Eureka: objective assessment of the empty pelvis syndrome to measure volumetric changes in pelvic dead space following pelvic exenteration.

BACKGROUND: Large tissue defects following pelvic exenteration (PE) fill with fluid and small bowel, leading to the empty pelvis syndrome (EPS). EPS causes a constellation of complications including pelvic sepsis and reduced quality of life. EPS remains poorly defined and cannot be objectively measured. Pathophysiology of EPS is multifactorial, with increased pelvic dead space potentially important. This study aims to describe methodology to objectively measure volumetric changes relating to EPS. METHODS: The true pelvis is defined by the pelvic inlet and outlet. Within the true pelvis there is physiological pelvic dead space (PDS) between the peritoneal reflection and the inlet. This dead space is increased following PE and is defined as the exenteration pelvic dead space (EPD). EPD may be reduced with pelvic filling and the volume of filling is defined as the pelvic filling volume (PFV). PDS, EPD, and PFV were measured intraoperatively using a bladder syringe, and Archimedes' water displacement principle. RESULTS: A patient undergoing total infralevator PE had a PDS of 50 ml. A rectus flap rendered the pelvic outlet watertight. EPD was then measured as 540 ml. Therefore there was a 10.8-fold increase in true pelvis dead space. An omentoplasty was placed into the EPD, displacing 130 ml; therefore, PFV as a percentage of EPD was 24.1%. CONCLUSIONS: This is the first reported quantitative assessment of pathophysiological volumetric changes of pelvic dead space; these measurements may correlate to severity of EPS. PDS, EPD, and PFV should be amendable to assessment based on perioperative cross-sectional imaging, allowing for potential prediction of EPS-related outcomes.

Exercise prehabilitation may lead to augmented tumor regression following neoadjuvant chemoradiotherapy in locally advanced rectal cancer.

  Purpose: We evaluate the effect of an exercised prehabilitation programme on tumour response in rectal cancer patients following neoadjuvant chemoradiotherapy (NACRT). Patients and Methods: Rectal cancer patients with (MRI-defined) threatened resection margins who completed standardized NACRT were prospectively studied in a post hoc, explorative analysis of two previously reported clinical trials. MRI was performed at Weeks 9 and 14 post-NACRT, with surgery at Week 15. Patients undertook a 6-week preoperative exercise-training programme. Oxygen uptake (VO2) at anaerobic threshold (AT) wasmeasured at baseline (pre-NACRT), after completion of NACRT and at week 6 (post-NACRT). Tumour related outcome variables: MRI tumour regression grading (ymrTRG) at Week 9 and 14; histopathological T-stage (ypT); and tumour regression grading (ypTRG)) were compared. Results: 35 patients (26 males) were recruited. 26 patients undertook tailored exercise-training with 9 unmatched controls. NACRT resulted in a fall in VO2 at AT -2.0 ml/kg-1/min-1(-1.3,-2.6), p < 0.001. Exercise was shown to reverse this effect. VO2 at AT increased between groups, (post-NACRT vs. week 6) by +1.9 ml/kg-1/min-1(0.6, 3.2), p = 0.007. A significantly greater ypTRG in the exercise group at the time of surgery was found (p = 0.02). Conclusion: Following completion of NACRT, exercise resulted in significant improvements in fitness and augmented pathological tumour regression.

Time from colorectal cancer diagnosis to laparoscopic curative surgery-is there a safe window for prehabilitation?

BACKGROUND: There is a growing interest in the adoption of formal prehabilitation programmes prior to elective surgery but regulatory targets mandate prompt treatment following cancer diagnosis. We aimed to investigate if time from diagnosis to surgery is linked to short- and long-term outcomes. METHODS: An exploratory analysis was performed utilising a dedicated, prospectively populated database. Inclusion criteria were biopsy-proven colorectal adenocarcinoma undergoing elective laparoscopic surgery with curative intent. Demographics, date of diagnosis and surgery was captured with patients dichotomised using 4-, 8- and 12-week time points. All patients were followed in a standardised pathway for 5 years. Overall survival was assessed with the Kaplan-Meier log-rank method. RESULTS: Six hundred sixty-eight consecutive patients met inclusion criteria. Mean time from diagnosis to surgery was 53 days (95% CI 48.3-57.8). Identified risk factors for longer time to surgery were males (OR 1.92 [1.2-3.1], p = 0.008), age ≤ 65 (OR 1.9 [1.2-3], p = 0.01), higher ASA scores (p = 0.01) stoma formation (OR 6.9 [4.1-11], p < 0.001) and neoadjuvant treatment (OR 5.06 [3.1-8.3], p < 0.001). There was no association between time to surgery and BMI (p = 0.36), conversion (16.3%, p = 0.5), length of stay (p = 0.33) and readmission or reoperation (p = 0.3). No differences in five-year survival were seen in those operated within 4, 8 and 12 weeks (p = 0.397, p = 0.962 and p = 0.611, respectively). Multivariate analysis showed time from diagnosis to surgery was not associated with five-year overall survival (HR 0.99, p = 0.52). CONCLUSION: Time from colorectal cancer diagnosis to curative laparoscopic surgery did not impact on overall survival. This finding may allow preoperative pathway alteration without compromising safety.

