Distinct responses in feed sorting, chewing behavior, and ruminal acidosis risk between primiparous and multiparous Simmental cows fed diets differing in forage and starch levels.

During early lactation, both primiparous (PP) and multiparous (MP) cows are commonly fed diets rich in starch and low in forages to support their high energy requirements. Yet, the PP cows experience this dietary challenge for the first time, which might result in higher odds for them to develop rumen and systemic health disorders. The primary objective of this study was to evaluate the effect of decreasing the amount of forages in the diet on chewing and sorting behaviors and rumen and systemic health variables in PP and MP dairy cows. Twenty-four lactating Simmental cows [8 PP, average dry matter intake (DMI) of 19.1 ± 1.1 kg/d; 16 MP, average DMI of 22.5 ± 1.1 kg/d] with a body weight of 737 ± 90 kg and 50 ± 22 days in milk were used in this study. Cows were first fed a total mixed ration with 60% forage and 40% concentrate [on a dry matter (DM) basis] considered marginal in forages for 2 wk. Then, cows were switched to a diet low in forages with 40% forage and 60% concentrate (on a DM basis) for 4 wk. Reticular pH was measured continuously with wireless pH-sensors inserted into the reticulum to calculate the subacute ruminal acidosis (SARA) index. Chewing activity was measured with noseband-sensor halters, and feed sorting was measured weekly. Blood samples were collected weekly and analyzed for metabolic and inflammation markers. Switching PP and MP cows from a marginal to low-forage diet decreased the time spent eating and ruminating per kilogram of DM. Primiparous cows chewed longer per kilogram of DMI than MP cows. Also, the PP cows sorted more pronounced for longer particles and against fine particles than MP cows did. Despite higher rumination activity per kilogram of DMI and the adaptive sorting behavior, the PP cows spent on average 4.6 h/d longer below a pH of 5.8 and had a higher SARA index (i.e., area pH <5.8/DMI) than MP cows, especially during the first week of the low-forage diet (9.5 vs. 4.8). The concentration of liver enzymes increased with the low-forage diet, which was especially pronounced in the PP cows. In conclusion, this study demonstrated greater susceptibility of PP cows to SARA and liver damage than MP cows fed the same diets. Although PP cows demonstrated greater chewing and ruminating activity per kilogram of DMI, as well as adapted sorting behavior in favor of large particles during the low-forage high-starch feeding, they developed more severe signs of SARA. This suggests higher forage fiber requirements for PP cows and the need for improved feeding strategies to mitigate rumen fermentation disorders during early lactation in these cows.

Feed-forward neural networks using cerebral MR spectroscopy and DTI might predict neurodevelopmental outcome in preterm neonates.

OBJECTIVES: We aimed to evaluate the ability of feed-forward neural networks (fNNs) to predict the neurodevelopmental outcome (NDO) of very preterm neonates (VPIs) at 12 months corrected age by using biomarkers of cerebral MR proton spectroscopy (1H-MRS) and diffusion tensor imaging (DTI) at term-equivalent age (TEA). METHODS: In this prospective study, 300 VPIs born before 32 gestational weeks received an MRI scan at TEA between September 2013 and December 2017. Due to missing or poor-quality spectroscopy data and missing neurodevelopmental tests, 173 VPIs were excluded. Data sets consisting of 103 and 115 VPIs were considered for prediction of motor and cognitive developmental delay, respectively. Five metabolite ratios and two DTI characteristics in six different areas of the brain were evaluated. A feature selection algorithm was developed for receiving a subset of characteristics prevalent for the VPIs with a developmental delay. Finally, the predictors were constructed employing multiple fNNs and fourfold cross-validation. RESULTS: By employing the constructed fNN predictors, we were able to predict cognitive delays of VPIs with 85.7% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 99.1% negative predictive value (NPV). For the prediction of motor delay, we achieved a sensitivity of 76.9%, a specificity of 98.9%, a PPV of 90.9% and an NPV of 96.7%. CONCLUSION: FNNs might be able to predict motor and cognitive development of VPIs at 12 months corrected age when employing biomarkers of cerebral 1H-MRS and DTI quantified at TEA. KEY POINTS: • A feed-forward neuronal network is a promising tool for outcome prediction in premature infants. • Cerebral proton magnetic resonance spectroscopy and diffusion tensor imaging can be used for the construction of early prognostic biomarkers. • Premature infants that would most benefit from early intervention services can be spotted at the time of optimal neuroplasticity.

