Fistula Samples Research Articles

Spatial Single Cell Profiling Using Imaging Mass Cytometry: Inflammatory Versus Penetrating Crohn's Disease.

Fistula formation is a major complication in Crohn's disease [CD] and the role of the immune cell compartment remains to be elucidated. Thus, we compared the immune cell compartment of CD fistula to inflammatory CD colitis using imaging mass cytometry [IMC] and immunofluorescence. A 36-marker panel including structural, functional, and lineage markers for use in IMC was established. This panel was applied to analyse paraffin-embedded CD fistula tract [n = 11], CD colitis [n = 10], and colon samples from non-inflamed controls [n = 12]. Computational methods for cell segmentation, dimensionality reduction, and cell type clustering were used to define cell populations for cell frequency, marker distribution, and spatial neighbourhood analysis. Multiplex immunofluorescence was used for higher resolution spatial analysis. Analysis of cell frequencies in CD fistulas compared to CD colitis and control colonic samples revealed a significant increase in neutrophils, effector cytotoxic T cells, and inflammatory macrophages in CD fistula samples, whereas regulatory T cells were decreased. Neutrophils in CD fistula expressed significantly more matrix metalloproteinase 9 [MMP9], correlating with extracellular matrix remodelling. Neighbourhood analysis revealed a strong association between MMP9+ neutrophils and effector cytotoxic T cells in both CD fistulas and colitis. This study presents the first highly multiplexed single cell analysis of the immune cell compartment of CD fistulas and their spatial context. It links immune cell dynamics, particularly MMP9+ neutrophils, to extracellular matrix remodelling in CD fistulas, offering insights into the complex network of cellular interactions and potential therapeutic targets for CD complications.

Microbiomic signatures of anal fistula and putative sources of microbes.

Anal fistula is a common perianal disease that typically develops from an abscess caused by in-flammation in the area. It has long been believed that intestinal microbes play a significant role in its development, considering its close relation to the intestinal environment. This work attempts to identify the microbiomic signatures of anal fistula, and putative sources of microbes by analyzing microbiomes of 7 anal fistula-associated sites in 12 patients. This study found that microbes in anal fistulas may originate from the skin surface in addition to the intestinal tract. This finding was further validated by NMDS analysis, which also indicated that the microbial communities in the inner and outer openings of the fistula were more similar to their surrounding environments. Using MaAslin2, the characteristics of the microbiome were examined, demonstrating a higher similarity between the abundant bacteria in the anal fistula samples and those found on the skin surface. Moreover, pin-to-pair analysis conducted on all subjects consistently showed a higher abundance of skin-sourced bacteria in anal fistulas. This study identifies the microbiomic signatures of anal fistula, and provides novel insights into the origin of microorganisms in anal fistulas.

Determination of the compatibility between arterial samples and blood samples from arteriovenous fistulas for ESRD patients

Arterial blood gases (ABG) is a common and gold standard procedure and is often used to assess acid-base status and electrolytes. Arterial blood sampling is technically difficult and painful and causes rare but serious complications for the patient (such as vascular injury or embolism). The blood gas analysis of samples drawn from the arteriovenous fistula (AVF) requires fewer punctures and is a relatively safer and less painful procedure for the patient. The study included (22) patients and a blood gas analyzer (ABL800 FLEX PLUS blood gas analyzer) was used. The results showed that there were no clinically significant differences between the arterial blood sample and the arteriovenous fistula sample.

Lack of anti-TNF drugs levels in fistula tissue - a reason for nonresponse in Crohn's perianal fistulating disease?