Systematic review: the impact of exercise on mesenteric blood flow and its implication for preoperative rehabilitation.

BACKGROUND: Exercise in the preoperative period, or prehabilitation, continues to evolve as an important tool in optimising patients awaiting major intra-abdominal surgery. It has been shown to reduce rates of post-operative morbidity and length of hospital stay. The mechanism by which this is achieved remains poorly understood. Adaptations in mesenteric flow in response to exercise may play a role in improving post-operative recovery by reducing rates of ileus and anastomotic leak. AIMS: To systematically review the existing literature to clarify the impact of exercise on mesenteric arterial blood flow using Doppler ultrasound. METHODS: PubMed, EMBASE and the Cochrane library were systematically searched to identify clinical trials using Doppler ultrasound to investigate the effect of exercise on flow through the superior mesenteric artery (SMA). Data were extracted including participant characteristics, frequency, intensity, timing and type of exercise and the effect on SMA flow. The quality of each study was assessed using the Downs and Black checklist. RESULTS: Sixteen studies, comprising 305 participants in total, were included. Methodological quality was generally poor. Healthy volunteers were used in twelve studies. SMA flow was found to be reduced in response to exercise in twelve studies, increased in one and unchanged in two studies. Clinical heterogeneity precluded a meta-analysis. CONCLUSION: The weight of evidence suggests that superior mesenteric arterial flow is reduced immediately following exercise. Differences in frequency, intensity, timing and type of exercise make a consensus difficult. Further studies are warranted to provide a definitive understanding of the impact of exercise on mesenteric flow.

Validation of preoperative cardiopulmonary exercise testing-derived variables to predict in-hospital morbidity after major colorectal surgery.

BACKGROUND: In single-centre studies, postoperative complications are associated with reduced fitness. This study explored the relationship between cardiorespiratory fitness variables derived by cardiopulmonary exercise testing (CPET) and in-hospital morbidity after major elective colorectal surgery. METHODS: Patients underwent preoperative CPET with recording of in-hospital morbidity. Receiver operating characteristic (ROC) curves and logistic regression were used to assess the relationship between CPET variables and postoperative morbidity. RESULTS: Seven hundred and three patients from six centres in the UK were available for analysis (428 men, 275 women). ROC curve analysis of oxygen uptake at estimated lactate threshold (V˙o2 at θ^L ) and at peak exercise (V˙o2peak ) gave an area under the ROC curve (AUROC) of 0·79 (95 per cent c.i. 0·76 to 0·83; P < 0·001; cut-off 11·1 ml per kg per min) and 0·77 (0·72 to 0·82; P < 0·001; cut-off 18·2 ml per kg per min) respectively, indicating that they can identify patients at risk of postoperative morbidity. In a multivariable logistic regression model, selected CPET variables and body mass index (BMI) were associated significantly with increased odds of in-hospital morbidity (V˙o2 at θ^L 11·1 ml per kg per min or less: odds ratio (OR) 7·56, 95 per cent c.i. 4·44 to 12·86, P < 0·001; V˙o2peak 18·2 ml per kg per min or less: OR 2·15, 1·01 to 4·57, P = 0·047; ventilatory equivalents for carbon dioxide at estimated lactate threshold (V˙E /V˙co2 at θ^L ) more than 30·9: OR 1·38, 1·00 to 1·89, P = 0·047); BMI exceeding 27 kg/m2 : OR 1·05, 1·03 to 1·08, P < 0·001). A laparoscopic procedure was associated with a decreased odds of complications (OR 0·30, 0·02 to 0·44; P = 0·033). This model was able to discriminate between patients with, and without in-hospital morbidity (AUROC 0·83, 95 per cent c.i. 0·79 to 0·87). No adverse clinical events occurred during CPET across the six centres. CONCLUSION: These data provide further evidence that variables derived from preoperative CPET can be used to assess risk before elective colorectal surgery.