Supplementation of a clay mineral-based product modulates plasma metabolomic profile and liver enzymes in cattle fed grain-rich diets.

Grain-rich diets often lead to subacute ruminal acidosis (SARA) impairing rumen and systemic cattle health. Recent data suggest beneficial effects of a clay mineral (CM)- based product on the rumen microbiome of cattle during SARA. This study sought to investigate whether the CM supplementation can counteract SARA-induced perturbations of the bovine systemic health. The study used an intermittent diet-induced SARA-model with eight dry Holstein cows receiving either no additive as control or CM via concentrates (n=8 per treatment). Cows received first a forage diet (Baseline) for 1 week, followed by a 1-week SARA-challenge (SARA 1), a 1-week recovery phase (Recovery) and finally a second SARA-challenge for 2 weeks (SARA 2). Cows were monitored for feed intake, reticular pH and chewing behavior. Blood samples were taken and analyzed for metabolites related to glucose and lipid metabolism as well as liver health biomarkers. In addition, a targeted electrospray ionization-liquid chromatography-MS-based metabolomics approach was carried out on the plasma samples obtained at the end of the Baseline and SARA 1 phase. Data showed that supplementing the cows' diet with CM improved ruminating chews per regurgitated bolus by 16% in SARA 1 (P=0.01) and enhanced the dry matter intake during the Recovery phase (P=0.05). Moreover, the SARA-induced decreases in several amino acids and phosphatidylcholines were less pronounced in cows receiving CM (P≤0.10). The CM-supplemented cows also had lower concentrations of lactate (P=0.03) and biogenic amines such as histamine and spermine (P<0.01) in the blood. In contrast, the concentration of acylcarnitines with key metabolic functions was increased in the blood of treated cows (P≤0.05). In SARA 2, the CM-cows had lower concentrations of the liver enzymes aspartate aminotransferase and γ-glutamyltransferase (P<0.05). In conclusion, the data suggest that supplementation of CM holds the potential to alleviate the negative effects of high-grain feeding in cattle by counteracting multiple SARA-induced perturbations in the systemic metabolism and liver health.

Supplementing phytogenic compounds or autolyzed yeast modulates ruminal biogenic amines and plasma metabolome in dry cows experiencing subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) causes ruminal dysbiosis, thereby increasing the risk of systemic metabolic disorders in cattle. We recently showed that supplementation with phytogenic compounds (PHY) or autolyzed yeast (AY) counteracted negative effects of SARA by improving ruminal pH and microbiome. This study investigated the effects of an intermittent SARA challenge on the ruminal concentration of biogenic amines (BA) and lipopolysaccharides (LPS), as well as on the blood metabolome. We also evaluated effects of PHY and AY on the latter variables. Eight rumen-cannulated nonlactating Holstein cows were arranged in an incomplete 4 × 3 Latin square design with 4 experimental runs and 3 treatment groups. During each run, cows were switched from an all-forage diet (baseline) to an intermittent concentrate-challenge diet with a forage:concentrate ratio of 35:65 (dry matter basis) to induce SARA for 1 (SARA1) or 2 (SARA2) wk, separated by 1 wk of forage-only feeding. The 3 treatment groups were no additive as control, PHY, or AY. During baseline, SARA1 and SARA2 rumen fluid samples were collected for analysis of BA and LPS. Blood samples were taken during baseline and SARA1 for a targeted metabolomics approach. High-concentrate feeding caused a 9-fold increase in ruminal LPS during SARA1 and an 11-fold increase in SARA2 compared with the baseline. Elevated concentrations of ruminal BA were found during both SARA periods, with histamine showing the strongest increase during SARA1. Moreover, a decrease in phosphatidylcholines, lysophosphatidylcholines, sphingomyelines, and several AA in the blood during SARA1 were detected. Supplementation of PHY decreased concentrations of LPS (-43%), histamine (-66%), pyrrolidine (-38%), and spermine (-54%) in SARA1 and cadaverine in SARA2 (-50%). Moreover, cows that received PHY had higher concentrations of cholesterol (+26%), several AA, and phosphatidylcholines in SARA1 compared with control cows. For AY, decreases in ruminal ethanolamine (-21%), methylamine (-52%), histamine (-54%), spermidine (-44%), and spermine (-80%) in SARA1 were observed, whereas in the blood an increase in tryptophan was noticed. In conclusion, the SARA was associated with markedly increased concentrations of LPS and BA in the rumen fluid and undesirable shifts in the plasma metabolome. Supplementation of PHY and AY counteracted some of these changes and therefore may help in attenuating negative effects of high-concentrate feeding in dairy cattle.

