Dietary supplementation with spray-dried porcine plasma has prebiotic effects on gut microbiota in mice.

In animal models of inflammation and in farm animals, dietary inclusion of spray-dried porcine plasma (SDP) reduces mucosal inflammation. Here, we study whether these effects could be mediated by changes in the intestinal microbiota and if these changes are similar to those induced by oral antibiotics. Weaned 21-day-old C57BL/6 mice were divided into 3 groups: the CTL group, fed the control diet; the COL group, administered low doses of neomycin and colistin; and the SDP group, supplemented with 8% SDP. After 14 days, analysis of the fecal microbiome showed that the microbiota profiles induced by SDP and the antibiotics were very different, thus, SDP has prebiotic rather than antibiotic effects. At the phylum level, SDP stimulated the presence of Firmicutes, considerably increasing the lactobacilli population. It also enhanced the growth of species involved in regulatory T-lymphocyte homeostasis and restoration of the mucosal barrier, as well as species negatively correlated with expression of pro-inflammatory cytokines. At the mucosal level, expression of toll-like receptors Tlr2, Tlr4 and Tlr9, and mucous-related genes Muc2 and Tff3 with regulatory and barrier stability functions, were increased. SDP also increased expression of Il-10 and Tgf-ß, as well as markers of macrophages and dendritic cells eventually promoting an immune-tolerant environment.

Efficacy of dietary spray dried plasma protein to mitigate the negative effects on performance of pigs fed diets with corn naturally contaminated with multiple mycotoxins.

The ability of spray dried plasma protein (SDPP) to reduce the negative effects of multiple mycotoxins from naturally contaminated corn on weaned pig performance and health was investigated (n = 180; 6.84 ± 0.11 kg). For 12 d after weaning, pigs were fed phase 1 nursery diets with either 0% SDPP (PP0) or 6% SDPP (PP6). After 12 d, pigs were fed phase 2 diets for 3 wk. Pigs fed PP0 in phase 1 continued to be fed a phase 2 diet with no SDPP (PP0/PP0) or were fed a diet including corn naturally contaminated with multiple mycotoxins (M), labeled PP0/PP0M. Pigs fed SDPP in phase 1 were fed either a diet with no SDPP (PP6/PP0), a diet with M and no SDPP (PP6/PP0M), a diet with M and 3% SDPP (PP6/PP3M), or a diet with M and 6% SDPP (PP6/PP6M). During phase 1, pigs fed PP6 had increased (P < 0.05) ADG, ADFI, and G:F, whereas immunological parameters were not altered. During phase 2, pigs consuming PP0/PP0M had reduced ADG (P < 0.01) and ADFI (P < 0.05) in contrast to pigs fed PP0/PP0, whereas the performance of pigs fed PP6/PP0M was intermediate to pigs fed PP0/PP0M and PP6/PP0. The ADG and ADFI did not differ for pigs fed PP0/PP0M and PP6/PP0M during phase 2. Performance of pigs fed PP6/PP3M in contrast to pigs fed PP6/PP0M during phase 2 did not differ; however, these pigs had lower (P < 0.05) tumor necrosis factor α and tended (P = 0.094) to have lower DNA damage. During phase 2, ADG and ADFI of pigs fed PP6/PP6M did not differ from pigs fed PP6/PP0M, but G:F tended (P = 0.067) to be increased in pigs fed PP6/PP6M. Over the entire study period, pigs fed PP0/PP0M had reduced (P < 0.05) ADG and tended (P = 0.067) to have reduced ADFI. During this time, pigs fed PP6/PP0M tended to have greater ADG and ADFI (P = 0.093 and P = 0.067, respectively) compared with pigs fed PP0/PP0M. Overall, feeding a diet with SDPP improved growth performance and feed intake of young pigs directly after weaning. Feeding multiple M had a negative impact on growth performance of pigs during this trial. This response was more significant when pigs were not fed SDPP in phase 1. Overall, when combining phase 1 and 2 performance data, daily gain and feed intake tended to be reduced when pigs were not fed 6% SDPP in phase 1. This study indicates that the composition of diets fed immediately after weaning may be important for pigs that subsequently are under a M challenge.

