMet, dLys, and dHis. Nevertheless, evaluation of the AA uptake relative to the cow's requirements showed that most EAA (exclusive Arg and Thr) were limiting over the whole experiment. Only dHis was sufficiently supplemented during the RP-AA period due to an overestimation of the diet's dMet and dLys supply in the beginning of the trial. The numerically increased milk urea N and urinary N excretion when RP-Met, RP-Lys, and RP-His were added to the low-protein diet suggest an increased catabolism of the excess His.

Exploring the microbial composition of Holstein Friesian and Belgian Blue colostrum in relation to the transfer of passive immunity.

For centuries, multicellular organisms have lived in symbiosis with microorganisms. The interaction with microorganisms has been shown to be very beneficial for humans and animals. During a natural birth, the initial inoculation with bacteria occurs when the neonate passes through the birth canal. Colostrum and milk intake are associated with the acquisition of a healthy gut flora. However, little is known about the microbial composition of bovine colostrum and the possible beneficial effects for the neonatal calf. In this prospective cohort study, the microbial composition of first-milking colostrum was analyzed in 62 Holstein Friesian (HF) and 46 Belgian Blue (BB) cows by performing amplicon sequencing of the bacterial V3-V4 region of the 16S rRNA gene. Calves received, 3 times, 2 L of their dam's colostrum within 24 h after birth. Associations between colostral microbial composition and its IgG concentration, as well as each calf's serum IgG levels, were analyzed. Colostrum samples were dominated by the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The 10 most abundant genera in the complete data set were Acinetobacter (16.2%), Pseudomonas (15.1%), a genus belonging to the Enterobacteriaceae family (4.9%), Lactococcus (4.0%), Chryseobacterium (3.9%), Staphylococcus (3.6%), Proteus (1.9%), Streptococcus (1.8%), Enterococcus (1.7%), and Enhydrobacter (1.5%). The remaining genera (other than these top 10) accounted for 36.5% of the counts, and another 8.7% were unidentified. Bacterial diversity differed significantly between HF and BB samples. Within each breed, several genera were found to be differentially abundant between colostrum of different quality. Moreover, in HF, the bacterial composition of colostrum leading to low serum IgG levels in the calf differed from that of colostrum leading to high serum IgG levels. Results of the present study indicate that the microbes present in colostrum are associated with transfer of passive immunity in neonatal calves.

Granulation strategies applied to industrial wastewater treatment: from lab to full-scale.

About one third of the industrial activated sludge (AS) plants worldwide suffer from bad settling sludge, often caused by filamentous bulking phenomena. The present study investigated the effectiveness of a sludge granulation/densification strategy, based only on a metabolic selection mechanism, to eliminate sludge bulking in a sequencing batch reactor (SBR) treating real industrial wastewater. The wastewater originated from a tank truck cleaning company transporting chocolate and beer. The proposed strategy involved the introduction of a slow unaerated/anaerobic feeding step in the SBR operation. On lab-scale, the new feeding strategy resulted in (1) excellent settling with a sludge volume index (SVI) decreasing from more than 300 mL·g-1 to 100 mL·g-1 and lower, (2) the elimination of sludge bulking genera and (3) the significant enrichment of glycogen-accumulating organisms (GAO), mainly Defluviicoccus and Candidatus Competibacter, and this in less than 80 days. The feeding strategy was then applied to the full-scale installation, yielding similar results: a stable average SVI of 37 mL·g-1 was reached after approximately 150 days. Full granulation was however not reached, which warrants further optimization. The present study shows that the proposed strategy can easily be applied in existing SBR systems to solve the problem of sludge bulking.

Invited review: MicroRNAs in bovine colostrum-Focus on their origin and potential health benefits for the calf.