High-grain diets supplemented with phytogenic compounds or autolyzed yeast modulate ruminal bacterial community and fermentation in dry cows.

The feeding of concentrate-rich diets may lead to microbial imbalances and dysfermentation in the rumen. The main objective of this study was to determine the effects of supplementing phytogenic compounds (PHY) or autolyzed yeast (AY) on rumen fermentation and microbial abundance in cows intermittently fed concentrate-rich diets. The experiment was carried out as an incomplete 3 × 4 Latin square design, with 8 nonlactating rumen-fistulated Holstein-Friesian cows. The cows were randomly assigned to a concentrate diet that was either not supplemented (CON), or supplemented with PHY or AY. Each of the 4 consecutive experimental periods was composed of a 1-wk roughage-only diet (RD), 6-d gradual concentrate increase, followed by 1 wk of 65% concentrate (dry matter basis; Conc I), and 1 wk of RD and a final 2-wk 65% concentrate (dry matter basis; Conc II) phase. Digesta samples were collected from the rumen mat for bacterial 16S rRNA gene Illumina MiSeq (Illumina, Balgach, Switzerland) sequencing, and samples of particle-associated rumen liquid were obtained for measuring short-chain fatty acids, lactate, ammonia, and pH during RD (d 6), Conc I (d 19), and Conc II (d 39). The concentrate feeding caused a decrease of overall bacterial diversity indices, especially during Conc I. The genera Ruminococcus, Butyrivibrio, and Coprococcus were decreased, whereas Prevotella, Megasphaera, Lachnospira, and Bacteroides were increased in abundance. Supplementation of both feed additives increased the abundance of gram-positive and decreased that of gram-negative bacteria. Supplementation of AY enhanced cellulolytic bacteria such as Ruminococcus spp., whereas PHY decreased starch and sugar fermenters including Bacteroides spp., Shuttleworthia spp., and Syntrophococcus spp. Moreover, PHY supplementation increased butyrate percentage in the rumen in both concentrate phases. In conclusion, intermittent high-concentrate feeding altered the digesta-associated rumen bacterial community and rumen fermentation with more significant alterations found in Conc I than in Conc II. The data also showed that both feed additives had the most significant modulatory effects on the bacterial community, and their subsequent fermentation, during periods of low pH.

Signals for identifying cows at risk of subacute ruminal acidosis in dairy veterinary practice.