The effect of neoadjuvant chemotherapy and chemoradiotherapy on exercise capacity and outcome following upper gastrointestinal cancer surgery: an observational cohort study.

BACKGROUND: In 2014 approximately 21,200 patients were diagnosed with oesophageal and gastric cancer in England and Wales, of whom 37 % underwent planned curative treatments. Potentially curative surgical resection is associated with significant morbidity and mortality. For operable locally advanced disease, neoadjuvant chemotherapy (NAC) improves survival over surgery alone. However, NAC carries the risk of toxicity and is associated with a decrease in physical fitness, which may in turn influence subsequent clinical outcome. Lower levels of physical fitness are associated with worse outcome following major surgery in general and Upper Gastrointestinal Surgery (UGI) surgery in particular. Cardiopulmonary exercise testing (CPET) provides an objective assessment of physical fitness. The aim of this study is to test the hypothesis that NAC prior to upper gastrointestinal cancer surgery is associated with a decrease in physical fitness and that the magnitude of the change in physical fitness will predict mortality 1 year following surgery. METHODS: This study is a multi-centre, prospective, blinded, observational cohort study of participants with oesophageal and gastric cancer scheduled for neoadjuvant cancer treatment (chemo- and chemoradiotherapy) and surgery. The primary endpoints are physical fitness (oxygen uptake at lactate threshold measured using CPET) and 1-year mortality following surgery; secondary endpoints include post-operative morbidity (Post-Operative Morbidity Survey (POMS)) 5 days after surgery and patient related quality of life (EQ-5D-5 L). DISCUSSION: The principal benefits of this study, if the underlying hypothesis is correct, will be to facilitate better selection of treatments (e.g. NAC, Surgery) in patients with oesophageal or gastric cancer. It may also be possible to develop new treatments to reduce the effects of neoadjuvant cancer treatment on physical fitness. These results will contribute to the design of a large, multi-centre trial to determine whether an in-hospital exercise-training programme that increases physical fitness leads to improved overall survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01325883 - 29(th) March 2011.

The Effect of beta-blockade on objectively measured physical fitness in patients with abdominal aortic aneurysms--A blinded interventional study.

BACKGROUND: Perioperative beta-blockade is widely used, especially before vascular surgery; however, its impact on exercise performance assessed using cardiopulmonary exercise testing (CPET) in this group is unknown. We hypothesized that beta-blocker therapy would significantly improve CPET-derived physical fitness in this group. METHODS: We recruited patients with abdominal aortic aneurysms (AAA) of <5.5 cm under surveillance. All patients underwent CPET on and off beta-blockers. Patients routinely prescribed beta-blockers underwent a first CPET on medication. Beta-blockers were stopped for one week before a second CPET. Patients not routinely taking beta-blockers underwent the first CPET off treatment, then performed a second CPET after commencement of bisoprolol for at least 48 h. Oxygen uptake (.VO2) at estimated lactate threshold and .VO2 at peak were primary outcome variables. A linear mixed-effects model was fitted to investigate any difference in adjusted CPET variables on and off beta-blockers. RESULTS: Forty-eight patients completed the study. No difference was observed in .VO2 at estimated lactate threshold and .VO2 at peak; however, a significant decrease in .VE/.VCO2 at estimated lactate threshold and peak, an increase in workload at estimated lactate threshold., O2 pulse and heart rate both at estimated lactate threshold and peak was found with beta-blockers. Patients taking beta-blockers routinely (chronic group) had worse exercise performance (lower .VO2 ). CONCLUSIONS: Beta blockade has a significant impact on CPET-derived exercise performance, albeit without changing .VO2 at estimated lactate threshold and.VO2 at peak. This supports performance of preoperative CPET on or off beta-blockers depending on local perioperative practice. CLINICAL TRIAL REGISTRATION: NCT 02106286.

Effect of prehabilitation on objectively measured physical fitness after neoadjuvant treatment in preoperative rectal cancer patients: a blinded interventional pilot study.