Estimate of serum immunoglobulin G concentration using refractometry with or without caprylic acid fractionation.

Objectives of this study were to develop a rapid calf-side test to determine serum IgG concentrations using caprylic acid (CA) fractionation, followed by refractometry of the IgG-rich supernatant and compare the accuracy of this method with results obtained using refractometry using raw serum. Serum samples (n=200) were obtained from 1-d-old calves, frozen (-20°C), and shipped to the laboratory. Samples were allowed to thaw for 1h at room temperature. Fractionation with CA was conducted by adding 1mL of serum to a tube containing 45, 60, or 75µL of CA and 0.5, 1.0, or 1.5mL of 0.06 M acetic acid. The tube contents were mixed well, allowed to react for 1 min, and then centrifuged at 3,300 × g for 0, 10, or 20 min at 25°C. The %Brix and refractive index of the fractionated supernatant were determined using a digital refractometer. Nonfractionated serum was analyzed for %Brix (BRn), refractive index (nDn), and IgG concentration by radial immunodiffusion. The mean serum IgG concentration was 19.0 mg/mL [standard deviation (SD)=9.7], with a range of 3.5 to 47.0 mg/mL. The mean serum BRn was 8.6 (SD=0.91), with a range of 6.8 to 11.0. The mean serum nDn was 1.34566 (SD=0.00140), with a range of 1.34300 to 1.34930. Serum nDn was positively correlated with IgG concentration (correlation coefficient=0.86; n=185). Fractionated samples treated with 1mL 0.6 M acetic acid and 60µL of CA and not centrifuged before analysis resulted in a strong relationship between the refractive index of the fractionated supernatant and IgG (correlation coefficient=0.80; n=45). Regression was used to determine cut points indicative of 10, 12, and 14 mg of IgG/mL to determine the sensitivity and specificity of refractometry to identify failure of passive transfer (serum IgG <10 mg/mL at 24 h old). The nDn were 1.34414, 1.34448, and 1.34480 to predict 10, 12, and 14 mg of IgG/mL of serum, respectively. The BRn cut points were 7.6, 7.8, and 8.0, respectively. The nDn cut points of 1.34448 and 1.34480 resulted in similar specificities (82.9%), whereas the 1.34414 cut point had a specificity of 60.0%. The BRn cut point of 7.6 and 7.8%Brix resulted in a similar percentage of correctly classified samples (89.7 and 90.8%, respectively); however, the 7.8% Brix cut point resulted in fewer false positives. These results suggest that Brix refractometry of nonfractionated calf serum provides a strong estimate of IgG concentration and 7.8% Brix may be used as the cut point to identify failure of passive transfer in 1-d-old calves.

Evaluation of the Brix refractometer to estimate immunoglobulin G concentration in bovine colostrum.

Refractometry using a Brix refractometer has been proposed as a means to estimate IgG concentration in bovine maternal colostrum (MC). The refractometer has advantages over other methods of estimating IgG concentration in that the Brix refractometer is inexpensive, readily available, less fragile, and less sensitive to variation in colostral temperature, season of the year and other factors. Samples of first-milking MC were collected from 7 dairy farms in Maine, New Hampshire, Vermont, and Connecticut (n=84) and 1 dairy farm in California (n=99). The MC was milked from the cow at 6.1 ± 5.6h postparturition and a sample was evaluated for Brix percentage by using an optical refractometer. Two additional samples (30 mL) were collected from the milk bucket, placed in vials, and frozen before analysis of total IgG by radial immunodiffusion (RID) using commercially available plates and by turbidimetric immunoassay (TIA). The second sample was analyzed for total bacterial counts and coliform counts at laboratories in New York (Northeast samples) and California (California samples). The Brix percentage (mean ± SD) was 23.8 ± 3.5, IgG concentration measured by RID was 73.4 ± 26.2g/L, and IgG concentration measured by TIA was 67.5 ± 25.0 g/L. The Brix percentage was highly correlated (r=0.75) with IgG analyzed by RID. The Brix percentage cut point to define high- or low-quality colostrum (50 g of IgG/L measured by RID) that classified more samples correctly given the proportion of high- (86%) and low-quality (14%) samples in this study was 21%, which is slightly lower than other recent estimates of Brix measurements. At this cut point, the test sensitivity, specificity, positive and negative predictive values, and accuracy were 92.9, 65.5, 93.5, 63.3, and 88.5%, respectively. Measurement of IgG by TIA correlated with Brix (r=0.63) and RID (r=0.87); however, TIA and RID methods of IgG measurement were not consistent throughout the range of samples tested. We conclude that Brix measurement of total solids in fresh MC is an inexpensive, rapid, and satisfactorily accurate method of estimating IgG concentration. A cut point of 21% Brix to estimate samples of MC >50 g/L was most appropriate for our data. Measurement of IgG in MC by TIA differed from measurement by RID.