Controlling rumen disorders is critical to ensure successful dairy herd health management. Lactation diets of dairy cows are commonly rich in concentrates and low in physically effective fibre. Feeding of these diets increases the risk of rumen disorders with far-reaching consequences for cattle health, welfare and sustainability of dairy production. The term subacute ruminal acidosis or SARA is often used as a synonym for poor rumen health. Being subclinical, SARA lacks of clear symptoms and is therefore difficult to diagnose and to control in the practice. This review article summarises common and identifies new direct and indirect cow signals related to SARA. We have performed a scientific evaluation and interpretation of each of these cow signals by highlighting their advantages and disadvantages from the practitioner's point of view. The gold standard of SARA cow signals still remains direct measurement of ruminal pH. However, continuous pH monitoring is cost-intensive and often biased by sensor drift. Single-point ruminal pH measurements by oral stomach tubing or rumenocentesis have strong limitations. Therefore, there is a need for reliable and robust markers of SARA that are easily accessible and inexpensive. Such indirect parameters are the observation of chewing and feeding activities, as well as the monitoring of milk, faecal, urine and blood variables. Also, novel technologies that allow rapid and non-invasive measurement of the rumen mucosa thickness and ruminal motility patterns might provide advantages in SARA diagnosis. Due to several constraints of these indirect diagnostic tools, such as limited specificity and sensitivity, we strongly recommend using a combination of the signals to reliably identify cows at risk of SARA in a dairy herd.

Technical note: Changes in rumen mucosa thickness measured by transabdominal ultrasound as a noninvasive method to diagnose subacute rumen acidosis in dairy cows.

Feeding high-grain diets leads to the release and accumulation of short-chain fatty acids in the rumen. The subsequent prolonged decline in ruminal pH can lead to subacute ruminal acidosis (SARA). Accumulation of short-chain fatty acids can cause proliferation of rumen papillae to increase absorption surface, subsequently leading to a thickening of the rumen mucosa. The aim of this study was to evaluate the appropriateness of continuous measurements of the rumen mucosa thickness (RMT) as a diagnostic tool for SARA in dairy cows compared with continuous measurements of ruminal pH. The study used 6 lactating Simmental cows switched from a moderate-grain (MG) diet with 40% concentrate (dry matter basis) for 1 wk to a high-grain (HG) diet with 60% concentrate (dry matter basis) for 4 wk. Reticuloruminal pH was recorded with indwelling sensors throughout the trial. Rumen mucosa thickness was measured by transabdominal ultrasound at 4 d during the MG diet and 23 d during the HG diet. Mean RMT increased from 4.7 ± 0.19 mm in the MG diet to 5.3 ± 0.17 mm in the HG diet, whereas daily mean reticular pH decreased from 6.8 ± 0.01 in the MG diet to 6.5 ± 0.01 in the HG diet. Older cows (>3 lactations) had increased RMT, associated with higher reticular pH throughout the experiment. The higher RMT and pH level in older cows underlines their lesser susceptibility to SARA during high-grain feeding. In conclusion, RMT can successfully be measured using linear ultrasound probes, commonly used by veterinary practitioners as rectal probes. By combining noninvasive RMT measurements with the lactation number of the individual cows in a herd, this study suggests that RMT is a viable option for diagnosing SARA. Further research, using a larger number of cows with different lactations numbers, is needed to establish a cut-off RMT indicating the risk of SARA.

Differences between pH of indwelling sensors and the pH of fluid and solid phase in the rumen of dairy cows fed varying concentrate levels.

Feeding of high-concentrate diets to cattle increases the risk of subacute ruminal acidosis (SARA). Indwelling wireless pH sensors have become popular as an early diagnostic tool for SARA recently. However, the recommended pH thresholds of SARA derive from measurements taken from free-rumen liquid (FRL) in the ventral rumen, and not from the reticulum, where the mentioned sensors are located. The aim of this study was to evaluate commercially available indwelling pH boli for the accuracy and precision in predicting ruminal pH of FRL and the particle-associated rumen liquid (PARL) under varying dietary concentrate levels. An additional aim was to define SARA pH thresholds of indwelled pH boli, which can be used for SARA diagnostics. The experiment was conducted with eight nonlactating rumen cannulated Holstein cows fed 0% or 65% concentrate. Data showed that the mean pH of indwelled boli was consistently higher than in FRL and PARL (pH 6.62 ± 0.02, 6.43 ± 0.02 and 6.18 ±0.02, respectively) across feeding regimens. Interestingly, the diurnal differences in pH among indwelled boli, FRL and PARL became greater during concentrate feeding, especially at 8 h after the morning feeding, suggesting that with high-concentrate diets a particular adjustment of reticular sensor pH vs. ruminal pH in FRL and PARL is needed. The concordance correlation coefficient analysis, representing the reproducibility of the bolus measurements, was high for bolus-FRL (0.733) and moderate for bolus-PARL (0.510) associations. Furthermore, the quantitative relationship of the pH in FRL and PARL to the pH of the boli was described by linear regression analysis. The study determined that the common SARA threshold of pH 5.8 in FRL corresponds to a bolus pH of 6.0.