Anti-TNF therapy is recommended as treatment for patients with Crohn´s perianal fistulas. However, a significant proportion of patients have a sub-optimal response to anti-TNF therapy. Higher serum levels of anti-TNF agents have been associated with improved outcomes in perianal Crohn's disease. Currently, it is unknown whether anti-TNF agent levels can be detected in tissue from fistula tracts themselves and whether this is associated with response. We undertook a pilot study to measure fistula tissue levels of anti-TNF medication (infliximab and adalimumab). We used a previously validated targeted proteomic technique, employing ultraperformance liquid chromatography-mass spectrometry, to detect/quantify anti-TNF drugs. Biopsies were obtained from fistula tracts of patients with Crohn's disease on maintenance treatment; with idiopathic (cryptoglandular) fistula tissues used as negative controls as well as positive controls (by spiking the latter tissues with anti-TNF drugs). Tissue was sampled from the fistula tracts of seven patients with Crohn's perianal disease (five patients were on adalimumab and two patients were on infliximab). The anti-TNF drugs, infliximab and adalimumab, were not detected in fistula samples from any of the Crohn's patients despite detection in 'spiked' positive control samples. Absence of detection of the anti-TNF drugs in fistula tissue raises the question on the role of tissue penetrance of anti-TNF drugs in response to therapy. Further work is required in a larger number of patients to validate the findings observed and investigate if any correlation exists between tissue and serum levels of anti-TNF and clinical outcome. Predicting response in Crohn's fistula patients on biologic therapy is difficult with no reliable biomarkers. This pilot study uses targeted proteomics to investigate the potential role of tissue drug levels in acting as a biomarker of treatment response.

A regression for estimating metabolizable glucose in diets of weaned piglets for optimal growth performance.

ObjectiveTwo experiments were conducted to provide a new approach for evaluating feed nutritional value by metabolizable glucose (MG) in piglet diets with different levels of starch and crude fiber. In Exp 1, a regression equation for MG was generated. In Exp 2, the equation was verified, and the optimal growth performance of piglets under appropriate MG levels was tested.MethodsIn Exp 1, 20 weaned piglets (7.74±0.81 kg body weight [BW]) were randomly assigned to 1 of 4 treatments, including the basal diet containing different levels of MG (starch, 25.80%, 31.67%, 45.71%, 49.36%; crude fiber, 1.23%, 1.35%, 1.80%, 1.51%). The piglets were implanted with an ileal fistula, cannulation of the carotid artery, portal vein, and mesenteric artery. The chyme from the ileum fistula and blood samples were collected. In Exp 2, 30 weaned piglets (8.96±0.50 kg BW) were randomly assigned to 1 of 5 treatments, including the experimental diets with different levels of MG (37.6, 132.5, 300.0, 354.3, and 412.5 g/kg). The piglets’ BW, and feed consumption were recorded to calculate growth performance during the 28-d experiment.ResultsIn Exp 1, the MG levels in 4 diets were 239.62, 280.68, 400.79, and 454.35 g/kg. The regression equation for the MG levels and dietary nutrients was: Y (MG) = 12.13×X1 (starch)+23.18×X2 (crude fiber)−196.44 (R2 = 0.9989, p = 0.033). In Exp 2, treatments with 132.5 and 300.0 g/kg MG significantly (p<0.05) increased average daily gain and feed conversion efficiency of weaned piglets, increased digestibility of crude fat, and had no effect on digestibility of crude protein compared to 300.0 to 412.5 g/kg MG.ConclusionThe pig model combining the ileum fistula and cannulation of blood vessels was successfully used to determine the dietary MG levels. The recommended MG level in weaned pig diets is 132.5 to 300.0 g/kg.

P345 Lack of anti-TNF drugs levels in fistula tissue: A reason for non-response in Crohn’s perianal fistula?

Abstract Background Anti-TNF therapy is recommended as a treatment for patients with Crohn ́s perianal fistulas. However, a significant proportion of patients have a sub-optimal response to anti-TNF therapy. Higher serum levels of anti-TNF agents have been associated with improved outcomes in perianal Crohn’s disease. Currently, it is unknown whether anti-TNF agent levels can be detected in tissue from fistula tracts themselves and whether this is associated with response. Methods We undertook a pilot study to develop a method to measure fistula tissue levels of anti-TNF medication (infliximab and adalimumab) using a targeted proteomic technique that employs ‘signature peptide detection’ following trypsin digestion called ultraperformance liquid chromatography–mass spectrometry (UPLC-MS), to quantify a protein. The targeted UPLC- MS/MS detection and quantification method implemented were previously validated. Biopsies were obtained from patients with Crohn’s disease who underwent an examination under anaesthesia for worsening fistula symptoms despite maintenance anti-TNF therapy. Idiopathic (cryptoglandular) tissues from purposively sampled matched (age/gender) patients were analysed as negative controls and these samples were spiked with anti-TNF drugs as positive controls. Results Tissue was sampled from the fistula tracts of seven patients with Crohn’s perianal disease (5 patients were on adalimumab and 2 patients were on infliximab). The limit of detection (LOD) and linearity range of the method was assessed for each drug in the spiked idiopathic fistula samples. Infliximab and adalimumab had a LOD of 0.004 and 2 μg/ml respectively with linearity demonstrated for both drugs. The anti-TNF drugs, infliximab and adalimumab, were not detected in fistula samples from any of the Crohn’s patients despite detection in ‘spiked’ positive control samples. In addition, to validate the result, samples were concentrated (x10) and still there was no detection of the drugs in the test samples. Conclusion The anti-TNF drugs adalimumab and infliximab were not detected in fistula biopsy samples from patients with Crohn ́s perianal fistulas with refractory symptoms despite maintenance therapy. This raises the question on the role of tissue penetrance of anti-TNF drugs in response to therapy. Further work is required in a larger number of patients to validate the findings observed and investigate whether any correlation exists between tissue and serum levels of anti-TNF and clinical outcome.

