Expression of metabolic sensing receptors in adipose tissues of periparturient dairy cows with differing extent of negative energy balance.

We recently showed that the mRNA expression of genes encoding for specific nutrient sensing receptors, namely the free fatty acid receptors (FFAR) 1, 2, 3, and the hydroxycarboxylic acid receptor (HCAR) 2, undergo characteristic changes during the transition from late pregnancy to lactation in certain adipose tissues (AT) of dairy cows. We hypothesised that divergent energy intake achieved by feeding diets with either high or low portions of concentrate (60% v. 30% concentrate on a dry matter basis) will alter the mRNA expression of FFAR 1, 2, 3, as well as HCAR2 in subcutaneous (SCAT) and retroperitoneal AT (RPAT) of dairy cows in the first 3 weeks postpartum (p.p.). For this purpose, 20 multiparous German Holstein cows were allocated to either the high concentrate ration (HC, n=10) or the low concentrate ration (LC, n=10) from day 1 to 21 p.p. Serum samples and biopsies of SCAT (tail head) and RPAT (above the peritoneum) were obtained at day -21, 1 and 21 relative to parturition. The mRNA abundances were measured by quantitative PCR. The concentrations of short-chain fatty acid (SCFA) in serum were measured by gas chromatography-flame ionisation detector. The FFAR1 and FFAR2 mRNA abundance in RPAT was higher at day -21 compared to day 1. At day 21 p.p. the FFAR2 mRNA abundance was 2.5-fold higher in RPAT of the LC animals compared to the HC cows. The FFAR3 mRNA abundance tended to lower values in SCAT of the LC group at day 21. The HCAR2 mRNA abundance was neither affected by time nor by feeding in both AT. On day 21 p.p. the HC group had 1.7-fold greater serum concentrations of propionic acid and lower concentrations of acetic acid (trend: 1.2-fold lower) compared with the LC group. Positive correlations between the mRNA abundance of HCAR2 and peroxisome proliferator-activated receptor γ-2 (PPARG2) indicate a link between HCAR2 and PPARG2 in both AT. We observed an inverse regulation of FFAR2 and FFAR3 expression over time and both receptors also showed an inverse mRNA abundance as induced by different portions of concentrate. Thus, indicating divergent nutrient sensing of both receptors in AT during the transition period. We propose that the different manifestation of negative EB in both groups at day 21 after parturition affect at least FFAR2 expression in RPAT.

Longitudinal changes in adipose tissue of dairy cows from late pregnancy to lactation. Part 1: The adipokines apelin and resistin and their relationship to receptors linked with lipolysis.

The transition from pregnancy to lactation is characterized by major changes in glucose and adipose tissue metabolism. Anti- and prolipolytic pathways mediated via the hydroxycarboxylic acid receptors 1 (HCAR1) and 2 (HCAR2) and tumor necrosis factor-α receptor 1 (TNFR1), as well as the adipokines apelin and resistin, are likely involved in regulating these processes. This study aimed to determine the mRNA abundance of the aforementioned receptors in both subcutaneous and visceral adipose tissue, to characterize the adipokine concentrations in serum, and to test the effects of feeding diets with either high or low portions of concentrate and a concomitant niacin supplementation from late gestation to early lactation. Twenty pluriparous German Holstein cows were all kept on the same silage-based diet until d 42 antepartum, when they were allocated to 2 feeding groups: until d 1 antepartum, 10 animals each were assigned to either a high-concentrate (60:40 concentrate-to-roughage ratio) or a low-concentrate diet (30:70). Both groups were further subdivided into a control and a niacin group, the latter receiving 24 g/d of nicotinic acid from d -42 until 24. From d 1 to 24 postpartum, the concentrate portion was increased from 30 to 50% for all cows. Biopsies of subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) were taken at d -42, 1, 21, and 100 relative to parturition. Blood samples were drawn along with the biopsies and on d -14, 3, 7, 14, and 42. The concentrations of the adipokines apelin and resistin in serum were measured via ELISA. The mRNA of the 3 receptors in AT was quantified as well as the protein abundance of HCAR2 by Western blot. The feeding regimen did not affect the variables examined. The concentrations of apelin remained fairly constant during the observation period, whereas the resistin concentrations increased toward parturition and decreased to precalving levels within 1 wk after calving. The mRNA abundance of HCAR1, HCAR2, and TNFR1 changed in SCAT and RPAT during the considered time period. For the HCAR2 protein, time-dependent changes were restricted to SCAT. The mRNA abundance of all receptors was greater in RPAT than in SCAT. The tissue-specific correlations observed between the receptors point to a link between these factors and may indicate different regulatory roles in the respective tissues. This study provides insight into the complex metabolic adaptations during the transition period and supports a differential regulation of lipolysis among SCAT and RPAT in dairy cows.

