Asymmetrical elevation of esophagogastric junction pressure suggests hiatal repair contributes to antireflux surgery dysphagia.

The radial distribution of esophago-gastric junction (EGJ) pressures with regard to troublesome dysphagia (TDysph) after antireflux surgery is poorly understood. Before and after antireflux surgery, end-expiratory and peak-inspiratory EGJ pressures were measured at eight angles of 45° radial separation in patients with reflux disease. All 34 patients underwent posterior crural repair, then either 90° anterior (N = 13) or 360° fundoplication (N = 21). Dysphagia was assessed prospectively using a validated questionnaire (score range 0-45) and TDysph defined as a dysphagia score that was ≥5 above pre-op baseline. Compared with before surgery, for 90° fundoplication, end-expiratory EGJ pressures were highest in the left-anterolateral sectors, the position of the partial fundoplication. In other sectors, pressures were uniformly elevated. Compared with 90° fundoplication, radial pressures after 360° fundoplication were higher circumferentially (P = 0.004), with a posterior peak. Nine patients developed TDysph after surgery with a greater increase in end-expiratory and peak-inspiratory EGJ pressures (P = 0.03 and 0.03, respectively) and significantly higher inspiratory pressure at the point of maximal radial pressure asymmetry (P = 0.048), compared with 25 patients without TDysph. Circumferential elevation of end-expiratory EGJ pressure after 90° and 360° fundoplication suggests hiatal repair elevates EGJ pressure by extrinsic compression. The highly localized focal point of elevated EGJ pressure upon inspiration in patients with TDysph after surgery is indicative of a restrictive diaphragmatic hiatus in the presence of a fundoplication.

Correlation of esophageal pressure-flow analysis findings with bolus transit patterns on videofluoroscopy.

Pressure-flow analysis quantifies the interactions between bolus transport and pressure generation. We undertook a pilot study to assess the interrelationships between pressure-flow metrics and fluoroscopically determined bolus clearance and bolus transport across the esophagogastric junction (EGJ). We hypothesized that findings of abnormal pressure-flow metrics would correlate with impaired bolus clearance and reduced flow across the EGJ. Videofluoroscopic images, impedance, and pressure were recorded simultaneously in nine patients with dysphagia (62-82 years, seven male) tested with liquid barium boluses. A 3.6 mm diameter solid-state catheter with 25 × 1 cm pressure/12 × 2 cm impedance was utilized. Swallowed bolus clearance was assessed using a validated 7-point radiological bolus transport scale. The cumulative period of bolus flow across the EGJ was also fluoroscopically measured (EGJ flow time). Pressure only parameters included the length of breaks in the 20 mmHg iso-contour and the 4 second integrated EGJ relaxation pressure (IRP4s). Pressure-flow metrics were calculated for the distal esophagus, these were: time from nadir impedance to peak pressure (TNadImp to PeakP) to quantify bolus flow timing; pressure flow index (PFI) to integrate bolus pressurization and flow timing; and impedance ratio (IR) to assess bolus clearance. When compared with controls, patients had longer peristaltic breaks, higher IRs, and higher residual EGJ relaxation pressures (break length of 8 [2, 13] vs. 2 [0, 2] cm, P = 0.027; IR 0.5 ± 0.1 vs. 0.3 ± 0.0, P = 0.019; IRP4s 11 ± 2 vs. 6 ± 1 mmHg, P = 0.070). There was a significant positive correlation between higher bolus transport scores and longer peristaltic breaks (Spearman correlation r = 0.895, P < 0.001) and with higher IRs (r = 0.661, P < 0.05). Diminished EGJ flow times correlated with a shorter TNadImp to PeakP (r = -0.733, P < 0.05) and a higher IR (r = -0.750, P < 0.05). Longer peristaltic breaks and higher IR correlate with failed bolus clearance on videofluoroscopy. The metric TNadImp to PeakP appears to be a marker of the period of time over which the bolus flows across the EGJ.

Inter- and intra-rater reproducibility of automated and integrated pressure-flow analysis of esophageal pressure-impedance recordings.