Colostrum is the first milk produced by a cow after she gives birth. Compared with mature milk, it has a high concentration of immunoglobulin G. Calves are born without circulating antibodies, thus ingestion of colostrum is necessary to protect the calf against pathogens in the first challenging weeks of life. In addition to the life-saving supply of antibodies, colostrum contains minerals, vitamins, growth factors, and immune cells. Recently, microRNAs (miRNAs) were added to that list. MicroRNAs are short, non-coding RNA molecules that can regulate gene expression at the post-transcriptional level. They are thought to act as key regulators of diverse biological and developmental processes. Colostrum contains higher amounts of miRNAs than mature milk; immune- and development-related miRNAs are prominent. Their expression pattern in milk is likely to be influenced by maternal nutrition and environment. The fat content of the maternal diet appears to have a major effect on expression of miRNAs in milk and in the neonate. The immunological state of the mammary gland seems to affect miRNA expression as well. In cows diagnosed with subclinical mastitis, alterations in the expression of miRNAs in milk have been observed. It is believed that miRNAs in colostrum and milk are signaling molecules passed from mother to newborn. They are packaged in extracellular vesicles, which makes them resistant to the harsh conditions in the gastrointestinal tract. Therefore, they can reach the small intestine, where they are absorbed and transferred into the bloodstream. MicroRNAs are important for the development of the intestines. For example, miRNAs stimulate cell viability, proliferation, and stem cell activity of the intestinal epithelium. Furthermore, miRNAs seem to act as key players in the development of the complete immune system. They can, among other things, regulate B- and T-cell differentiation and affect interleukin production of macrophages. The abundance of miRNAs in colostrum and milk and the possibility for their absorption in the intestines of the neonate supports the hypothesis that these tiny molecules are important for the development of the newborn. The probable relation of diet to the expression of miRNAs by the mother creates a possible avenue to optimize expression of miRNAs and improve neonatal maturation.

Reduction of Mycobacterium avium ssp. paratuberculosis in colostrum: Development and validation of 2 methods, one based on curdling and one based on centrifugation.

The aim of this study was to develop and validate 2 protocols (for use on-farm and at a central location) for the reduction of Mycobacterium avium ssp. paratuberculosis (MAP) in colostrum while preserving beneficial immunoglobulins (IgG). The on-farm protocol was based on curdling of the colostrum, where the IgG remain in the whey and the MAP bacteria are trapped in the curd. First, the colostrum was diluted with water (2 volumes colostrum to 1 volume water) and 2% rennet was added. After incubation (1 h at 32°C), the curd was cut and incubated again, after which whey and curd were separated using a cheesecloth. The curd was removed and milk powder was added to the whey. Approximately 1 log reduction in MAP counts was achieved. A reduction in total proteins and IgG was observed due to initial dilution of the colostrum. After curd formation, more than 95% of the immunoglobulins remained in the whey fraction. The semi-industrial protocol was based on centrifugation, which causes MAP to precipitate, while the IgG remain in the supernatant. This protocol was first developed in the laboratory. The colostrum was diluted with skimmed colostrum (2 volumes colostrum to 1 volume skimmed colostrum), then skimmed and centrifuged (at 15,600 × g for 30 min at room temperature). We observed on average 1.5 log reduction in the MAP counts and a limited reduction in proteins and IgG in the supernatant. To obtain a semi-industrial protocol, dairy pilot appliances were evaluated and the following changes were applied to the protocol: after 2:1 dilution as above, the colostrum was skimmed and subsequently clarified, after which the cream was heat treated and added to the supernatant. To investigate the effect of the colostrum treatment on the nutritional value and palatability of the colostrum and the IgG transfer, an animal experiment was conducted with 24 calves. Six received the dam's colostrum, 6 were given untreated purchased colostrum (control), and 2 groups of 6 calves received colostrum treated according to both of the above-mentioned methods. No significant differences were found between the test groups and the dam's colostrum group in terms of animal health, IgG uptake in the blood serum, milk, or forage uptake. Two protocols to reduce MAP in colostrum (for use on-farm or at a central location) were developed. Both methods preserve the vital IgG.

The effect of maize silage type on the performances and methane emission of dairy cattle.