BACKGROUND: Patients requiring surgery for locally advanced rectal cancer often additionally undergo neoadjuvant chemoradiotherapy (NACRT), of which the effects on physical fitness are unknown. The aim of this feasibility and pilot study was to investigate the effects of NACRT and a 6 week structured responsive exercise training programme (SRETP) on oxygen uptake [Formula: see text] at lactate threshold ([Formula: see text]) in such patients. METHODS: We prospectively studied 39 consecutive subjects (27 males) with T3-4/N+ resection margin threatened rectal cancer who completed standardized NACRT. Subjects underwent cardiopulmonary exercise testing at baseline (pre-NACRT), at week 0 (post-NACRT), and week 6 (post-SRETP). Twenty-two subjects undertook a 6 week SRETP on a training bike (three sessions per week) between week 0 and week 6 (exercise group). These were compared with 17 contemporaneous non-randomized subjects (control group). Changes in [Formula: see text] at [Formula: see text] over time and between the groups were compared using a compound symmetry covariance linear mixed model. RESULTS: Of 39 recruited subjects, 22 out of 22 (exercise) and 13 out of 17 (control) completed the study. There were differences between the exercise and control groups at baseline [age, ASA score physical status, World Health Organisation performance status, and Colorectal Physiologic and Operative Severity Score for the Enumeration of Mortality and Morbidity (CR-POSSUM) predicted mortality]. In all subjects, [Formula: see text] at [Formula: see text] significantly reduced between baseline and week 0 [-1.9 ml kg(-1) min(-1); 95% confidence interval (CI) -1.3, -2.6; P<0.0001]. In the exercise group, [Formula: see text] at [Formula: see text] significantly improved between week 0 and week 6 (+2.1 ml kg(-1) min(-1); 95% CI +1.3, +2.9; P<0.0001), whereas the control group values were unchanged (-0.7 ml kg(-1) min(-1); 95% CI -1.66, +0.37; P=0.204). CONCLUSIONS: NACRT before rectal cancer surgery reduces physical fitness. A structured exercise intervention is feasible post-NACRT and returns fitness to baseline levels within 6 weeks. CLINICAL TRIAL REGISTRATION NCT: 01325909.

Cardiopulmonary exercise testing for the prediction of morbidity risk after rectal cancer surgery.

BACKGROUND: This study investigated the relationship between objectively measured physical fitness variables derived by cardiopulmonary exercise testing (CPET) and in-hospital morbidity after rectal cancer surgery. METHODS: Patients scheduled for rectal cancer surgery underwent preoperative CPET (reported blind to patient characteristics) with recording of morbidity (recorded blind to CPET variables). Non-parametric receiver operating characteristic (ROC) curves and logistic regression were used to assess the relationship between CPET variables and postoperative morbidity. RESULTS: Of 105 patients assessed, 95 (72 men) were included; ten patients had no surgery and were excluded (3 by choice, 7 owing to unresectable metastasis). Sixty-eight patients had received neoadjuvant treatment. ROC curve analysis of oxygen uptake (V˙o2 ) at estimated lactate threshold (θ^L ) and peak V˙o2 gave an area under the ROC curve of 0·87 (95 per cent confidence interval 0·78 to 0·95; P < 0·001) and 0·85 (0·77 to 0·93; P < 0·001) respectively, indicating that they can help discriminate patients at risk of postoperative morbidity. The optimal cut-off points identified were 10·6 and 18·6 ml per kg per min for V˙o2 at θ^L and peak respectively. CONCLUSION: CPET can help predict morbidity after rectal cancer surgery.

Cardiopulmonary exercise variables are associated with postoperative morbidity after major colonic surgery: a prospective blinded observational study.

BACKGROUND: Postoperative complications are associated with reduced fitness. Cardiopulmonary exercise testing (CPET) has been used in risk stratification. We investigated the relationship between preoperative CPET and in-hospital morbidity in major colonic surgery. METHODS: We prospectively studied 198 patients undergoing major colonic surgery (excluding neoadjuvant cancer therapy), performing preoperative CPET (reported blind to clinical state), and recording morbidity (assessed blind to CPET), postoperative outcome, and length of stay. RESULTS: Of 198 patients, 62 were excluded: 11 had emergency surgery, 25 had no surgery, 23 had incomplete data, and three were unable to perform CPET. One hundred and thirty-six (89 males, 47 females) were available for analysis. The median age was 71 [inter-quartile range (IQR) 62-77] yr. Sixty-five patients (48%) had a complication at day 5 after operation. Measurements significantly lower in patients with complications than those without were O2 uptake (VO2) at estimated lactate threshold (θ(L)) [median 9.9 (IQR 8.3-12.7) vs 11.2 (9.5-14.2) ml kg(-1) min(-1), P<0.01], VO2 at peak [15.2 (12.6-18.1) vs 17.2 (13.7-22.5) ml kg(-1) min(-1), P=0.01], and ventilatory equivalent for CO2 (V(E)/VCO2) at θ(L) [31.3 (28.0-34.8) vs 33.9 (30.0-39.1), P<0.01]. A final multivariable logistic regression model contained VO2 at θ(L) {one-point change odds ratio (OR) 0.77 [95% confidence interval (CI) 0.66-0.89], P<0.0005; two-point change OR 0.61 (0.46-0.81) and gender [OR 4.42 (1.78-9.88), P=0.001]}, and was reasonably able to discriminate those with and without complications (AUC 0.71, CI 0.62-0.80, 68% sensitivity, 65% specificity). CONCLUSIONS: CPET variables are associated with postoperative morbidity. A multivariable model with VO2 at θ(L) and gender discriminates those with complications after colonic surgery.