Modulation of chewing behavior and reticular pH in nonlactating cows challenged with concentrate-rich diets supplemented with phytogenic compounds and autolyzed yeast.

Feeding of concentrate-rich diets impairs chewing behavior and leads to rumen acidosis in cattle. Because of their modulatory effects on ruminal fermentation, phytogenic compounds (PHY) and autolyzed yeast derivatives (AY) may alleviate the negative consequences of high-concentrate diets. Therefore, this research investigated if chewing behavior and the reticular pH dynamics are modulated by AY and PHY supplementation during repeated concentrate-rich challenges used to simulate intermittent rumen acidotic insults. Eight rumen-cannulated, dry, and nonpregnant Holstein cows were assigned to an incomplete double 4 × 3 Latin square design with 3 treatments and 4 experimental runs (n = 8/treatment). Cows were fed concentrates either not supplemented (CON) or supplemented with PHY or AY. Initially, cows were fed a pure forage diet (FD) and switched to a 65% concentrate diet on DM basis for 1 (CONC 1) and 2 (CONC 2) wk. Between CONC 1 and CONC 2, the cows were fed the FD for 1 wk. Chewing activity was measured using noseband sensors and reticular pH by wireless pH sensors. Data showed that cows spent less time ruminating in CONC 1 than in CONC 2. In agreement, reticular pH drop was more pronounced during CONC 1 than during CONC 2. Cows fed with PHY spent 4 h less with reticular pH <6.0 during CONC 1 and 3 h less with pH <6.0 h in CONC 2 as compared with CON cows. Similarly, PHY supplementation extended rumination time with 88 min/d compared with CON cows during CONC 1. The AY supplementation increased DMI by 20% resulting in a longer eating time compared with CON diet during CONC 1. Enhancement of ruminating by PHY and eating time by AY supplementation resulted in longer total chewing time for PHY (474 min/d) and AY (466 min/d) as compared with CON (356 min/d) in CONC 1. In conclusion, cows experiencing 2 intermittent concentrate-rich challenges increased their ruminating behavior during the second challenge, and this effect was associated with higher reticular pH readings. The PHY supplementation enhanced rumination as well as reticular pH during CONC 1. However, the enhanced pH of cows fed with PHY during CONC 2 was not related to greater rumination, suggesting that influencing factors beyond rumination seemed to play a role in modulating reticular pH in PHY cows during CONC 2. The AY supplementation increased DMI without depressing rumination or reticular pH. Effects of both feed additives were more pronounced during CONC 1 challenge when reticular pH was lower.

High diversity of viable bacteria isolated from lymph nodes of slaughter pigs and its possible impacts for food safety.