To examine whether type of maize silage is important for milk production performances, maize silage LG30224 (LG) was compared with Falkone (FA), the latter having a 4.0% points lower rumen NDF digestibility and 19 g/kg dry matter (DM) more starch. To bridge the lower energy content of FA, a third treatment was involved by adding maize meal (MM) in a ratio of 92/8 on DM (FA+MM). Maize and grass silage were fed ad libitum in a ratio of 65/35 on DM basis. Concentrates were supplemented individually to meet energy and protein requirements. The experiment was set up as a Latin square with three groups of nine Holstein cows during three periods of 3 weeks. In the last 2 weeks of each period, DM intake (DMI) and milk performances were measured. Each group included one cannulated cow to study effects on rumen fermentation. During the last 4 days of each period, two cows from each group were placed in gas exchange chambers to measure nutrient digestibility and methane production. Total DMI was higher (p < 0.05) for FA+MM (20.8 kg/day) than for FA (20.3 kg/day), while DMI for LG was intermediate (20.6 kg/day). Treatment did not affect milk production nor composition, whereas fat-protein-corrected milk was higher for LG (30.5 kg/day) and FA+MM (30.3 kg/day) than for FA (29.9 kg/day). The ration did not affect pH nor volatile fatty acid composition in the rumen. Further, total tract digestibility of OM, crude protein, NDF and starch did not differ among treatments. The ration with LG gave higher methane production per day and per kg NDF intake than both rations with FA, but the difference was not significant when expressed per kg DMI or FPCM. Thus, maize silage type is of little importance for milk production if energy and physical structure requirements are met.

The importance of adequate fixation for immunofluorescent staining of bovine embryos.

Immunofluorescent staining is often used to investigate the expression of specific proteins in pre-implantation embryos. The success of this method is determined by the specificity of the antibodies, but also by the protocol used for fixation and permeabilization of the samples. In this study, different fixatives are compared in combination with immunofluorescent staining of caudal-type homeobox 2 (CDX2), fibronectin 1 (FN1) and integrins (ITGs) on bovine blastocysts. For both CDX2 and the ITGs, the outcome of the staining was largely dependent on the fixation methods. Paraformaldehyde fixation was best for the intracellular CDX2 protein, whereas acetone fixation gave the best results for the transmembrane ITGs. No difference was observed for the FN1 staining between samples fixed with paraformaldehyde or acetone. These examples demonstrate that the choice of fixation and permeabilization agents is very important for the outcome of the experiment, and this choice is dictated by the (extra)cellular location of the protein under investigation. Inappropriate fixation and/or permeabilization methods can lead to erroneous conclusions regarding the site and amount of protein expression.

mRNA expression of Bcl-2, Bax, caspase-3 and -7 cannot be used as a marker for apoptosis in bovine blastocysts.

At present the most widely used technique for apoptosis detection in embryos remains the in situ visualization of DNA fragmentation by terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) assay although this technique may be prone to artefacts. The aim of the present study was to investigate if the mRNA expression of a set of genes involved in apoptosis (Bax, Bcl-2, caspase-3 and -7) at an earlier point in the apoptotic cascade could be a good marker for apoptosis in in vitro produced bovine embryos. After normalization to the geometric mean of three reference genes, GAPD, YWHAZ and SDHA, mRNA expression levels of Bax, Bcl-2, caspase-3 and -7 were compared in embryos treated with an apoptosis inducer, staurosporine and in non-treated embryos. None of the genes were differently expressed in treated in comparison with non-treated embryos. In conclusion, mRNA expression of Bax, Bcl-2, caspase-3 and-7 cannot be used as a reliable apoptosis detection method. Immunofluorescent staining of caspase-3 and -7 is a better choice where as for Bcl-2 no reliable and practicable alternative is available at the moment.

Gamete origin in relation to early embryo development.