Longitudinal changes in adipose tissue of dairy cows from late pregnancy to lactation. Part 2: The SIRT-PPARGC1A axis and its relationship with the adiponectin system.

The transition period in dairy cows is characterized by major changes in glucose and adipose tissue metabolism. The Sirtuin-1 (SIRT1) PPARγ co-activator 1α (PPARGC1A) axis might be related to the adiponectin (ADIPOQ) system to orchestrate the regulation of these processes. We aimed to assess the mRNA abundance of the aforementioned components in one visceral and one subcutaneous fat depot, together with the ADIPOQ concentrations in serum of dairy cows from late gestation to early lactation. In addition, the effect of 2 diets differing in energy density was tested. Twenty pluriparous German Holstein cows were all kept on the same silage-based diet until d 42 antepartum. From then on until d 1 antepartum, 10 animals each were assigned to either high-concentrate (60:40 concentrate:roughage) or low-concentrate (30:70) diets. Both groups were further subdivided into a control and a niacin group, the latter receiving 24 g/d nicotinic acid from d -42 until d 24. From d 1 postpartum (p.p.) to d 24 p.p., the concentrate portion was increased from 30 to 50% for all cows. Biopsies of subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) were taken at d -42, 1, 21, and 100 relative to parturition. Blood samples were drawn along with the biopsies as well as on d -21, -14, -7, -3, 1, 3, 7, 14, 21, 28, 35, 42, 63, 82, and 100 relative to calving. Quantification of target mRNA was done using quantitative PCR and serum ADIPOQ concentration was measured via ELISA. The feeding regimen did not affect the variables examined. Serum ADIPOQ concentrations decreased toward parturition, returned to precalving levels within 1 wk after parturition, and remained on a constant level until the end of the experiment. The mRNA abundance of SIRT1, PPARGC1A, NAMPT, and the ADIPOQ receptors 1 (ADIPOR1) and 2 (ADIPOR2) changed in SCAT and RPAT during the considered time period. Comparing SCAT and RPAT, the mRNA of SIRT1, ADIPOR1, and ADIPOR2 were more abundant in RPAT, whereas PPARGC1A and NAMPT were expressed more highly in SCAT. The protein abundance of SIRT1 tended to increase from d -42 to 21. At d 21 we detected more PPARGC1A protein in the low-concentrate group as compared with the high-concentrate group. The correlations observed point to a link between these factors and might hint to a functional role of the variables in the regulation of glucose metabolism. This study substantiates the existence of the SIRT1-PPARGC1A-axis and indicates a functional relationship between SIRT1 and ADIPOR1 in bovine adipose tissue.

Changes in lipid metabolism and ß-adrenergic response of adipose tissues of periparturient dairy cows affected by an energy-dense diet and nicotinic acid supplementation.