AIMS: Ileocaecal lymph nodes (ICLNs) of pigs, the key immune inductive site for bacterial systemic invasion, were examined in this study with emphasis on viable and cultivable bacteria. METHODS AND RESULTS: Asymptomatic and pathologically altered ICLNs of slaughter pigs (n = 16) with hyperplasia, purulence or granulomatous formations were aerobically and anaerobically cultivated. In total, 209 isolates were collected and the near full-length 16S rRNA gene from each isolate was sequenced. Taxonomic classification revealed that 68% of the isolates belonged to Proteobacteria, 27% to Firmicutes and 5% to Actinobacteria. Purulent and granulomatous ICLNs generally tended to contain more Proteobacteria than asymptomatic and enlarged ICLNs (P = 0·061). The isolates could be assigned to 25 species belonging to 17 genera including Escherichia, Carnobacterium, Lactobacillus, Staphylococcus and Acinetobacter. Furthermore, pathogens such as Streptococcus suis and Salmonella enterica were detected. The most abundant isolate (57%) was most similar (>99%) to Escherichia coli. Random amplified polymorphic DNA (RAPD) profiling revealed a high genetic diversity among E. coli isolates and 24% of these isolates were positive for at least one gene associated with enterohemorrhagic disease (eae, fliC, stx1 or hlyA). Compared with a recently published DNA-based high-throughput sequencing data set including the same ICLNs, 4% of species detected were cultivable. CONCLUSIONS: The presence of viable, commensal and pathogenic bacterial phylotypes could be proven in ICLNs with Proteobacteria being dominant. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that broadly characterizes viable bacteria from ICLNs of pigs. The presence of bacteria in lymph nodes of farm animals has practical relevance for host colonization and possible chronic infection. It is also of great interest for basic research investigating translocation of bacteria from the gastrointestinal tract to ICLNs.

Amplitude-integrated electroencephalography shows mild delays in electrocortical activity in preterm infants born small for gestational age.

AIM: Being born small for gestational age (SGA) seems to be a relevant risk factor for long-term neurologic deficits. We compared the differences between amplitude-integrated electroencephalography (aEEG) signals in very preterm infants born small for gestational age (SGA) and those in age-matched infants born appropriate size for gestational age (AGA). METHODS: We performed serial aEEG recording on 305 infants: 255 (83.6%) were AGA, and 50 (16.3%) were SGA. RESULTS: The number of bursts per hour decreased over time in both groups, but was higher in the SGA group at every time point. On day one, it was significantly higher in the SGA group (17.4) than in the AGA group (10.1) (p = 0.016). The total Burdjalov score increased with post-natal age and tended to be lower in SGA infants, but did not reach statistical significance at any time point. The percentage of continuous background patterns increased with post-natal age in both groups, with no significant difference between the groups. CONCLUSION: Very preterm infants born SGA showed normal maturation of aEEG signals during post-natal life, but they also showed mild delays in electrocortical activity compared to age-matched AGA infants. The predictive value of these findings on neurodevelopmental outcome needs to be further evaluated.

Neuroprotective effects of the sigma-1 receptor ligand PRE-084 against excitotoxic perinatal brain injury in newborn mice.

Excessive glutamate release followed by N-methyl-D-aspartate receptor (NMDAR) activation plays a crucial role in perinatal brain injury. We have previously shown that dextromethorphan, a low-affinity NMDAR antagonist with anti-inflammatory properties, is neuroprotective against neonatal excitotoxic brain injury. Of interest, dextromethorphan is also a sigma-1 receptor (σ1R) agonist. The pharmacologic class of σ1R agonists has yielded propitious results in various animal models of adult central nervous system pathology. In an established neonatal mouse model of excitotoxic brain injury, we evaluated the effect of the selective σ1R agonist 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate (PRE-084). A single intraperitoneal injection of 0.1 µg/g (low dose) or 10 µg/g (high dose) bodyweight (bw) PRE-084, given 1h after the excitotoxic insult, significantly reduced lesion size in cortical gray matter 24 h and 120 h after the insult. Repetitive injections of 0.1 µg/g PRE-084 proved to be equally effective. PRE-084 treatment resulted in a decrease in cell death indicated by reduced TUNEL positivity and caspase-3 activation. Furthermore, it lowered the number of isolectin B4-positive, activated microglial cells. PRE-084 had no effect on developmental apoptosis in the undamaged brain. In vitro findings in primary hippocampal neurons suggest that PRE-084 treatment provides partial protection against glutamate induced morphological and functional changes. For excitotoxicity as playing a crucial role in the pathogenesis of perinatal brain injury, we demonstrate for the first time that systemic treatment with the highly selective σ1R agonist PRE-084 protects against NMDAR-mediated excitotoxic brain damage.

Feasibility of cerebral MRI in non-sedated preterm-born infants at term-equivalent age: report of a single centre.