Fertilization in vivo requires a complex series of selection events to occur in order to guarantee that only the fittest gametes take part in the fusion process and give rise to a viable embryo. Conventional practice in bovine in vitro fertilization however is to select oocytes and sperm by quite crude procedures. It is therefore not inconceivable that essentially unfit gametes may drive aberrant embryo development in vitro. Abnormal embryonic cells are being removed by apoptosis, which is a physiological process in embryos. Only an excess or a lack of apoptosis can lead to embryonic death or abnormal development. Suboptimal culture conditions undoubtedly contribute to undue embryonic apoptosis, but the intrinsic quality of the oocyte may also be a causative factor. It is generally accepted that the oocyte is in control of early embryogenesis, but is it also in control of future embryonic suicide? Is a compromised follicular environment predestining the oocyte to a dire fate? What is the contribution of the cumulus cells to oocyte quality, and can they rescue it from early demise? And what can be said about the origin of the spermatozoa? Research in human in vitro fertilization has definitely shown that factors such as paternal age, smoking and other sperm stressors can contribute to abnormal embryo development and even diseased offspring. This review will address the questions raised above, and will describe what is known about the cellular and molecular biology that may account for abnormal bovine embryo development caused by gamete origin.

A patient with a 'typical presentation' of Wernicke encephalopathy was found to have sporadic Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) has a significant degree of clinical heterogeneity that is especially found in the features at onset. Here we present a patient with the sporadic form of CJD mimicking Wernicke encephalopathy. We first treated him with a high dose of thiamine; however, the vitamin B1 levels proved to be normal, which ruled out Wernicke encephalopathy. Meanwhile, his clinical condition progressively worsened and he developed a rapidly progressive cognitive disorder, mutism and myoclonus of the muscles. At this point, the diagnosis of CJD was most likely. The patient died two months after the first symptoms. Autopsy showed prion-protein depositions in several regions. Genetic analysis was negative for familial CJD. Those findings confirmed the diagnosis of 'sporadic Creutzfeldt-Jakob disease'. CJD presents in a wide range of sequences and clinical symptoms. Therefore, recognition in the early stage can be difficult.

Technical note: validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes.

Analysis of gene expression is becoming more important in all areas of biological research to evaluate gene expression during physiological and pathological conditions (e.g., mastitis), not the least in the field of animal research. Presently, real-time gene expression analysis is considered to be the method of choice for accurate and sensitive quantification of mRNA transcripts. Because comparison of gene expression levels is frequently the aim of these experiments, there is a critical need to validate internal control genes. When studying gene expression in bovine polymorphonuclear leukocytes, special attention should be paid to this validation, because polymorphonuclear leukocytes are subjected to numerous physiological influences, depending on the stage of lactation. In this study, 8 commonly used reference genes (ACT, GAPD, H2A, TBP, HPRT1, SDHA, YWHAZ, and 18S rRNA) were evaluated in bovine polymorphonuclear leukocytes. The transcription levels of 6 reference genes were determined using real-time PCR. By geometrically averaging the expression levels of these genes, SDHA, YWHAZ, and 18S rRNA were selected as being the most stable genes for accurate normalization of real-time results of bovine polymorphonuclear leukocytes.

Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP.

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.

Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase.

The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT1) has been studied by mutagenesis and Fourier transform infrared analysis. We have shown that a CAT mutant truncated by seven amino acid residues folds into active protein. In this study, the last three residues of this truncated CAT mutant were randomized to detect structural information required for achieving a native enzyme conformation. Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CAT fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure. This minimal length corresponds to a protein with full-length alpha5-helix in the three-dimensional crystal structure of CAT type III. The amino acid preferences at the carboxy terminal in the randomization experiments suggest that this helix also forms completely in the shortened CAT mutants. In addition correct folding and/or stabilization requires the formation of a hydrophobic + microdomain at the end of the alpha5-helix. The role of this hydrophobic interaction in CAT folding and structure stabilization is discussed.

Androgens decrease and retinoids increase the expression of insulin-like growth factor-binding protein-3 in LNcaP prostatic adenocarcinoma cells.

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Clinical optimization and multicenter validation of antigen-specific cut-off values on the INNO-LIA ANA update for the detection of autoantibodies in connective tissue disorders.