BACKGROUND: Automated impedance manometry pressure-flow analysis (AIM analysis) determines pressure measurements relative to bolus flow and has to date shown subtle variations in esophageal motility in relation to dysphagia. In this study, we assessed intra- and inter-rater reproducibility of AIM metrics derived using purpose designed software. METHODS: Fifty patients referred for evaluation of gastro-esophageal reflux symptoms (33 men, age 52 ± 1.9 years) underwent combined high-resolution impedance manometry and completed a dysphagia questionnaire. From 10 liquid and 10 viscous swallows, a subset of four swallows (two saline and two viscous) was systematically selected from each patient for manual and AIMplot analysis, which was performed twice by five observers (two experts, three non-experts). Intra- and inter-rater agreement were determined using intraclass correlation coefficients. KEY RESULTS: AIMplot-based analysis showed high intra-rater and inter-rater reproducibility for all metrics (mean ICCs of 0.95 and 0.94, respectively). Reproducibility of metrics derived for liquid and viscous did not differ (ICCs of 0.96 and 0.91 for liquid and viscous, respectively). In addition, metrics derived by experts had an equivalent level of reproducibility compared to non-experts (ICCs of 0.96 and 0.94, respectively). Variables that could be derived with commercial software (ManoView™) correlated highly with variables from AIMplot-based analysis, such as 4-s integrated relaxation pressure (r = 0.85) and the 20-mmHg isobaric contour defect (r = 0.92). CONCLUSIONS & INFERENCES: Esophageal AIM analysis is highly reproducible, independent of an observer's level of experience in esophageal motility. Therefore, AIM analysis produces data that are reliable for clinical and research purposes.

Susceptibility to dysphagia after fundoplication revealed by novel automated impedance manometry analysis.

BACKGROUND: Conventional measures of esophageal pressures or bolus transport fail to identify patients at risk of dysphagia after laparoscopic fundoplication. METHODS: Liquid and viscous swallows were evaluated with impedance/manometry in 19 patients with reflux disease before and after surgery. A new method of automated impedance manometry (AIM) analysis correlated esophageal pressure with impedance data and automatically calculated a range of pressure and bolus movement variables. An iterative analysis determined whether any variables were altered in relation to dysphagia. Standard measures of esophago-gastric junction pressure, bolus presence time, and total bolus transit time were also evaluated. KEY RESULTS: At 5 months postop, 15 patients reported some dysphagia, including 7 with new-onset dysphagia. For viscous boluses, three AIM-derived pressure-flow variables recorded preoperatively varied significantly in relation to postoperative dysphagia. These were: time from nadir esophageal impedance to peak esophageal pressure (TNadImp-PeakP), median intra-bolus pressure (IBP, mmHg), and the rate of bolus pressure rise (IBP slope, mmHgs(-1) ). These variables were combined to form a dysphagia risk index (DRI=IBP×IBP_slope/TNadImp-PeakP). DRI values derived from preoperative measurements were significantly elevated in those with postoperative dysphagia (DRI=58, IQR=21-408 vs no dysphagia DRI=9, IQR=2-19, P<0.02). A DRI >14 was optimally predictive of dysphagia (sensitivity 75% and specificity 93%). CONCLUSIONS & INFERENCES: Before surgery, a greater and faster compression of a swallowed viscous bolus with less bolus flow time relates to postoperative dysphagia. Thus, susceptibility to postfundoplication dysphagia is related to a pre-existing sub-clinical variation of esophageal function.

Post-fundoplication contrast studies: is there room for improvement?

OBJECTIVE: Since the mid-1990s, laparoscopic fundoplication for gastro-oesophageal reflux disease has become the surgical procedure of choice. Several surgical groups perform routine post-operative contrast studies to exclude any (asymptomatic) anatomical abnormality and to expedite discharge from hospital. The purpose of this study was to determine the accuracy and interobserver reliability for surgeons and radiologists in contrast study interpretation. METHODS: 11 surgeons and 13 radiologists (all blinded to outcome) retrospectively reviewed the contrast studies of 20 patients who had undergone a laparoscopic fundoplication. Each observer reported on fundal wrap position, leak or extravasation of contrast and contrast hold-up at the gastro-oesophageal junction (on a scale of 0-4). A κ coefficient was used to evaluate interobserver reliability. RESULTS: Surgeons were more accurate than radiologists in identifying normal studies (specificity = 91.6% vs 78.9%), whereas both groups had similar accuracy in identifying abnormal studies (sensitivity = 82.3% vs 85.2%). There was higher agreement amongst surgeons than amongst radiologists when determining wrap position (κ = 0.65 vs 0.54). Both groups had low agreement when classifying a wrap migration as partial or total (κ = 0.33 vs 0.06). Radiologists were more likely to interpret the position of the wrap as abnormal (relative risk = 1.25) while surgeons reported a greater degree of hold-up of contrast at the gastro-oesophageal junction (mean score = 1.17 vs 0.86). CONCLUSION: Radiologists would benefit from more information about the technical details of laparoscopic anti-reflux surgery. Standardised protocols for performing post-fundoplication contrast studies are needed.

Impact of ineffective oesophageal motility and wrap type on dysphagia after laparoscopic fundoplication.

BACKGROUND: Laparoscopic 360° fundoplication is the most common operation for gastro-oesophageal reflux disease, but is associated with postoperative dysphagia in some patients. Patients with ineffective oesophageal motility may have a higher risk of developing postoperative dysphagia, but this remains unclear. METHODS: From 1991 to 2010, 2040 patients underwent primary laparoscopic fundoplication for gastro-oesophageal reflux disease and met the study inclusion criteria; 343 had a 90°, 498 a 180° and 1199 a 360° fundoplication. Primary peristalsis and distal contraction amplitude during oesophageal manometry were determined for 1354 patients. Postoperative dysphagia scores (range 0-45) were recorded at 3 and 12 months, then annually. Oesophageal dilatations and/or reoperations for dysphagia were recorded. RESULTS: Preoperative oesophageal motility did not influence postoperative dysphagia scores, the need for dilatation and/or reoperation up to 6 years. Three-month dysphagia scores were lower after 90° and 180° compared with 360° fundoplication (mean(s.e.m.) 8·0(0·6) and 9·8(0·5) respectively versus 11·9(0·4); P < 0·001 and P = 0·003), but these differences diminished after 6 years of follow-up. The incidence of dilatation and reoperation for dysphagia was lower after 90° (2·6 and 0·6 per cent respectively) and 180° (4·4 and 1·0 per cent) fundoplications than with a 360° wrap (9·8 and 6·8 per cent; both P < 0·001 versus 90° and 180° groups). CONCLUSION: Tailoring the degree of fundoplication according to preoperative oesophageal motility by standard manometric parameters has no long-term impact on postoperative dysphagia. There is, however, a proportionate increase in short-term dysphagia scores with increasing degree of wrap, and a corresponding proportionate increase in dilatations and reoperations for dysphagia. These differences in dysphagia scores diminish with time.

Long-term outcomes of revisional surgery following laparoscopic fundoplication.

BACKGROUND: A small proportion of patients who have laparoscopic antireflux procedures require revisional surgery. This study investigated long-term clinical outcomes. METHODS: Patients requiring late revisional surgery following laparoscopic fundoplication for gastro-oesophageal reflux were identified from a prospective database. Long-term outcomes were determined using a questionnaire evaluating symptom scores for heartburn, dysphagia and satisfaction. RESULTS: The database search found 109 patients, including 98 (5.6 per cent) of 1751 patients who had primary surgery in the authors' unit. Indications for surgical revision were dysphagia (52 patients), recurrent reflux (36), mechanical symptoms related to paraoesophageal herniation (16) and atypical symptoms (five). The median time to revision was 26 months. Outcome data were available for 104 patients (median follow-up 66 months) and satisfaction data for 102, 88 of whom were highly satisfied (62.7 per cent) or satisfied (23.5 per cent) with the outcome. Patients who had revision for dysphagia had a higher incidence of poorly controlled heartburn (20 versus 2 per cent; P = 0.004), troublesome dysphagia (16 versus 6 per cent; P = 0.118) and a lower satisfaction score (P = 0.023) than those with recurrent reflux or paraoesophageal herniation. CONCLUSION: Revisional surgery following laparoscopic fundoplication can produce good long-term results, but revision for dysphagia has less satisfactory outcomes.

Symptoms experienced during 24-h pH monitoring and their relationship to outcome after laparoscopic total fundoplication.

Some patients having a 24-h pH monitoring test prior to laparoscopic fundoplication experience no symptoms at all in spite of having a positive test, and other patients experience only atypical symptoms in spite of having a positive test. This study investigates the postoperative outcome of such patients. All patients underwent esophageal manometry and 24-h esophageal pH monitoring before laparoscopic total fundoplication. Patients were divided into three groups based on their symptom profile recorded during a positive 24-h pH monitoring: those with typical symptoms (n = 104), those with atypical symptoms (n = 28) and those who experienced no symptoms at all (n = 23). The outcomes measured were heartburn score (0-10), dysphagia composite score (0-45) and satisfaction score (0-10) at 12 months after surgery. Outcome analysis reveals the heartburn scores were significantly reduced postoperatively for all groups of patients. At 1 year after surgery, there was no difference among the three groups of patients in terms of heartburn score and dysphagia composite scores, nor the experience of bloating, belching, or their willingness to repeat surgery. Despite one group experiencing no symptoms, and another group atypical symptoms during a positive pH study, the postoperative satisfaction scores for these two groups was good, but significantly less (P = 0.03, P = 0.02, respectively) than the group of patients with a typical symptom index. In conclusion, patients who experience only atypical symptoms or no symptoms at all during their preoperative positive 24-h pH monitoring may still obtain a good result from antireflux surgery. However, these symptom profiles should alert the surgeon that such patients may have an outcome which is not as good as patients who experience only typical symptoms during a pH study.

Esophageal ileus following laparoscopic fundoplication.

Early postoperative dysphagia occurs in most patients following laparoscopic fundoplication. Whether dysphagia is associated with a change in esophageal motor function and/or a change in gastroesophageal junction characteristics is unknown. Esophageal motility in the early postoperative period has not been evaluated previously. Esophageal motility was studied on the first postoperative day in 10 patients who underwent laparoscopic Nissen fundoplication and 10 patients who underwent laparoscopic cholecystectomy (control group), using standard perfusion manometry. Primary peristalsis on water swallows following fundoplication elicted a median response of 5% successful peristalsis compared with median response of 100% successful peristalsis following cholecystectomy (P = 0.05). The fundoplication was associated with failure of primary esophageal peristalsis in 7/10 patients, compared to 2/10 patients who underwent cholecystectomy (P = 0.068 Fisher's exact test). Three months after fundoplication, in nine patients studied, primary peristalsis was similar to peristalsis observed preoperatively in seven patients and two patients still had an aperistaltic esophagus. In this study, esophageal manometry 1 day after surgery demonstrated grossly disturbed esophageal motility in most patents following laparoscopic fundoplication, compared to normal motility following laparoscopic cholecystectomy. Peristalsis improved at 3 months or more following surgery. This suggests that an 'esophageal ileus' occurs during the early period after laparoscopic fundoplication.

Preoperative gastric emptying and patterns of reflux as predictors of outcome after laparoscopic fundoplication.

BACKGROUND: Some studies have suggested that patients with predominantly upright reflux have a poor outcome after laparoscopic antireflux surgery. It has been proposed that this might be related to gastric emptying. The aim of this study was to evaluate the relationship between preoperative upright reflux pattern, gastric emptying rate and outcome after laparoscopic fundoplication. METHODS: Patients presenting for antireflux surgery underwent 24-h ambulatory pH testing and dual-isotope radionuclide gastric emptying studies. Reflux pattern was determined by the acid exposure in the distal oesophagus (percentage of time at pH < 4) during supine and upright posture. Outcome after fundoplication was assessed by a standardized postal questionnaire administered at 3 months, and 1, 2 and 5 years after surgery. RESULTS: Of 372 patients identified from a prospectively maintained database, 109 had upright reflux, 57 supine reflux and 206 bipositional reflux. Overall patient satisfaction was not influenced by reflux pattern at any time after surgery. Delayed gastric emptying was documented in 31.0 per cent of patients, but there was no relationship with reflux pattern. CONCLUSION: Patients with upright reflux did not have a less favourable outcome after fundoplication. Categorizing patients according to reflux pattern is of no value in predicting outcome or selecting patients for surgery.

Illness behavior and laparoscopic antireflux surgery: tailoring the wrap to suit the patient.

Psychological factors are believed to play a role in gastroesophageal reflux disease. It has previously been shown that preoperative illness behavior influences the outcome after laparoscopic Nissen fundoplication. Between August 2001 and June 2004 we considered a partly subjective assessment of illness behavior when selecting patients with gastroesophageal reflux disease for laparoscopic anterior partial (n = 77) or total fundoplication (n = 90). A prospective questionnaire study of illness behavior was also undertaken and the results were correlated with clinical follow up after 12 months. There was a statistically significant difference in age (P < 0.001), primary esophageal peristalsis on manometry (P = 0.037) and two illness behavior category scores related to hypochondriasis (P = 0.041 and P = 0.025) between laparoscopic anterior partial fundoplication and Nissen total fundoplication groups. Despite these differences, there was no significant correlation between preoperative illness behavior score and patient satisfaction in either group. There was a statistically significant negative correlation between the ability to express personal feelings and postoperative heartburn score in those who had a laparoscopic anterior partial fundoplication (P = 0.048). The clinical outcome in both groups was good to excellent in terms of postoperative heartburn and satisfaction scores. A tailored approach in the choice of wrap, taking into account psychological factors preoperatively, is an appropriate strategy for laparoscopic fundoplication.

Nasopharyngeal pH monitoring in chronic sinusitis patients using a novel four channel probe.

OBJECTIVE: To determine the prevalence of acid reflux into the nasopharynx in patients with chronic sinusitis. STUDY DESIGN: Prospective study of patients presenting to a specialist rhinology practice with chronic sinusitis. METHODS: Forty patients with chronic sinusitis underwent ambulatory 24 hour pH testing. The mean age of the patients was 56.3 years (25 Female, 15 Male). The studies were performed using a specially developed bifurcated 4 channel pH probe, incorporating 2 circumferential sensors positioned at the naso- and hypo-pharynx, and 2 unidirectional sensors positioned at the proximal and distal esophagus. RESULTS: The circumferential band sensors yielded a stable recording largely free of artefact allowing meaningful recordings to be obtained from thirty-seven patients. Twelve patients (32.4%) were diagnosed with gastroesophageal reflux. A total of 809 reflux episodes were recorded. Of these, 596 (73.7%) reached the distal esophagus, with 187 (23.1%) and 24 (3.0%) reaching the proximal esophagus and hypopharynx respectively. Only 2 episodes (0.2%) were recorded in the nasopharynx. This occurred in 2 of 37 patients (5%). CONCLUSIONS: Acid reflux into the nasopharynx is a rare event in patients with chronic sinusitis even though a significant proportion (32.4%) have abnormal 24 hour pH studies. It is likely that alternative mechanisms other than direct acid contact are involved in the pathogenesis of chronic sinusitis.

Postoperative mortality following oesophagectomy and problems in reporting its rate.

BACKGROUND: There have been three previous reviews of the world literature describing postoperative mortality rate following oesophagectomy. The first documented rates in the first half of the last century, the second the period 1960-1979 and the third the interval 1980-1988. The aim of this review was to document the rate for the period 1990-2000. METHODS: Reports were sourced through PubMed and/or Medline listings. RESULTS: The number of papers included in the review was 312, involving 70,756 patients. The overall mortality rate was 6.7 per cent. The 30-day mortality rate was 4.9 per cent and the in-hospital mortality rate 8.8 per cent. Survival rates, where reported, were 62.7 per cent at 1 year and 27.9 per cent at 5 years. CONCLUSION: Operative mortality rates following oesophagectomy have continued to fall. However, the true rate is almost certainly higher than that reported here, for a variety of reasons. The 1-year survival of patients was only reported in about a quarter of the papers. It may be a more meaningful figure than postoperative mortality rate.

Metallothionein in human oesophagus, Barrett's epithelium and adenocarcinoma.

The potential of the metal-binding protein, metallothionein, in assessing the progression of normal oesophagus through Barrett's to adenocarcinoma was investigated. Metallothionein was quantitatively determined in resected tissues from patients undergoing oesophagectomy for high grade dysplasia/adenocarcinoma and in biopsies from patients with Barrett's syndrome. In 10 cancer patients, metallothionein concentrations in adenocarcinoma were not significantly different from normal oesophagus, although six had elevated metallothionein concentrations in the metaplastic tissue bordering the adenocarcinoma. In 17 out of 20 non-cancer patients with Barrett's epithelium, metallothionein was significantly increased by 108% (P<0.004). There was no association between the metallothionein levels in Barrett's epithelium and the presence of inflammatory cells, metaplasia or dysplasia. Metallothionein is a marker of progression from normal to Barrett's epithelium but is not increased in oesophageal adenocarcinoma.

Laparoscopic Nissen fundoplication: five-year results and beyond.

HYPOTHESIS: Laparoscopic Nissen fundoplication provides long-term relief of symptoms of gastroesophageal reflux disease. DESIGN: Prospectively evaluated case series. SETTING: University teaching hospital. PATIENTS: From September 1991 to December 1999, we performed more than 900 laparoscopic antireflux procedures. The outcome for patients who underwent surgery between September 1991 and June 1994 (178 cases) was determined. This included all patients having laparoscopic Nissen fundoplication, from the first procedure onward. INTERVENTIONS: Long-term follow-up for 5 or more years after laparoscopic Nissen fundoplication was obtained by an independent investigator who interviewed patients using a structured questionnaire. MAIN OUTCOME MEASURES: Prospective evaluation of clinical symptoms using a structured questionnaire. RESULTS: Outcome data covering a period of 5 or more years after surgery was available for 176 patients (99%), with 2 patients lost to follow-up. Nine patients died (8 of unrelated causes) at some stage following surgery, and the outcome was difficult to determine in 1 patient with cerebral palsy. Hence, questionnaire data were available for 166 patients at a median follow-up of 6 years (range, 5-8 years). Three patients (1.7%) underwent revision surgery for recurrent reflux; 87% of the 176 patients remained free of significant reflux. Reoperation was required for dysphagia in 7 patients (3.9%), 2 for a tight wrap and 5 for a tight diaphragmatic hiatus. In addition, reoperation was necessary for a paraesophageal hiatus hernia in 13 patients (7.3%). Of the reoperations, 56% were performed within 12 months of the original procedure, and 22% during the second year of follow-up. Further surgery was uncommon after 2 years. The long-term outcome was considered "good or excellent" by 90% of patients. CONCLUSIONS: The long-term outcome of laparoscopic Nissen fundoplication is similar to that following open fundoplication. Good results are obtained in most patients.

Detection of bile reflux: in vivo validation of the Bilitec fibreoptic system.

The Bilitec ambulatory bile reflux monitor is advocated for the assessment of bile reflux. However, it has only been validated by bench top (in vitro) studies. This in vivo study used controlled 'iatrogenic' episodes of bile reflux to determine the ability of the Bilitec system to detect bile acids in the oesophageal lumen. At least 10 ml of fluid was aspirated from the stomach of each of 32 patients, and analysed quantitatively for total bile acids (TBA) and total bilirubin (TB). An in vitro assessment of this fluid with the Bilitec probe was performed initially. The Bilitec probe was then sited in the distal oesophagus, and the gastric fluid was introduced into the mid-oesophagus, through a naso-oesophageal tube, as a series of 10-ml volumes of varying dilutions (1:8, 1:4, 1:2, undiluted) to simulate gastro-oesophageal reflux events. Intraoesophageal bilirubin absorbance was compared with TBA and TB for each dilution. Both TBA and TB values correlated strongly with bilirubin absorbance in vitro (r = 0.83, p < 0.0001 and r = 0.82, p < 0.0001 respectively). However, this relationship was weaker in vivo (r = 0.64, p = 0.0001 and r = 0.68, p < 0.0001 respectively). Of the dilutions which contained potentially injurious concentrations of bile acid (>1 mmol/L), 77% were associated with absorbance >0.14 (currently accepted absorbance threshold for oesophageal bile reflux). Although in vitro sensitivity of the Bilitec probe to bile acids was excellent, in vivo sensitivity was less reliable, and 23% of significant 'bile reflux' episodes did not exceed the currently recommended Bilitec absorbance threshold for bile reflux. In contrast, false-positive results were unlikely. These findings suggest that the overall accuracy of the Bilitec system is probably sufficient for clinical use, although this method is not sufficiently reliable to be regarded as the gold standard for the evaluation of duodeno-oesophageal reflux.

Basement membrane zone type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan in human tissues and cultured cells.

Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The alpha1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino- and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences for O-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the alpha chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size >/=400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250- and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa alpha chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of approximately 8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the approximately 19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and Ständker, L. (1999) Biochemistry 38, 10217-10224), which may be liberated from the alpha1(XV) chain.

Type XV collagen in human colonic adenocarcinomas has a different distribution than other basement membrane zone proteins.

In situ carcinomas must penetrate their own basement membrane to be classified as invasive, and subsequently infiltrate surrounding connective tissue and cross vascular basement membranes to metastasize hematogenously. Accordingly, in many studies, integral basement membrane components, including type IV collagen, laminin, and heparan sulfate proteoglycan, have been localized in a spectrum of tumors to gain insight into their role in neoplasia. A number of recently identified extracellular matrix molecules and isoforms of the aforementioned proteins have been localized to the basement membrane zone, illustrating another level of biochemical heterogeneity in these structures. As the complexity of these matrices becomes more apparent, their roles in maintaining homeostasis and in tumor biology falls into question. Of the new group of collagens localized to the basement membrane zone, type XV was the first to be characterized (Cell Tissue Res, 286:493-505, 1996). This nonfibrillar collagen has a nearly ubiquitous distribution in normal human tissues via a strong association with basement membrane zones, suggesting that it functions to adhere basement membrane to the underlying stroma. To begin investigation of this protein in malignant tumors, we have localized type XV in human colonic adenocarcinomas and compared its distribution with that of type IV collagen and laminin. Collagens XV and IV and laminin were found in all normal and colonic epithelial, muscle, fat, neural, and vascular basement membrane zones, as shown previously. In moderately differentiated, invasive adenocarcinomas, laminin and type IV collagen were sometimes observed as continuous, linear deposits around some of the malignant glands, but more often they were seen in either discontinuous deposits or were completely absent. In contrast, type XV collagen was characterized as virtually absent from the basement membrane zones of malignant glandular elements in moderately differentiated tumors. Nevertheless there were also similarities; all 3 proteins were usually present in the stroma and adjacent vascular basement membrane zones surrounding invasive glands. The loss of type XV collagen from these malignant epithelial basement membrane zones and its increased interstitial expression suggests a role for this protein in the invasive process and the possibility that it may provide a sensitive indicator of tumor invasion.

Purification, crystallization and x-ray analysis of crystals of pectate lyase A from Exwinia chrysanthemi.

Pectate lyase A is secreted by Erwinia chrysanthemi and is a virulence factor for soft rot diseases in plants. Crystals of pectate lyase A were obtained by vapor-diffusion techniques in the presence of polyethylene glycol. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 48.96, b = 148.86, c = 78.61 A, beta = 97.32 degrees. The crystals contain two protein molecules of 38 kDa per asymmetric unit and diffract to 2.4 A using Cu Kalpha radiation.

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.