AIM: MRI is gaining in importance as an imaging tool for brain development and injury in preterm infants. The aim of this study was to evaluate the feasibility of performing MRI in non-sedated preterm-born infants at term-equivalent age (TEA). METHODS: A total of 89 infants born before 32 gestational weeks were recruited. Infants were scanned without sedation. Duration of the entire examination including scan repetition and interruptions was registered. RESULTS: Of the 89 infants, 56 (63%) underwent MRI at TEA. Out-patients required a significantly shorter total MR examination time than did in-patients (32 ± 12 vs. 54 ± 10 min, p < 0.01). Of the 56 infants, 39 (69.6%) were examined without interruption. Only four (7.2%) of the 56 scans were unusable because of motion artefacts. Mean duration of all scans was 36 ± 14 min. In cases with no interruptions, sessions were completed within 32 ± 12 min; MR sessions with interruption lasted 45 ± 13 min. CONCLUSION: A well-trained team is indispensable in obtaining best-quality images as a prerequisite for good counselling. From our experience, we worked out a guideline to ensure that scans in stable non-sedated preterm-born infants at TEA run smoothly and provide high-quality images.

[Patients' perception on attention received from Health Centres non-sanitary staff]. / Percepción de los pacientes sobre la atención recibida por el personal no sanitario en los centros de salud.

AIMS: To know patients' perceptions about relational aspects and technical procedures when they are attended by the administrative staff in Health Centres. To assess the utility of two ways for measuring satisfaction. MATERIAL AND METHODS: Cross-sectional study carried out in people attending the administrative sections of Health Centres for diverse reasons. Just after the interaction with the administrative they were interviewed using two different questions for assessing their opinions and satisfaction with communicational and technical aspects related with their demands. Descriptive analysis. Significant differences among mean was explored by χ(2) test. Open-ended questions were grouped in categories in a process involving three researchers independently. RESULTS: Over than 90% (360) of the attendees declared to be satisfied or very satisfied with the service received from the staff personal. Nevertheless, among 18-36% gave suggestions for improving the service after their consultation. Independently the domain explored, people suggested the communicational, personal capability, quality and quantity of explanations and waiting time as the main aspects to be improved. CONCLUSIONS: Surveys with open-ended questions are more useful to assess the quality of the attention the citizens receive from no-sanitary staff in Health Centres. These type of questions are also more useful for detecting problems and planning new interventions. Relational and informative issues seem to be the most prioritary areas to improve in this section of Health Centres.

Systemic G-CSF treatment does not improve long-term outcomes after neonatal hypoxic-ischaemic brain injury.

Hypoxia-ischaemia (HI) is a major factor in the pathogenesis of developmental brain injury, leading to cognitive deficits and motor disabilities in preterm infants. The haematopoietic growth factor granulocyte colony-stimulating factor (G-CSF) has been shown to exert a neuroprotective activity in rodent models of ischaemic stroke and is currently subject to phase I/II clinical trials in adults. Results of studies examining the effect of G-CSF in perinatal brain damage have been contradictory. We have previously shown that G-CSF increases NMDAR-mediated excitotoxic brain injury in the neonatal mouse brain. In this study, we evaluated the effect of G-CSF on long-term outcomes after HI. On postnatal day 5, mice pubs were first randomly assigned to a sham operation or HI and then divided into four treatment groups: i) G-CSF; ii) phosphate buffered saline (PBS) 1h after injury; iii) G-CSF and iv) PBS 60 h after injury. G-CSF (200 µg/kg BW) was administered five times within a 24h interval. Neuromotor and cognitive outcomes were assessed by open-field, novel object recognition tests and rotarod tests starting on P90, with subsequent histological analyses of brain injury. G-CSF treatment did not improve either neurobehavioural outcomes or brain injuries. Interestingly, the application of PBS and G-CSF in the acute phase increased brain damage in the hippocampus. We could not confirm the neuroprotective properties of G-CSF in neonatal HI brain damage. The exacerbation of injury by the administration of substances in the acute phase might indicate a heightened state of neurological sensitivity that is specific to mechanisms of secondary neurodegeneration and influenced by unidentified external factors possibly associated with the treatment protocol during the acute phase. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

Neurodevelopmental outcome following congenital cytomegalovirus infection in preterm infants with twin-to-twin transfusion syndrome: a case report.

Twin-to-twin transfusion syndrome and conital cytomegalovirus infection bear the risk of brain damage. In the 27th week of gestation of a twin pregnancy a Caesarean section was performed because of pathological cardiotocogram and Doppler ultrasonography of the second twin (recipient). Both infants presented with severe, persistent thrombocytopenia, elevated liver enzymes and direct hyperbilirubinemia. Primary congenital CMV infection was diagnosed. Both twins showed severe neuropathological symptoms, pathological aEEG with seizure activity and severe neurodevelopmental delay at corrected age of 12 months. The severity of brain pathology, the complex etiology, its consequence for neurotion with extreme prematurity make this case of special interest.

Analysis of a non-ras 21-kDa protein in patients with metastatic testicular germ-cell tumors.

A novel protein of 21 kDa (p21) has been detected in the sera of patients with different solid tumors. The serum levels of this p21 protein were measured in seven patients with metastatic testicular germ-cell tumors before and after chemotherapy using an enzyme-linked immunosorbent assay. In five out of six patients who responded to chemotherapy a concomitant decrease of p21 serum levels was found. The decrease of p21 was in accordance with the decline of the established tumor markers alpha-fetoprotein, human chorionic gonadotropin beta-subunit and lactate dehydrogenase in three patients with non-seminomatous tumors and with the decline of lactate dehydrogenase and the clinical response in two patients with seminoma. In one patient the predicted decline of p21 did not occur despite the patient's clinical response to chemotherapy. In the seventh patient, who relapsed directly after chemotherapy, no decline of either p21 levels or tumor markers was observed. The absolute amount of the p21 protein prior to chemotherapy did not correlate with the patients' tumor burden. Elevated levels of p21 were found in patients with seminomatous and non-seminomatous germ-cell tumors. Since seminoma patients do not secrete tumor markers like alpha-fetoprotein or human chorionic gonadotropin beta, the determination of p21 levels may help to evaluate the efficacy of chemotherapy in patients with seminomatous as well as in patients with marker-negative non-seminomatous germ-cell tumors. The biological role of p21 and its clinical significance will be further investigated.

Regulation of the proto-oncogenes c-sis, c-fos, c-myc and c-myb in acute myeloid leukemias.

RNA transcriptional levels of the proto-oncogenes c-sis, c-fos, c-myb and c-myc were measured in peripheral blood leukemic blast cells of 16 patients with acute myeloid leukemia (AML) of different FAB subtypes, 8 being at diagnosis and 8 upon relapse. The studied proto-oncogenes were found to be regulated but varied considerably within morphologically identical subtypes. This is consistent with the clinically observable diverse behavior of seemingly identical AMLs as to the course and outcome of the individual disease. Overexpression of c-sis and c-myc was found more often in AML upon relapse than at diagnosis and in two cases overexpression not found at diagnosis was present at relapse. This implies alterations of biological behavior in the course of antileukemic drug therapy. A decline of c-myb expression was observed in one patient studied throughout therapy which was found to be associated with a complete but transient hematological remission after chemotherapy.

The isolation and partial characterization of linked alpha A- and alpha D-globin genes from a duck DNA recombinant library.

Genomic DNA from an adult duck (Cairina moschata) was used to construct a library of cloned DNA fragments in the vector lambda Charon 4A. Screening of the DNA library resulted in the isolation of a recombinant, D alpha G-1, which carries both the adult duck alpha A- and alpha D-globin genes. The two globin genes are separated by approx. 2.2 kb of DNA, they are encoded by the same DNA strand and their orientation with respect to the direction of transcription is 5'- alpha D- alpha A-3'. Partial sequence analyses indicate that the two alpha-globin genes contain intervening sequences at positions homologous to those in chicken and mammalian alpha-globin genes.