Dairy cattle will mobilize large amounts of body fat during early lactation as an effect of decreased lipogenesis and increased lipolysis. Regulation of lipid metabolism involves fatty acid synthesis from acetate and ß-adrenergic-stimulated phosphorylation of hormone-sensitive lipase (HSL) and perilipin in adipocytes. Although basic mechanisms of mobilizing fat storage in transition cows are understood, we lack a sufficiently detailed understanding to declare the exact regulatory network of these in a broad range of dairy cattle. The objective of the present study was to quantify 1) protein abundance of fatty acid synthase (FAS), 2) extent of phosphorylation of HSL and perilipin in vivo, and 3) ß-adrenergic stimulated lipolytic response of adipose tissues in vitro at different stages of the periparturient period. We fed 20 German Holstein cows an energy-dense or an energetically adequate diet prepartum and 0 or 24 g/d nicotinic acid (NA) supplementation. Biopsy samples of subcutaneous and retroperitoneal adipose tissue were obtained at d 42 prepartum (d -42) and at d 1, 21, and 100 postpartum (d +1, d +21, d +100, respectively). To assess ß-adrenergic response, tissue samples were incubated with 1 µ isoproterenol for 90 min at 37°C. The NEFA and glycerol release, as well as HSL and perilipin phosphorylation, was measured as indicators of in vitro stimulated lipolysis. In addition, protein expression of FAS and extent of HSL and perilipin phosphorylation were measured in fresh, nonincubated samples. There was no effect of dietary energy density or NA on the observed variables. The extent of HSL and perilipin phosphorylation under isoproterenol stimulation was strongly correlated with the release of NEFA and glycerol, consistent with the functional link between ß-adrenergic-stimulated protein phosphorylation and lipolysis. In the nonincubated samples, FAS protein expression was decreased at d +1 and d +21, whereas HSL and perilipin phosphorylation increased from d -42 to d +1 and remained at an increased level throughout the first 100 d of lactation. In vitro lipolytic response was significant in prepartum samples at times when in vivo lipolysis was only minimally activated by phosphorylation. These data extend our understanding of the complex nature of control of lipolysis and lipogenesis in dairy cows and could be useful to the ongoing development of systems biology models of metabolism to help improve our quantitative knowledge of the cow.

Diagnostic opportunities for evaluation of the exposure of dairy cows to the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN): reliability of blood plasma, bile and follicular fluid as indicators.

To investigate the usefulness of follicular fluid (FF) in relation to blood plasma and bile as indicators of exposure of dairy cows to ZEN, DON and their metabolites, a dose-response study was performed with 30 dairy cows. The cows, 10 in each group (named CON; FUS-50, FUS-100), received a diet with three different concentrations of Fusarium toxin-contaminated maize. Thereby, the following dietary concentration were reached: CON (0.02 mg ZEN and 0.07 mg DON, per kg dry matter, DM), FUS-50 (0.33 mg ZEN and 2.62 mg DON, per kg DM) and FUS-100 (0.66 mg ZEN and 5.24 mg DON, per kg DM). ZEN, DON and de-epoxy-DON (de-DON) were detected in FF. Based on the linear regression between toxin concentration in plasma and FF, it seems that about 50% (m = 0.5) of ZEN present in plasma is present in FF while an increase of 1 ng/ml DON or de-DON in plasma is paralleled by an increase of 1.5 ng/ml DON or 1.1 ng/ml de-DON in FF. ZEN, DON and their metabolites, except zearalenone (ZAN), were also detected in bile. Contrary to DON and de-DON, ZEN and its metabolites were accumulated in bile so that the concentration of ZEN and metabolites was much higher than for DON and de-DON. The main compound was ß-zearalenol (ß-ZEL). The biliary ZEN, α-zearalenol (α-ZEL) and ß-ZEL concentration correlated linearly with each other with an uncertainty of <15% (r(2) ≥ 0.86), whereas the ratio between ZEN: α-ZEL: ß-ZEL was about 1.5:1:11. With the help of established linear relationship between toxin intake and toxin concentration, bile could be used as diagnostic indicator to assess the exposure of cows.

Effect of increasing body condition on key regulators of fat metabolism in subcutaneous adipose tissue depot and circulation of nonlactating dairy cows.