A Distinct Gut Microbiota Exists Within Crohn's Disease–Related Perianal Fistulae

BackgroundGut bacteria are strongly suspected to play a key role in the pathogenesis of Crohn's disease (CD). Studies have demonstrated alterations in the gut microbiota in this patient population. The purpose of this study was to characterize the gut microbiota of fistulizing perianal CD. Materials and MethodsStool and fistula samples were obtained from patients undergoing surgery for CD-related anorectal fistulae. Microbial compositions of matched stool and fistula samples were characterized using 16S rRNA gene profiling. The effect of sample type, patient gender, disease classification (Montreal A/B), disease activity (Harvey Bradshaw Index), antibiotic use, and presence of active proctitis on microbial composition was assessed. ResultsSamples were obtained from 18 patients. Bacteroides was the most abundant genera across all samples collected, followed by Streptococcus and Bifidobacterium. Bifidobacterium was present at significantly higher levels in fecal samples than fistula samples, whereas Achromobacter and Corynebacterium were present at significantly higher levels in fistula samples. Antibiotic, but not thiopurine or antitumor necrosis factor medication, exposure affected the gut microbial composition. Patient gender, disease classification, disease activity, and presence of active proctitis did not alter stool or fistula microbiota. ConclusionsOur data show that the gut microbiota within CD-related anorectal fistulae is distinct from that in stool samples obtained from the same patients. We also observe a dysbiosis in patients treated with antibiotics compared with those not treated with antibiotics.

Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-β1/SMAD and myocardin/serum response factor.

Tetraspanins (TSPANs) comprise a large family of 4-transmembrane domain proteins. The importance of TSPANs in vascular smooth muscle cells (VSMCs) is unexplored. Given that TGF-β1 and myocardin (MYOCD) are potent activators for VSMC differentiation, we screened for TGF-β1 and MYOCD/serum response factor (SRF)-regulated TSPANs in VSMC by using RNA-seq analyses and RNA-arrays. TSPAN2 was found to be the only TSPAN family gene induced by TGF-β1 and MYOCD, and reduced by SRF deficiency in VSMCs. We also found that TSPAN2 is highly expressed in smooth muscle-enriched tissues and down-regulated in in vitro models of VSMC phenotypic modulation. TSPAN2 expression is attenuated in mouse carotid arteries after ligation injury and in failed human arteriovenous fistula samples after occlusion by dedifferentiated neointimal VSMC. In vitro functional studies showed that TSPAN2 suppresses VSMC proliferation and migration. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that TSPAN2 is regulated by 2 parallel pathways, MYOCD/SRF and TGF-β1/SMAD, via distinct binding elements within the proximal promoter. Thus, we identified the first VSMC-enriched and MYOCD/SRF and TGF-β1/SMAD-dependent TSPAN family member, whose expression is intimately associated with VSMC differentiation and negatively correlated with vascular disease. Our results suggest that TSPAN2 may play important roles in vascular disease.-Zhao, J., Wu, W., Zhang, W., Lu, Y. W., Tou, E., Ye, J., Gao, P., Jourd'heuil, D., Singer, H. A., Wu, M., Long, X. Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-β1/SMAD and myocardin/serum response factor.

MYOSLID Is a Novel Serum Response Factor-Dependent Long Noncoding RNA That Amplifies the Vascular Smooth Muscle Differentiation Program.

Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, an lncRNA that seems to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the VSMC-specific lncRNAs regulated by myocardin (MYOCD)/serum response factor, the master switch for VSMC differentiation, is unknown. To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs that overexpress MYOCD. Several novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced Smooth muscle LncRNA, Inducer of Differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/serum response factor and transforming growth factor-β/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula samples compared with healthy veins. Although MYOSLID did not affect gene expression of transcription factors, such as serum response factor and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated transforming growth factor-β1-induced SMAD2 phosphorylation. We have demonstrated that MYOSLID, the first human VSMC-selective and serum response factor/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and transforming growth factor-β/SMAD pathways.

Studies on acacia gums: Part VII effect of exudates form and tree age on the characteristics of Acacia seyal var. seyal and Acacia seyal var. fistula

Authenticated samples of Acacia seyal var. seyal and Acacia seyal var. fistula have been investigated. Samples of each variety from young (5–7 years) and old trees (15–20 years) were collected during the same season from the Eldamazeen area of Sudan. For each variety, two types of gum exudates (seep and nodule form) were collected. The solubility, optical rotation, sugar composition, colour Gardner, viscosity, and molecular weights were measured. The rhamnose and uronic acid contents were higher in var. seyal than var. fistula but the amount of arabinose is greatest in var. fistula. The galactose content is almost the same for all samples with the possible exception of var. seyal with a higher content in older trees. The weight average molecular weight (Mw) for var. fistula is almost double that of var. seyal and generally the proportion of the high molecular fraction increases with age. The average value for the Mw of the arabinogalactan moiety (AG) is 6.2 × 105 and 9.3 × 105 g/mol for seyal and fistula respectively. Tannin content is highest in seep (∼0.8 wt%), lowest in the nodule form (0.08 wt%) and is a contaminant resulting directly from contact of the gum exudate with the tree bark. Tannin content increases with the age of tree for seyal var. seyal but not for var. fistula samples. These results give a clear difference between these two Acacia gum varieties, both of which are currently regulatory approved as gum arabic for food use.

Bacteriological analysis of necrotic pulp and fistulae in primary teeth

Objectives Primary teeth work as guides for the eruption of permanent dentition, contribute for the development of the jaws, chewing process, preparing food for digestion, and nutrient assimilation. Treatment of pulp necrosis in primary teeth is complex due to anatomical and physiological characteristics and high number of bacterial species present in endodontic infections. The bacterial presence alone or in association in necrotic pulp and fistula samples from primary teeth of boys and girls was evaluated. Material and Methods Necrotic pulp (103) and fistula (7) samples from deciduous teeth with deep caries of 110 children were evaluated. Bacterial morphotypes and species from all clinical samples were determined. Results A predominance of gram-positive cocci (81.8%) and gram-negative coccobacilli (49.1%) was observed. In 88 out of 103 pulp samples, a high prevalence of Enterococcus spp. (50%), Porphyromonas gingivalis (49%), Fusobacterium nucleatum (25%) and Prevotella nigrescens (11.4%) was observed. Porphyromonas gingivalis was detected in three out of seven fistula samples, Enterococcus spp. in two out of seven samples, and F. nucleatum, P. nigrescens and D. pneumosintes in one out of seven samples. Conclusions Our results show that Enterococcus spp. and P. gingivalis were prevalent in necrotic pulp from deciduous teeth in boys from 2 to 5 years old, and that care of the oral cavity of children up to five years of age is important.

Evaluation of a thin-layer chromatographic technique for identification of bush species browsed by goats

Diet selection by herbivores is a major determinant of animal and plant production and has a direct bearing on the sustainability of stock farming generally. Fistulated goats were confined in designated plots on the University of Fort Hare research farm and fistula samples were collected by the fistula valve technique. Leaves of preferred bush species were also sampled. Methanol extracts of both fistula and bush samples were prepared and analysed by thin-layer chromatography. Chromatoplates, when visualised under ultraviolet light, revealed a number of fluorescent compounds, some of which were common in both the fistula and bush sample extracts. By comparing the presence of characteristic compounds in the fistula sample with those that appear identical in particular bush species, dietary preferences of browsers can be established. However, any results of this nature need to be confirmed by field observations of the particular animal species.