OBJECTIVES: The INNO-LIA ANA Update is a qualitative multiparameter line immunoassay for detection of autoantibodies to several different antigens associated with connective tissue disorders. We sought to optimize and validate the cut-off values for its antigen-specific components: SmB, SmD, RNP-70k, RNP-A, RNP-C, SSA/Ro52, SSA/Ro60, SSB/La, Cenp-B, Topo-I, Jo-1, ribosomal P, and histones. Our aim was to achieve 98% specificity for each of the markers, with respect to differential disease controls, while maintaining sensitivity. METHODS: For optimization, the cut-off value of the different antigen lines was fixed to achieve this specificity using an in-house set of 955 patient samples. Specificity was validated at multiple sites using a different set of 330 samples obtained from 158 apparently healthy blood donors, 100 patients with a variety of infections, 20 each with Wegener's granulomatosis, inflammatory bowel disease, and primary antiphospholipid syndrome, and 12 with psoriatic arthritis. Sensitivity was evaluated, using this optimized cut-off control, in 147 patients with scleroderma, 93 with Sjögren's disease, 40 with systemic lupus erythematosus, 40 with rheumatoid arthritis, 39 with mixed connective tissue disease, and 19 with polymyositis. Sensitivity and specificity of the INNO-LIA ANA Update were determined using the clinical diagnosis as reference. RESULTS: The optimized cut-off values resulted in a specificity 98% or more for all LIA markers except one (histones 97.8%) in the validation set of 330 samples. The sensitivity for each marker tested in 378 samples from the target patient groups was comparable to that reported in the literature. CONCLUSION: The INNO-LIA ANA Update shows uniformly high specificities combined with sensitivities very similar to those of reference assays, in a single test format.

Perceptual changes in the anterior maxilla after placement of endosseous implants.

BACKGROUND: The innervation of skin and oral mucosa plays a major physiologic role in exteroception. It is also of interest clinically, as illustrated by sensory changes after neurosurgical procedures. PURPOSE: The goal of this study was to assess the perception of mechanical stimuli applied to the anterior maxilla in denture wearers and subjects rehabilitated with osseointegrated implants compared with that in subjects with a natural dentition. MATERIALS AND METHODS: Five groups of subjects were selected and tested in the maxillary region only. Test groups included patients with a complete denture, an implant-supported fixed prosthesis (full or partial), or a single-tooth replacement. Light-touch sensation and two-point discrimination were performed on the buccal side of the alveolar mucosa and vibrotactile function was determined for natural teeth, full dentures, or implant-supported prostheses. RESULTS: For light-touch sensation, no significant differences could be found between the groups. For two-point discrimination, full-denture patients showed higher threshold levels than the other groups. The threshold levels for vibrotactile function were higher in both full dentures and implant-supported prostheses compared with natural dentitions. CONCLUSION: Natural dentitions offer superior vibrotactile function compared to any other dental status. Full dentures often show a stronger deterioration of the (vibro)tactile function compared with implant-supported prostheses.

Correlation between androstenedione and 17-hydroxyprogesterone levels in the saliva and plasma of patients with congenital adrenal hyperplasia.

INTRODUCTION: Congenital adrenal hyperplasia (CAH) or adrenogenital syndrome is the most common cause of female ambiguous genitalia. Management of such patients involves medical treatment using glucocorticoids such as hydrocortisone, prednisone or dexamethasone. Monitoring is done by measurement of 17-hydroxyprogesterone (17-OHP) or androstenedione in serum, plasma or saliva. The aim of this study was to develop a system of monitoring steroid treatment in CAH patients using only saliva. METHODS: We studied the saliva of 24 CAH patients who received glucocorticoid replacement therapy. The patients were asked to collect saliva upon awakening, and in the afternoon and evening. The levels of 17-OHP and androstenedione in the saliva as well as in serum were then measured by immunoassay. RESULTS: There was a significant positive correlation between 17-OHP in serum and in saliva (R equals 0.929, p-value less than 0.01). A significant positive correlation between androstenedione level in saliva and serum was also found (R equals 0.611, p-value less than 0.01). This study also revealed a significant positive correlation between androstenedione and 17-OHP in serum (R equals 0.647, p-value less than 0.01) and saliva (R equals 0.799, p-value less than 0.01). All patients showed increased level of 17-OHP and androstenedione in the sample collected upon awakening. CONCLUSION: Determination of salivary androstenedione and 17-OHP in CAH patients could be a useful alternative to the measurement of these hormones in serum.