Estimate of colostral immunoglobulin G concentration using refractometry without or with caprylic acid fractionation.

Our objectives were to evaluate the use of refractometry as a means of estimating immunoglobulin G (IgG) concentration of bovine maternal colostrum (MC) and determine if fractionation of MC using caprylic acid (CA) improved estimates of IgG. Samples (n=85) of MC were collected from a single dairy in California and used to determine the method of CA fraction that produced the best prediction of IgG based on CA fractionation followed by refractometry. Subsequently, samples of MC (n=827) were collected from 67 farms in 12 states to compare refractometry with or without CA fractionation as methods to estimate IgG concentration. Samples were collected from the feeding pool and consisted of fresh (n=196), previously frozen (n=479), or refrigerated (n=152) MC. Samples were further classified by the number freeze-thaw cycles before analysis. Fractionation with CA was conducted by adding 1 mL of MC to a tube containing 75 µL of CA and 1 mL of 0.06 M acetic acid. The tube was shaken and allowed to react for 1 min. Refractive index of the IgG-rich supernatant (nDf) was determined using a digital refractometer. Whole, nonfractionated MC was analyzed for IgG by radial immunodiffusion (RID) and refractive index (nDw). The relationship between nDf and IgG (r=0.53; n=805) was weak, whereas that between nDw and IgG was stronger (r=0.73; n=823). Fresh samples analyzed by refractometry that subsequently went through 1 freeze-thaw cycle before RID analysis resulted in the strongest relationship between IgG and nDf or nDw (r=0.93 and 0.90, respectively). The MC samples collected fresh on the farm but frozen 2 or more times before analysis by refractometry or RID had low correlations between IgG and nDf and nDw (r=0.09 and 0.01). Samples refrigerated or frozen on the farm before analysis had weaker relationships between RID and nDf or nDw (r=0.38 to 0.80), regardless of the number of freeze-thaw cycles. Breed and lactation number did not affect the accuracy of either test. These results indicated that refractometry, without or with CA fractionation, was an accurate and rapid method to determine IgG concentration when samples of MC were not previously stored before refractometry and frozen only once before RID analysis.

Evaluation of immunoglobulin G absorption from colostrum supplements gavaged to newborn piglets.

Absorption of energy and IgG at birth from colostrum may improve survival and immune competency of newborn piglets. Adequate intake of colostrum may be difficult for piglets due to low birth weight, birth order, or viability. This study was designed to evaluate orally fed colostrum supplements with different energy sources and IgG from porcine plasma on piglet serum IgG content and absorption of IgG compared to pooled sow colostrum. Ninety-six newborn piglets from 12 sows with an average birth weight of 1,288 g were used. Eight piglets were removed from each sow immediately at birth, prior to suckling, and randomly allotted to receive either pooled sow colostrum or 1 of 3 colostrum supplements (A, B, and C) fed at 2 dosing schemes. Piglets received their allotted treatment as either one 30-mL dose at 0 h or three 10-mL doses at 0, 2, and 4 h. Piglets received ad libitum access to water at 2-h intervals after receiving their last treatment dose. Twelve hours after the first dose, piglets were weighed and 4 mL of blood was collected. Plasma IgG content, apparent efficiency of absorption, hematocrit, protein, and glucose were determined. Birth weight and final BW did not differ between treatments; however, pigs fed sow colostrum lost more weight (-72 g) than pigs fed colostrum supplements (-40 g; P < 0.001). Differences in hematocrit or serum glucose were not detected. Serum protein was higher (P < 0.05) in piglets fed colostrum supplements than in pigs fed sow colostrum. Serum IgG content did not differ among treatments. Apparent efficiency of IgG absorption was greatest for sow colostrum followed by colostrum supplements B, A, and C (28.5, 27.6, 25.5, and 24.7%, respectively). The single and multiple dose regimes delivered comparable serum IgG whereas the single dose yielded better piglet hydration as noted by less weight loss. In conclusion, all colostrum supplements were comparable in delivering absorbable IgG to the neonatal piglet.