Bone morphogenetic protein expression patterns in human esophageal atresia with tracheoesophageal fistula

The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in bone morphogenetic protein (BMP) signaling, since BMPs are critical to proper lung development and branching. With IRB approval, portions of newborn human proximal esophageal pouch and distal fistula samples were recovered at the time of surgical repair of EA/TEF. The tissues were processed for immunohistochemistry. Commercially available fetal tissues were used as controls. In control tissues, BMP ligands (BMP 2, 4, and 7) were all present in the esophagus but absent in the trachea. BMPRIA was absent in both tissues. BMPRIB was detected in trachea but not in esophagus and BMPRII was detected in esophagus but not in trachea. In the EA/TEF specimens, all BMP ligands were present in the proximal esophageal pouch but absent in the fistula tract. BMPRIA and BMPRIB were not detected in either tissue. However, BMPRII was found in both fistula tract and proximal pouch. The submucosa of the fistula appears to maintain a mixed (identical neither to lung, esophagus, or trachea) BMP signaling pattern, providing one mechanism which could potentially explain the esophageal dismotility and lack of lung branching seen in the fistula/distal esophagus.

Altered BMP signaling in human EA/TEF

Abstract Introduction: The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in Bone Morphogenetic Protein (BMP) signaling, since BMP's are critical to proper lung development and branching. Methods: With IRB approval, portions of newborn human proximal esophageal pouch and distal fistula samples were recovered at the time of surgical repair of EA/TEF. The tissues were processed for immunohistochemistry. Commercially available fetal tissues were used as controls. Results: BMP ligands (BMP2, −4, −7) and receptors (BMPRIA, BMPRIB, BMPRII) were all present in these tissues. BMP2 and BMP4 were expressed in the epithelial/sub-epithelial gland region of the proximal pouch, as well as in normal esophagus, whereas they were absent in fistula and trachea. BMP7, however, showed strong staining only in the glands of the trachea. BMPRIA was negative in the fistula, but positive in the tracheal epithelium. BMPRIB was positive in submucosal glands of the fistula and trachea. BMPRII was expressed at the base of the fistula and proximal pouch epithelium. Conclusions: Thus, the submucosa of the fistula appears to maintain a trachea-like BMP signaling pattern, which would explain its lack of esophageal motility and lack of lung branching.

A role for sonic hedgehog signaling in the pathogenesis of human tracheoesophageal fistula

Background/Purpose: Many theories of the pathogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) have been proposed, but no specific mechanism has been demonstrated. The authors previously reported data suggesting a respiratory origin of the fistula tract in the rat model and in humans. Sonic hedgehog (Shh) “knockout” mice have the VACTERL association, and thus it was hypothesized that defects in Shh signaling may exist in the human neonatal EA/TEF fistula tract. Methods: With IRB approval, human proximal esophageal pouch and distal fistula samples were removed at the time of standard repair of EA/TEF in accordance with what the surgeons deemed appropriate in preparation for anastomosis. Tissues were processed for HE, reverse-transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry. Normal embryonic lung cDNA was used as a positive control for the RT-PCR reactions. Results: As expected, Shh was present by immunohistochemistry in the proximal esophageal pouch, but was specifically absent in the distal fistula tract. Gli-1, -2, and -3 (all intracellular mediators of Shh signaling) were present in the proximal pouch and distal esophagus by RT-PCR. Conclusions: The absence of Shh signaling in the developing fistula tract of the human neonate was surprising given that Shh normally is present in esophagus and other gut components. These results support the conclusion that the fistula tract is not an esophaguslike structure, despite both its histologic appearance and its use as an esophageal replacement. Also, like in Shh-null mutant mice, aberrant Shh signaling may play a critical role in the pathogenesis of EA/TEF in humans. J Pediatr Surg 38:465-468. Copyright 2003, Elsevier Science (USA). All rights reserved.

The quantification of grazing capacity from grazing — and production values for forage species in semi-arid grasslands of southern Africa

The relation between rangeland condition and grazing capacity was determined along a degradation gradient. In studying agronomic values of forage species, the average production per tuft was combined with its grazing preferences, to link grazing values for species in the semi-arid grasslands of southern Africa. A production index, based on mean mass per tuft, was first compiled for species. Preference utilisation ratio of species was estimated for each of cattle and sheep, from the proportion of each species found in the diet relative to the proportion of the species found in the forage on offer. A microhistological technique was applied to oesophageal fistula samples to determine the proportion of each species found in the diet. Grazing values and grazing index were determined from the production and preference utilisation ratio of species. Where grazing values of species were determined subjectively in the past, in this study species were objectively classified based on estimated grazing values. Meaningful relationships between rangeland condition and grazing capacity were determined, along a degradation gradient, with the long-term coefficient of forecasting more than 75%. The financial implication with respect to sustainable animal production and rangeland condition variation was also calculated.