Oesophageal cancer: The effect of early nutrition support on clinical outcomes.

BACKGROUND AND AIMS: Malnutrition is prevalent in oesophageal cancer. Evidence for the use of nutrition support and prehabilitation in this cohort is variable. The aim of this study was to examine the effect of early nutrition support and functional measures of nutritional status on post-operative outcomes in adult patients with oesophageal cancer. METHODS: Retrospective review of adults with oesophageal cancer undergoing oesophagectomy (n = 151). Early nutrition support was defined as: oral or enteral nutrition supplementation during neoadjuvant treatment. Late nutrition support defined as: oral or enteral nutrition supplementation prescribed post-operatively. Nutrition outcome measures were; percentage weight loss from 3 to 6 months prior to diagnosis, peri- and post-operatively, and pre-operative assessment of handgrip-strength (HGS). RESULTS: Pre-operative weight loss ≥10% was a significant predictor of mortality at 1 year (OR 2.84, 95%CI 1.03-7.83, p = 0.04) independent of tumour stage, adjuvant treatment, age and gender. Adults prescribed early nutrition support during neoadjuvant treatment experienced less weight loss at 12-months post-oesophagectomy compared to adults prescribed late oral nutrition support (p=<0.05). Pre-operative HGS measurements were not a useful predictor of postoperative complications (p = 0.2), length of stay (p = 0.9) or 90-day mortality (p = 0.6). CONCLUSIONS: Pre-operative weight loss ≥10% was associated with mortality. Early nutrition support was associated with less weight loss at 12-months post-operatively. Pre-operative HGS measures did not have prognostic value as a stand-alone measure. Future work should investigate the efficacy of early nutrition support in reducing both pre- and post-operative weight loss to improve nutritional status and surgical outcomes as part of a multimodal prehabilitation programme in adults with oesophageal cancer.

An L-arginine-dependent mechanism mediates Kupffer cell inhibition of hepatocyte protein synthesis in vitro.

The hepatic failure associated with severe sepsis is characterized by specific, progressive, and often irreversible defects in hepatocellular metabolism (1). Although the etiologic microbe can often be identified, the direct causes and mechanisms of the hepatocellular dysfunction are poorly understood. We have hypothesized that Kupffer cells (KC), which interact with ambient septic stimuli, respond by providing signals to adjacent hepatocytes (HC) in sepsis . Furthermore, we have provided evidence (2, 3) that KC activated by LPS from Gram-negative bacteria can induce profound changes in the function of neighboring HC in coculture. In our model, coculture of either KC (2) or peritoneal macrophages (Mphi)(3) with HC normally promotes HC protein synthesis ([(3)H]leucine incorporation). The addition of LPS or killed Escherichia colt' to such cocultures induces a profound decrease in HC protein synthesis, as well as qualitative changes ([(35)S]methionine, SDS-gel electrophoresis) in protein synthesis without inducing HC death (2, 3) . In this report we show that the inhibition in protein synthesis is mediated via an L-arginine-dependent mechanism. The metabolism of L-arginine by activated Mphi to substances with cytostatic and even lethal effects on target cells is a relatively recent discovery. After the description by Stuehr and Marletta (4, 5) that LPS- triggered Mphi produced nitrite/nitrate (NO(2)(-)/NO(3)(-)), Hibbs et al. (6, 7) and Iyengar et al. (8) demonstrated that L-arginine was the substrate for the formation of both these nitrogen end products and citrulline. A role for the arginine-dependent mechanism in Mphi tumor cytotoxicity (6, 7) and microbiostatic activity (9) has been suggested. However, the in vivo functions of this novel Mphi mechanism have not yet been defined, but it is possible that there are both physiologic as well as pathologic roles. Our in vitro results raise the possibility that some metabolic responses to microbial invasion maybe partially mediated by the L-arginine-dependent mechanism. What other metabolic responses are affected and the possible pathologic consequences remain to be studied.