Half-life of porcine antibodies absorbed from a colostrum supplement containing porcine immunoglobulins.

Absorption of immunoglobulins (Ig) at birth from colostrum is essential for piglet survival. The objective was to evaluate the half-life of antibodies absorbed in the bloodstream of newborn piglets orally fed a colostrum supplement (CS) containing energy (fat and carbohydrates) and IgG from porcine plasma. Viable piglets (n = 23; 900 to 1,800 g BW) from 6 sows were colostrum deprived and blood sampled and within the next 2 h of life randomly allocated to either control group (n = 9) providing 30 mL of Ig-free milk replacer or a group (n = 14) receiving 30 mL of CS by oral gavage. Piglets were transported to a Biosafety Level 3 facility (Centre de Recerca en Sanitat Animal, Spain) and fed Ig-free milk replacer every 3 to 4 h for 15 d. Survival, weight, plasma IgG content by radial immunodiffusion (RID), and antibodies against porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome (PRRS), Mycoplasma hyopneumoniae (Mhy), and swine influenza virus (SIV) were determined by specific ELISA before treatment administration, at 24 h, and weekly for 56 d. Clinical symptoms were not observed for either group. Mortality index was lower (17 vs. 38%; P < 0.02) and BW higher (17.7 vs. 15.3 kg; P = 0.035) for pigs supplemented with CS than piglets in the control group. At 24 h postadministration, the CS group had a plasma IgG mean of 7.6 ± 0.06 vs. 0.14 ± 0.03 mg/mL for the control group. The IgG levels in the CS group decayed until day 21 when de novo synthesis of IgG was detected in 25% of piglets. Half-life of antibody concentration (HLAC) by RID was 6.2 d. In the CS group, efficiency of PCV2 and PPV antibody transfer was high. For PCV2, all animals remained positive by day 56 and the calculated HLAC was 17.7 d. For PPV, 72.7% of piglets were ELISA positive by day 35 and HLAC was 12.0 d. For PRRS, all piglets remained positive by day 14 and the calculated HLAC was 11.9 d. For Mhy and SIV the calculated HLAC were 8.4 and 3.0 d. In summary, half-life of antibodies derived from blood plasma in the bloodstream of newborn piglets varied from 3.0 to 17.7 d. The study also confirm that antibodies derived from porcine plasma were well absorbed and can be an useful tool for providing protection against several or specific pathogens and can be a good alternative to formulate CS for newborn piglets.

Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia.

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.

Genomic analysis of insulin-like growth factor-I gene transfer in thermally injured rats.

Thermal trauma causes tissue damage by membrane destabilization and energy depletion at the cellular level, resulting in tissue necrosis and inflammation leading to delayed cell death. One therapeutic approach is to block the immediate triggering of the inflammatory cascade that results in prolonged hypermetabolic responses and immune dysfunction while promoting the expression of growth factors. In the present study, we determined hepatic gene expression responses to insulin-like growth factors-i (IGF-I) gene transfer to burned rats using high-density DNA microarray assays. The expression of 123 out of approximately 8,800 genes assayed (1.4% of total) were significantly altered. Of these, 42 genes were altered irrespective of treatment by burn trauma (p < 0.05). Changes in gene expression were confirmed by measuring mRNA levels using reverse transcription-polymerase chain reaction and protein levels by Western blot assays. DNA microarray analyses showed two major mechanisms that mediated beneficial outcomes after IGF-I gene transfer in the burned rat livers. These mechanisms were the stimulation of IGF binding protein potentiation of peripheral IGF-I and the inhibition of the burn-augmented pro-apoptotic and oxidative mitochondrial metabolites stimulated by thermal trauma.

Initial stages of microbiologically influenced tarnishing on titanium after 20 months of immersion in freshwater.