In response to negative energy balance, overconditioned cows mobilize more body fat than thin cows and subsequently are prone to develop metabolic disorders. Changes in adipose tissue (AT) metabolism are barely investigated in overconditioned cows. Therefore, the objective was to investigate the effect of increasing body condition on key regulator proteins of fat metabolism in subcutaneous AT and circulation of dairy cows. Nonlactating, nonpregnant dairy cows (n=8) investigated in the current study served as a model to elucidate the changes in the course of overcondition independent from physiological changes related to gestation, parturition, and lactation. Cows were fed diets with increasing portions of concentrate during the first 6wk of the experiment until 60% were reached, which was maintained for 9wk. Biopsy samples from AT of the subcutaneous tailhead region were collected every 8wk, whereas blood was sampled monthly. Within the experimental period cows had an average BW gain of 243±33.3 kg. Leptin and insulin concentrations were increased until wk 12. Based on serum concentrations of glucose, insulin, and nonesterified fatty acids, the surrogate indices for insulin sensitivity were calculated. High-concentrate feeding led to decreased quantitative insulin sensitivity check index and homeostasis model assessment due to high insulin and glucose concentrations indicating decreased insulin sensitivity. Adiponectin, an adipokine-promoting insulin sensitivity, decreased in subcutaneous AT, but remained unchanged in the circulation. The high-concentrate diet affected key enzymes reflecting AT metabolism such as AMP-activated protein kinase and hormone-sensitive lipase, both represented as the proportion of the phosphorylated protein to total protein, as well as fatty acid synthase. The extent of phosphorylation of AMP-activated protein kinase and the protein expression of fatty acid synthase were inversely regulated throughout the experimental period, whereas the extent of phosphorylation of hormone-sensitive lipase was consistently decreasing by the high-concentrate diet. Overcondition in nonpregnant, nonlactating dairy cows changed the expression of key regulator proteins of AT metabolism and circulation accompanied by impaired insulin sensitivity, which might increase the risk for metabolic disorders.

Agonists of the G protein-coupled receptor 109A-mediated pathway promote antilipolysis by reducing serine residue 563 phosphorylation of hormone-sensitive lipase in bovine adipose tissue explants.

A balanced lipolytic regulation in adipose tissues based on fine-tuning of prolipolytic and antilipolytic pathways is of vital importance to maintain the metabolic health in dairy cows. Antilipolytic pathways, such as the G protein-coupled receptor 109A (GPR109A)-mediated pathway and the insulin signaling pathway in bovine adipose tissues may be involved in prohibiting excessive lipomobilization by reducing triglycerol hydrolysis. This study aimed to evaluate the in vitro antilipolytic potential of the mentioned pathways in bovine adipose tissue explants. Therefore, subcutaneous and retroperitoneal adipose tissue samples (approximately 100mg) of German Holstein cows were treated for 90 min ex vivo with nicotinic acid (2, 8, or 32 µM), nicotinamide (2, 8, or 32 µM), ß-hydroxybutyrate (0.2, 1, or 5mM), or insulin (12 mU/L), with a concurrent lipolytic challenge provoked with 1 µM isoproterenol. Lipolytic and antilipolytic responses of the adipose tissues were assessed by measuring free glycerol and nonesterified fatty acid release. To identify molecular components of the investigated antilipolytic pathways, protein abundance of GPR109A and the extent of hormone-sensitive lipase (HSL) phosphorylation at serine residue 563 were detected by Western blotting. Treatment with nicotinic acid or ß-hydroxybutyrate decreased the lipolytic response in adipose tissue explants and concurrently reduced the extent of HSL phosphorylation, but treatment with nicotinamide or insulin did not. Subcutaneous adipose tissue constitutively expressed more GPR109A protein, but no other depot-specific differences were observed. This study provides evidence that the GPR109A-mediated pathway is functionally existent in bovine adipose tissues, and confirms that HSL phosphorylation at serine residue 563 is also important in antilipolytic regulation in vitro. This antilipolytic pathway may be involved in a balanced lipid mobilization in the dairy cow.

Effects of dexamethasone-21-isonicotinate on peripheral insulin action in dairy cows 5 days after surgical correction of abomasal displacement.

BACKGROUND: Dexamethasone frequently is used for treatment of ketosis in dairy cows, but its effects are not fully understood. HYPOTHESIS: Dexamethasone treatment affects whole body insulin sensitivity. ANIMALS: Twelve German Holstein cows, 2-4 weeks postpartum, 5 days after omentopexy to correct left abomasal displacement. METHODS: Randomized, blinded, case-control study. Treatment with dexamethasone-21-isonicotinate (DG; 40 µg/kg IM; n = 6) or saline (control group [CG], 15 mL IM, n = 6) on day 0 (d0). Blood samples were obtained before (d0) and after treatment (d1 and d2), and analyzed for glucose, insulin, and nonesterified fatty acid (NEFA) concentrations. Hepatic triglycerides (TAG) were measured in liver samples taken on d0 and d2. Five consecutive hyperinsulinemic-euglycemic clamps (HEC-I-V; insulin dosages: 0.1, 0.5, 2, 5, 10 mU/kg/min, respectively) were performed on d1 and steady state glucose infusion rate (SSGIR), insulin concentration (SSIC), insulin sensitivity index (ISI = SSGIR/SSIC), and plasma NEFA concentration (SSNEFA) were assessed. RESULTS: Compared with CG-cows, DG-cows on d1 had higher plasma glucose (P = .004) and insulin (P < .001) concentrations, decreased SSGIR (HEC-II, P = .002; HEC-IV, P = .033), ISI (HEC-I, P < .015; HEC-II, P = .004), and insulin-stimulated decrease in SSNEFA (HEC-II, P = .006; HEC-III, P = .01; HEC-IV, P = .003; HEC-V, P = .011). Decrease in hepatic TAG content in DG-cows was higher compared with CG-cows (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Dexamethasone decreases whole body insulin sensitivity and affects glucose and lipid metabolism in early lactating dairy cows.

Lipolysis in early lactation is associated with an increase in phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)α1 in adipose tissue of dairy cows.

Adenosine monophosphate-activated protein kinase (AMPK)α1 is activated in the context of triacylglycerol hydrolysis in adipose tissue in monogastric animals. This study describes AMPKα1 protein expression and the occurrence of its phosphorylated form (pAMPKα1) in different adipose tissue depots as influenced by time and postpartum diet in dairy cows. Biopsy samples were obtained from subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissues of 20 Holstein cows 21 d prepartum (ap) and 1 and 21 d postpartum (pp). After d 1 pp, cows were randomly assigned to 2 groups (n=10) and fed different amounts of concentrate until the third biopsy sampling at 21 d pp. Protein expression of AMPK and the extent of its phosphorylation in adipose tissue were measured by semiquantitative Western blotting. Results were not influenced by postpartum feeding. Therefore, both groups were pooled and data analyzed together. Expression of AMPKα1 in SCAT showed a decrease over time, resulting in lower expression at 1d pp compared with 21 d ap. Expression in RPAT was maintained over time. Phosphorylation increased in SCAT, showing a greater extent of phosphorylation at d 21 pp compared with 21 d ap. In RPAT, this could be seen as a trend. The proportion of pAMPKα1 to AMPKα1 significantly increased over time in both tissues. In the first adipose tissue sampling (21 d ap), AMPKα1 protein expression and extent of phosphorylation were significantly higher in RPAT than in SCAT. Lipolysis in early lactation of dairy cows was associated with an increase in phosphorylation of AMPKα1 and ratio of pAMPKα1 to AMPKα1 in bovine adipose tissues. This indicates that AMPKα1 may be involved in the regulation of energy metabolism of bovine adipose tissues.

Hormone-sensitive lipase protein expression and extent of phosphorylation in subcutaneous and retroperitoneal adipose tissues in the periparturient dairy cow.

Lipomobilization is essential for dairy cows to balance the energy requirement for milk production in early lactation. This study aimed to determine the role of hormone-sensitive lipase (HSL) and its activation by phosphorylation at Ser 660 (HSLp660) and 563 (HSLp563) in different adipose tissue depots as influenced by time and postpartum diet in dairy cows. Biopsy samples were obtained from s.c. (SCAT) and retroperitoneal (RPAT) adipose tissues of 20 Holstein cows 21 d prepartum, and 1 and 21 d postpartum. After d 1 postpartum, cows were randomly assigned to 2 groups (n=10). Groups received diets with either a concentrate-to-roughage ratio on a dry matter basis of 30:70% (low-concentrate, LC, group) or 60:40% (high-concentrate group), fed until the third biopsy sampling 21 d postpartum. Dry matter intake, milk yield, and milk composition were recorded. Blood samples were taken weekly, starting 21 d prepartum and analyzed for nonesterified fatty acids, ß-hydroxybutyrate (BHBA), glucose, and insulin. Protein expression of HSL and its extent of phosphorylation in adipose tissue were measured by semiquantitative Western blotting. Total HSL expression was lower in both adipose tissues 1 d after calving compared with prepartum sampling (SCAT: 4.10±0.5 vs. 2.4±0.3; RPAT: 11.1±1.3 vs. 6.6±1.1). Phosphorylation at Ser 660 was higher 21 d postpartum compared with 21 d prepartum in RPAT (2.9±0.3 vs. 4.6±0.6). Phosphorylation at Ser 563 was higher 21 d postpartum than 21 d prepartum in SCAT (0.6±0.1 vs. 3.9±1.1), and in RPAT a difference was observed between 21 d prepartum and 1 d postpartum (1.0±0.1 vs. 3.3. ± 0.6). On d 21 postpartum, the LC group showed a lower extent of Ser 563 phosphorylation in RPAT (3.9±0.8 vs.10.0±1.9) and a higher concentration of serum BHBA (0.77±0.05 vs. 0.47±0.11) than did the high-concentrate group. An inhibitory influence of higher BHBA concentrations on HSL phosphorylation in the LC group could be a possible explanation. On comparing RPAT to SCAT, HSL expression and the extent of Ser 660 and 563 phosphorylation was higher in RPAT at 21 d prepartum (HSL: 4.1±0.5 vs. 11.1±1.2; HSLp660 1.3±0.2 vs. 2.9±0.3; HSLp563: 0.6±0.1 vs. 1.0±0.1). In conclusion, the postpartum feeding regimen influenced the phosphorylation pattern, especially in RPAT, implying a regulatory role for different phosphorylation sites in adaptive lipolysis of dairy cows. It is suggested that RPAT is more sensitive to periparturient challenges than is SCAT.

Mutations in the C-terminal region of the HIV-1 reverse transcriptase and their correlation with drug resistance associated mutations and antiviral treatment.

OBJECTIVE: replication of HIV-1 after cell entry is essentially dependent on the reverse transcriptase (RT). Antiretroviral drugs impairing the function of the RT currently aim at the polymerase subunit. One reason for failure of antiretroviral treatment is the evolvement of resistance-associated mutations in the viral genome. For RT inhibitors, almost all identified mutations are located within the polymerase; therefore, general genotyping confines to investigate this subunit. Recently several studies have shown that substitutions within the RNase H and the connection domain increase antiviral drug-resistance in vitro, and some of them are present in patient isolates. AIM: the aim of the present study was to investigate the prevalence of these substitutions and their association with mutations in the polymerase domain arising during antiretroviral treatment. MATERIAL AND METHODS: we performed genotypic analyzes on seventy-four virus isolates derived from treated and untreated patients, followed at the HIV Centre of the Johann Wolfgang Goethe University Hospital (Frankfurt/Main, Germany). We subsequently ana?lysed the different substitutions in the c-terminal region to evaluate whether there were associations with each other, n-terminal substitutions or with antiretroviral treatment. RESULTS: We identified several primer grip substitutions, but almost all of them were located in the connection domain. This is consistent with other in-vivo studies, in which especially the primer grip residues located in the RNase H were unvaried. Furthermore, we identified other substitutions in the connection domain and in the RNase H. Especially E399D seemed to be associated with an antiretroviral treatment and N-terminal resistance-delivering mutations. CONCLUSION: some of the identified substitutions were associated with antiviral treatment and drug resistance-associated mutations. Due to the low prevalence of C-terminal mutations and as only a few of them could be associated with antiviral treatment and N-terminal resistance-delivering mutations, we would not recommend routinely testing of the C-terminal RT region.

Intensification of a failing regimen with zidovudine may cause sustained virologic suppression in the presence of resensitising mutations including K65R.

OBJECTIVES: The reverse transcriptase (RT)-mutation K65R limits further therapeutic options and has been selected by unfavorable RT-combinations, e.g. tenofovir in combination with abacavir and/or didanosine. METHODS: We identified HIV-1 infected patients from a large treatment cohort who experienced virological failure (HIV-1 RNA >1000 copies/mL) with evidence of resistance mutations including the K65R, but without thymidine analogue mutations (TAMs) in genotypic resistance assay. Phenotype was performed from previously collected frozen plasma. The patients were followed for clinical and resistance outcome after treatment intensification with only zidovudine. RESULTS: Five patients had experienced antiretroviral treatment failure on various nucleoside analogue combinations, containing abacavir, didanosine, lamivudine, nevirapine, reverset and/or tenofovir. RT-sequence revealed mutations at position K65R in combination with other non-TAMs. The patients' median viral load prior to zidovudine intensification was 3.551 Log10 (range 3.053-4.681) and despite evidence for resistance to the failing drug regimen, all responded within 4 weeks to undetectable levels (<1.699 Log10 or <50 copies/mL) and remained virologically suppressed during follow-up (20 months through 6.5 years). CONCLUSIONS: In virologically failing patients due to K65R- and other non-thymidine-mutations, simple regimen intensification with zidovudine resulted in sustained HIV-1 suppression. The finding of re-sensitized HIV-1 in patients may be clinically relevant.

Diagnostic accuracy of d-dimer and other peritoneal fluid analysis measurements in dairy cows with peritonitis.

BACKGROUND: Peritoneal fluid analysis in cattle traditionally includes the classic parameters despite the fact that they have only moderate diagnostic accuracy and often fail to identify the pathogenesis or etiological factors. Therefore additional parameters recently have been established to improve diagnostic precision. In a recent study, reference ranges for several of these parameters have been proposed in dairy cows. HYPOTHESIS/OBJECTIVES: The aim of this observational study was to assess the diagnostic value of D-Dimer and other measurements of peritoneal fluid analysis in dairy cows with peritonitis. ANIMALS: The study included 110 Holstein-Friesian cows grouped into cows with peritonitis (n = 47) and cows without peritonitis (n = 63). METHODS: Peritoneal fluid was obtained by abdominocentesis. Total protein, albumin, glucose, cholesterol, fibrinogen, l-lactate, D-Dimer, lactate dehydrogenase (LDH), alkaline phosphatase, creatine phosphokinase, white blood cell, and red blood cell were determined in peritoneal fluid and venous blood. Serum-ascites albumin gradient (SAAG) and ratios of peritoneal fluid-venous blood were calculated. Sensitivity (SN) and specificity (SP) were calculated and receiver operating characteristic curve analysis performed. RESULTS: Peritoneal fluid D-Dimer was most accurate in diagnosing peritonitis in cows (SN and SP>95.0%). Total protein concentration, LDH and LDH ratio, and SAAG had sensitivities between 49.0 and 67.1%, and specificities between 88.4 and 95.5%. A low-peritoneal fluid glucose concentration was found to be highly indicative of septic peritonitis. CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of the recently introduced parameters may increase the diagnostic value of peritoneal fluid analysis and provide additional specific information. Therefore these measurements should be included in the routine procedure.

Biochemical constituents of peritoneal fluid in cows.

Samples of peritoneal fluid and jugular venous blood were taken simultaneously from 95 clinically healthy Holstein-Friesian cows. The concentrations of total protein, albumin, glucose, cholesterol, fibrinogen, L-lactate and D-dimer, the activities of lactate dehydrogenase (LDH), alkaline phosphatase and creatine kinase, and the white blood cell count were determined in the samples. Light's criteria, the serum-ascites albumin gradient (SAAG) and the ratios of the concentration of each parameter in peritoneal fluid to its concentration in blood were calculated. The mean concentrations of total protein, albumin and D-dimer, the activity of LDH and the SAAG were different from the reference values for monogastric animals and human beings.

First-line ritonavir/indinavir 100/800 mg twice daily plus nucleoside reverse transcriptase inhibitors in a German multicentre study: 48-week results.

OBJECTIVES: To evaluate safety and efficacy of the protease inhibitor combination ritonavir/indinavir 100/800 mg twice daily plus 2-3 nucleoside reverse transcriptase inhibitors (NRTI) in antiretroviral-naive patients. METHODS: Within this open-label, uncontrolled multicentre trial, antiretroviral-naive patients (n = 57) with median baseline HIV-RNA of 308,000 copies/mL (range 170-3.01 million copies/mL) and median CD4 cell count of 50 cells/microL (range 0-853 cells/microL) were started on 2-3 NRTIs plus ritonavir/indinavir 100/800 mg twice daily. CD4 cell counts and HIV-RNA were determined at weeks 0, 4, 8, 12, 16, 20, 24 and 48. Statistical analysis was performed on treatment as well as intent-to-treat. RESULTS: Viral load decreased by a median of 3.79 log10 copies/mL (range 2.0-4.60 log10 copies/mL) until week 48. At week 48, 23/57 (40%, intent-to-treat) patients showed a viral load