Nutrient selection by cattle from grass and grass/legume pastures

Esophageal fistula and hand-clipped samples were collected from a 32-yr-old crested wheatgrass (Agropyron cristatum L.) pasture and an alfalfa (Medicago sativa L.)/meadow bromegrass (Bromus biebersteinii Roem &amp; Schult.)/Russian wildrye (Psathyrostachys juncea [Fisch.] Nevski) pasture to compare estimates of nutritional quality. Samples were analyzed for crude protein (CP), in vitro digestible organic matter (IVDOM) and selected minerals. Fistula samples had greater (P &lt; 0.05) CP and IVDOM contents than clipped samples at all times except that CP did not differ (P &gt; 0.05) on two occasions and IVDOM did not differ (P &gt; 0.05) on one occasion. Differences in mineral content between clipped and esophageal samples were less consistent, but when they occurred the mineral contents of esophageal samples usually exceeded (P &lt; 0.05) those of clipped samples. We conclude that clipped pasture samples mostly provide biased estimates of the nutritional quality of herbage consumed by cattle grazing grass and grass/legume pastures and should be reported with caution. Key words: Pasture, selection, cattle, protein, digestibility, minerals

An evaluation of the equipment for the oesophageal fistula valve technique

The remote control system for the collection of multiple oesophageal fistula samples was evaluated under range conditions. A total of 1027 fistula valve samples were collected from three fistulated goats over a period of 1 year, totalling 105 animal sampling days. On average, 9.9 extrusa samples were collected per animal per day. One hundred (9.7%) of the 1027 sampling attempts failed, due to blockage of the valve. In addition to the blockages, a total of 138 equipment failures were recorded which resulted from electrical disconnection (68%), dislodgement of the valve (20%), and battery or antenna problems (12%). The majority of the electrical problems were due to disconnection of the in-line plugs, whose reassembly restored full function to the valve. Generally, a higher incidence of blockages was recorded during grazing, while equipment failure mainly occurred during periods dominated by browsing activity. Notwithstanding the relatively high rate of equipment failure, this technique provides a practical method for the collection of multiple oesophageal fistula samples throughout the day without disturbing the animal's normal feeding behaviour.

A comparison of rumen fluid sampled by oro-ruminal probe versus rumen fistula

Ten adult (height 63–85 cm) rumen-fistulated sheep were used to test the usefulness of an oro-ruminal probe and a suction pump for the acquisition of ruminal fluid. Use of these instruments in 50 samplings rendered 200 ml of ruminal fluid each time. Introduction of the probe to a length of 91.3 ± 3.5 cm took 18 ± 6.5 s. Collection of 200 ml of ruminal fluid took 7.4 ± 2.2 s. Sampling location was the ventral ruminal sack. Comparing fluid taken by the oro-ruminal probe with samples taken via ruminal fistula by a tube-like probe, no significant differences in pH, total acidity, reduction potential, ammonia, acetate, L-lactate, sodium and chloride concentrations were found. However, significant differences between oro-ruminal probe samples and fistula samples were observed with regard to the concentrations of total volatile fatty acids, propionate, n-butyrate, i-butyrate, n-valerate, i-valerate, calcium, phosphate, magnesium and potassium. The differences were independent of the sampling order (oro-ruminal probe prior to fistula and vice versa). These results indicate that samples taken by the oro-ruminal probe do not contain sampling-dependent saliva residues. Those samples collected via the fistula had consistently higher concentrations of total volatile fatty acids, propionate, n-butyrate, i-butyrate, n-valerate, and i-valerate, as well as higher concentrations of calcium, phosphate, magnesium and potassium than samples taken with the oro-ruminal probe. These results indicate that samples taken via fistula originated from more dorsal regions than the samples taken by oro-ruminal probe from the ventral ruminal sack. The oro-ruminal probe and the suction pump used proved to be useful for the collection of ruminal fluid from the ventral ruminal sack in adult sheep.

Protein profile of human perilymph: in search of markers for the diagnosis of perilymph fistula and other inner ear disease.

Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) beta 2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins--apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with SP40/40, or cytolysis inhibitor--were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use beta 2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.