This paper studies the initial stages of iridescent tarnishes on titanium heat exchanger tubes in contact with running freshwater on the river Tagus in Spain for up to 20 months. Electrochemical impedance spectroscopy (EIS), scanning electron microscopy [(SEM with energy dispersive X-ray (EDX)] and X-ray photoelectron spectroscopy (XPS) in conjunction with argon-ion sputtering were the techniques used. The EIS data indicated a capacitive behavior, showing a semicircle that was better defined as the experimental time increased, indicating a decreasing tarnishing resistance of titanium. XPS and EDX results indicated that the main elements identified were calcium, phosphorus, nitrogen, and iron. The amount of these elements was higher on the tarnished titanium specimens than on the untarnished specimens. SEM analysis showed the presence of diatoms in the iridescent tarnishes on titanium tubes.

Fatal familial insomnia: clinical, neuropathological, and genetic description of a Spanish family.

The clinical presentation and evolution, neuropathological findings, and genotyping of three members of a Spanish family affected with fatal familial insomnia are reported. The mother and two of her offspring developed a rapidly evolving disease with insomnia and behavioural disorders as the initial symptoms and died between 5 and 10 months after the onset of the illness. Frontal brain biopsy in the mother disclosed only non-significant spongiosis, and full neuropathological examination of her offspring showed thalamic and olivary degeneration with isolated focal cortical spongiosis. Genetic examination could only be performed in the contemporary patients and both harboured the prion protein (PrP) 178Asn mutation and homozygous 129 Met/Met genotype.

Neuroimaging diagnosis of Tolosa-Hunt syndrome: MRI contribution.

OBJECTIVE: To present our experience in the neuroradiological diagnosis of six patients with Tolosa-Hunt syndrome. METHODS: Computerized tomograms and MRI, with and without contrast enhancement, of the cranium and orbits of patients fulfilling IHS criteria for the diagnosis of Tolosa-Hunt syndrome were analyzed. RESULTS: Standard CT scan, with and without contrast enhancement, disclosed an enlarged cavernous sinus in one patient and was normal in the remaining five. In comparison, MRI was clearly abnormal in the four patients on whom it was performed, showing a convex enlargement of the symptomatic cavernous sinus by an abnormal tissue isointense with gray matter on short TR/TE images and iso-hypointense on long TR/TE scans. This abnormal tissue markedly increased in signal intensity after contrast injection and, in two patients, extended into contiguous regions, mainly the orbital apex and subtemporal fossa ipsilaterally. One patient had follow-up studies after successful treatment with corticosteroids. Although diminished in size, the abnormal tissue was still visible on MRI after 3 months of treatment and only disappeared after 6 months of treatment. CONCLUSIONS: These MRI findings help in the differential diagnosis of the Tolosa-Hunt syndrome from conditions such as meningioma, lymphoma, and sarcoidosis, as well as confirming the similarities of the Tolosa-Hunt syndrome and orbital pseudotumor. In the presence of painful ophthalmoplegia, the finding by MRI of cavernous sinus enlargement, with the herein described signal and extension characteristics and slow resolution with corticosteroid treatment, is highly suggestive of the Tolosa-Hunt syndrome.

[Infiltrating bladder carcinoma: influence of complementary treatment on local control and survival]. / Carcinoma vesical infiltrante: influencia del tratamiento complementario en el control local y en la supervivencia.

We analyzed 82 patients with transitional bladder cancer stages (T2-T4) M0. According to treatment, 3 different groups were considered: Group 1: 25 patients treated with TUR and radical cystectomy. Group 2: 33 patients also received external beam radiotherapy (45-60 Gy/5 weeks) prior cystectomy. Group 3: 24 patients were also treated with intraoperative radiotherapy (15 Gy) and 3 courses of neoadjuvant chemotherapy. Our results suggest that local control rate increases significantly with the intensity of treatment (Group 3: 62% patients were PT0). Similar results were achieved in multivariate analysis. Univariate analysis of survival shows ganglionar status, vascular embolism and treatment intensity as covariates associated. Multivariate analysis demonstrate that renal function and type of complementary treatment were the covariates associated with survival. Taking both groups 2 and 3, survival was related to pathologic stage, vascular embolism and renal function. In conclusion, the significant influence of complementary treatment on the local control and survival suggest strongly their systematic utilization in infiltrating bladder cancer.