Parvovirus-associated cerebellar hypoplasia and hydrocephalus in day old broiler chickens.

Cerebellar hypoplasia and hydrocephalus were identified in day old broiler chickens showing nervous signs, impaired mobility, and diarrhea. At postmortem examination, brains of chickens were misshapen and cerebellums were smaller than normal. Microscopically, cerebellar folia were reduced in size and irregularly shaped, and the ventricles were widely distended. Affected cerebellums had focal areas along the base of folia where the internal granular cell layer had been lost, and Purkinje cells were disorganized and located within the molecular layer. Parvovirus DNA was detected by polymerase chain reaction in three of nine brains with oligonucleotide primers designed for amplification of chicken and turkey parvoviruses. On the basis of phylogenetic analyses, the detected virus was most closely related to chicken parvoviruses. These findings suggest that a chicken parvovirus might cause a neurologic disease of young chickens characterized by cerebellar hypoplasia and hydrocephalus; however, its role as the cause of the disease remains to be confirmed.

Why women are not small men: sex-related differences in perioperative cardiopulmonary exercise testing.

BACKGROUND: The use of preoperative cardiopulmonary exercise testing (CPET) to evaluate the risk of adverse perioperative outcomes is increasingly prevalent. CPET-derived information enables personalised perioperative care and enhances shared decision-making. Sex-related differences in physical fitness are reported in non-perioperative literature. However, little attention has been paid to sex-related differences in the context of perioperative CPET. AIM: We explored differences in the physical fitness variables reported in a recently published multi-centre study investigating CPET before colorectal surgery. We also report the inclusion rate of females in published perioperative CPET cohorts that are shaping guidelines and clinical practice. METHODS: We performed a post hoc analysis of the trial data of 703 patients who underwent CPET prior to major elective colorectal surgery. We also summarised the female inclusion rate in peer-reviewed published reports of perioperative CPET. RESULTS: Fitness assessed using commonly used perioperative CPET variables-oxygen consumption at anaerobic threshold (AT) and peak exercise-was significantly higher in males than in females both before and after correction for body weight. In studies contributing to the development of perioperative CPET, 68.5% of the participants were male. CONCLUSION: To our knowledge, this is the first study to describe differences between males and females in CPET variables used in a perioperative setting. Furthermore, there is a substantial difference between the inclusion rates of males and females in this field. These findings require validation in larger cohorts and may have significant implications for both sexes in the application of CPET in the perioperative setting.

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells.

Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF-stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.

The role of ADP-ribosylation factor and phospholipase D in adaptor recruitment.

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTPgammaS stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093-1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTPgammaS-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPgammaS loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTPgammaS. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTPgammaS or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.

A novel adaptor-related protein complex.

Coat proteins are required for the budding of the transport vesicles that mediate membrane traffic pathways, but for many pathways such proteins pathways, but for many pathways such proteins have not yet been identified. We have raised antibodies against p47, a homologue of the medium chains of the adaptor complexes of clathrin-coated vesicles (Pevsner, J., W. Volknandt, B.R. Wong, and R.H. Scheller. 1994. Gene (Amst.). 146:279-283), to determine whether this protein might be a component of a new type of coat. p47 coimmunoprecipitates with three other proteins: two unknown proteins of 160 and 25 kD, and beta-NAP, a homologue of the beta/beta'-adaptins, indicating that it is a subunit of an adaptor-like heterotetrameric complex. However, p47 is not enriched in preparations of clathrin-coated vesicles. Recruitment of the p47-containing complex onto cell membranes is stimulated by GTP gamma S and blocked by brefeldin A, indicating that, like other coat proteins, its membrane association is regulated by an ARF. The newly recruited complex is localized to non-clathrin-coated buds and vesicles associated with the TGN. Endogenous complex in primary cultures of neuronal cells is also localized to the TGN, and in addition, some complex is associated with the plasma membrane. These results indicate that the complex is a component of a novel type of coat that facilitates the budding of vesicles from the TGN, possibly for transporting newly synthesized proteins to the plasma membrane.

Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation.

BACKGROUND: Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells. RESULTS: We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis. CONCLUSIONS: Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.