Hypothalamic involvement in the activation of the pituitary-adrenocortical axis by nerve growth factor.

Intravenous injection of nerve growth factor (NGF) into rats produces a dose-dependent (from 0.1 to 5 nmol/kg) increase in circulating concentrations of adrenocorticotropin (ACTH) and corticosterone. We have investigated whether this effect is produced through a direct action on a component of the hypothalamo-pituitary-adrenocortical axis. NGF (50 and 500 nM), added to the incubation medium of in vitro isolated pituitary segments or dispersed adrenal cells, did not modify either basal and stimulated release of biologically active or immunoreactive ACTH or release of corticosterone, respectively. The presence of NGF in the incubation medium of in vitro isolated hypothalami produced a dose-dependent (from 150 to 600 nM) increase of both release and content of some material with corticotropin-releasing bioactivity. The nature of this corticotropin-releasing bioactivity was determined directly by radioimmunoassays. Results have indicated that NGF induced an increase of both release and content of hypothalamic arginine-vasopressin (AVP), while no changes were observed in the release and content of hypothalamic corticotropin-releasing hormone (CRH). These results suggest that adrenocortical stimulation by NGF in vivo could be mediated by the release of hypothalamic AVP rather than CRH. The finding that in vivo NGF stimulatory effect was not abolished by the specific CRH antagonist alpha-helical CRH(9-41), while it was accompanied by an increase in circulating AVP levels, supports this interpretation. However, the fact that the hypothalamus is stimulated in vitro by NGF concentrations higher than those expected to reach this structure after systemic injection of active doses raises the possibility that other brain areas such as the hippocampus participate in NGF-induced adrenocortical activation.

Ionic channel currents in cultured neurons from human cortex.

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 microM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na+ current, two types of Ca2+ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K+ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca(2+)-activated K+ channels with slope conductances of 146 and 23 pS (K+i/K+o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca2+. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K+ and Ca2+ channel currents, but Na+ currents were not detected in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of somatotopic organization of the sensory thalamus based on infarction in the nucleus ventralis posterior.

BACKGROUND: This is to describe a restricted sensory syndrome of unique distribution due to thalamic infarct. CASE DESCRIPTION: We report a case of pure sensory disturbance involving the left intraoral and perioral regions and the tips of the thumb and forefinger of the left hand. Magnetic resonance imaging revealed a small infarct in the contralateral thalamus, presumably affecting the nucleus ventralis posterior. CONCLUSIONS: This patient provides an excellent correlation between clinical findings and thalamic representation of body surface as established during stereotactic procedures.

Life-threatening cervico-mediastinal haematoma after carotid sinus massage.

Retropharyngeal and cervico-mediastinal haematomas are rare but potentially fatal entities. The potential for airway compression necessitates rapid assessment and treatment. A cervico-mediastinal haematoma following carotid sinus massage which required cervical drainage is presented. This is a life-threatening event demanding a high index of suspicion. A review of the literature and indications for treatment are discussed.

[Effect of complementary treatment (radiotherapy, chemotherapy) on the survival of patients with T3 tumors of the bladder treated with radical surgery]. / Influencia del tratamiento complementario (radioterapia, quimioterapia) en la supervivencia de los tumores de vejiga T3 sometidos a cirugía radical.

We present the results obtained from the study of 65 patients with vesical carcinoma stage T3 treated with radical surgery with or without supplementary radiotherapy, with or without supplementary chemotherapy. The multivaried study of survival (Cox's model) shows that it depends significantly on the surgical complications and on the pathological state at the time of the cystectomy. Univaried analysis indicates that patients with descent from post-radiotherapy stage and those subjected to IOR (intraoperative radiotherapy) have a significantly better survival than the rest and suggests that radiotherapy improves overall survival. Influence of supplementary treatment (radiotherapy, chemotherapy) in the survival of T3 bladder tumours subjected to radical surgery.

Evaluation of methods for determining 6-hydroxydopamine cytotoxicity.

The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y(SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 micrograms/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations.