Long-term outcome of infliximab treatment in chronic active ulcerative colitis: a Swedish multicentre study of 250 patients.

BACKGROUND: Real-life long-term data on infliximab treatment in ulcerative colitis are limited. AIM: To study the long-term efficacy and safety of infliximab in chronic active ulcerative colitis and possible predictors of colectomy and response were also examined. METHODS: A retrospective multi-centre study of infliximab treatment in 250 patients with chronic active ulcerative colitis with inclusion criteria: age ≥18 years, ambulatory treated, steroid-dependent or intolerant and/or immunomodulator refractory or intolerant. RESULTS: Steroid-free clinical remission was achieved by 123/250 patients (49.2%) at 12 months and in 126/250 patients at a median follow-up of 2.9 years (50.4%). Primary response at 3 months was achieved by 190/250 (76.0%) patients and associated with a high probability of response 168/190 (88.4%) at 12 months and 143/190 (75.3%) at follow-up. Long-term rate of colectomy in primary responders was 6/190 (3.2%) at 12 months and 27/190 (14.2%) at last follow-up. Failure to achieve response at 3 months was associated with a high risk of subsequent colectomy, 29/60 (48.3%) at 12 months and 41/60 (68.3%) at follow-up. Response at 12 months was associated with a low risk of subsequent colectomy, 14/181 (7.7%) compared with non-response 19/34 (55.9%) (P < 0.0001). Non-response at 3 months was an independent predictor of subsequent colectomy (HR = 9.40, 95% CI = 5.10-17.35, P < 0.001). Concomitant azathioprine therapy did not influence outcome in terms of colectomy. CONCLUSIONS: Long-term efficacy of infliximab treatment in chronic active ulcerative colitis is excellent especially in patients who respond to induction treatment. Conversely, non-response at 3 months predicts a poor outcome, with a high risk of subsequent colectomy.

Tumor-associated GM-CSF overexpression induces immunoinhibitory molecules via STAT3 in myeloid-suppressor cells infiltrating liver metastases.

Assumptions that liver immune cells and immunosuppressive pathways are similar to their counterparts in other spaces have led to gaps in our understanding of intrahepatic neoplasm aggressiveness. Myeloid-derived suppressor cells (MDSCs) are potent inhibitors of antitumor immunity and pose a major obstacle to solid tumor treatment. Liver MDSCs (L-MDSCs) associated with liver metastases (LM) are particularly problematic by contributing to intrahepatic immunosuppression that promotes tumor progression. L-MDSCs have been reported to expand in response to granulocyte-macrophages colony-stimulating factor (GM-CSF) and suppress antitumor immunity in LM. To extend these findings, we examined mechanisms of intrahepatic immunosuppression exploited by L-MDSCs. We found that the majority of L-MDSCs co-expressed GM-CSF receptor (GM-CSF-R), indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PD-L1), while demonstrating high levels of signal transducer and activator of transcription factor 3 (STAT3) activation. GM-CSF-secreting tumor cells induced STAT3 phosphorylation in L-MDSCs in addition to expression of IDO and PD-L1. GM-CSF or GM-CSF-R blockade markedly reduced L-MDSC IDO and PD-L1 expression, implicating tumor-derived GM-CSF in supporting L-MDSC-immunoinhibitory molecule expression. Small-molecule inhibitors of Janus-activated kinase 2 (JAK2) and STAT3 also dramatically diminished IDO and PD-L1 expression in L-MDSCs. We determined that STAT3 exerts transcriptional control over L-MDSC IDO and PD-L1 expression by binding to the IDO1 and PD-L1 promoters. Our data suggest that the GM-CSF/JAK2/STAT3 axis in L-MDSCs drives immunosuppression in a model of LM and blockade of this pathway may enable rescue of intrahepatic antitumor immunity.

Regional CAR-T cell infusions for peritoneal carcinomatosis are superior to systemic delivery.

Metastatic spread of colorectal cancer (CRC) to the peritoneal cavity is common and difficult to treat, with many patients dying from malignant bowel obstruction. Chimeric antigen receptor T cell (CAR-T) immunotherapy has shown great promise, and we previously reported murine and phase I clinical studies on regional intrahepatic CAR-T infusion for CRC liver metastases. We are now studying intraperitoneal (IP) delivery of CAR-Ts for peritoneal carcinomatosis. Regional IP infusion of CAR-T resulted in superior protection against carcinoembryonic antigen (CEA+) peritoneal tumors, when compared with systemically infused CAR-Ts. IP CAR-Ts also provided prolonged protection against IP tumor re-challenges and demonstrated an increase in effector memory phenotype over time. IP CAR-Ts provided protection against tumor growth at distant subcutaneous (SC) sites in association with increases in serum IFNγ levels. Given the challenges posed by immunoinhibitory pathways in solid tumors, we combined IP CAR-T treatment with suppressor cell targeting. High frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) were found within the IP tumors, with MDSC expressing high levels of immunosuppressive PD-L1. Combinatorial IP CAR-T treatment with depleting antibodies against MDSC and Treg further improved efficacy against peritoneal metastases. Our data support further development of combinatorial IP CAR-T immunotherapy for peritoneal malignancies.

Evidence for the secondary sexual development of the anal fin in female kokanee salmon Oncorhynchus nerka.

This study examines whether the anal fin undergoes secondary sexual development similar to other reproductive traits in salmonids. This hypothesis was tested by comparing the anal-fin size of female kokanee salmon Oncorhynchus nerka that were in the early and late stages of sexual development. Females in an advanced stage of maturation had significantly larger anal fins relative to females in an early state of maturation (+4-7%), indicating that the anal fin undergoes secondary sexual development. The magnitude of this secondary growth was comparable with snout length (+9-10%), which is known to undergo secondary sexual development in female salmonids. When morphological trait dimensions were compared between the sexes, the anal fin was the only morphological trait found to have a female-biased sexual size dimorphism. This is the first study to show that the anal fin of female salmonids undergoes secondary sexual development.

Neutrophil:lymphocyte ratios and serum cytokine changes after hepatic artery chimeric antigen receptor-modified T-cell infusions for liver metastases.

Our phase I Hepatic Immunotherapy for Metastases (HITM) trial tested the safety of chimeric antigen receptor-modified T-cell (CAR-T) hepatic artery infusions (HAI) for unresectable carcinoembryonic antigen (CEA)+ liver metastases (LM). High neutrophil:lymphocyte ratios (NLR) predict poor outcome in cancer patients and we hypothesized that NLR changes would correlate with early responses to CAR-T HAI. Six patients completed the protocol. Three patients received CAR-T HAI in dose escalation (1 × 10(8), 1 × 10(9) and 1 × 10(10) cells) and the remainder received three doses (1 × 10(10) cells) with interleukin (IL)2 support. Serum cytokines and NLR were measured at multiple time points. The mean NLR for all patients was 13.9 (range 4.8-38.1). NLR increased in four patients following treatment with a mean fold change of 1.9. Serum IL6 levels and NLR fold changes demonstrated a trend towards a positive correlation (r=0.77, P=0.10). Patients with poor CEA responses were significantly more likely to have higher NLR level increases (P=0.048). Increased NLR levels were associated with poor responses following CAR-T HAI. NLR variations and associated cytokine changes may be useful surrogates of response to CAR-T HAI.

Inter-observer reproducibility of semi-automatic tumor diameter measurement and volumetric analysis in patients with lung cancer.

OBJECTIVES: Therapy monitoring in oncologic patient requires precise measurement methods. In order to improve the precision of measurements, we used a semi-automated generic segmentation algorithm to measure the size of large lung cancer tumors. The reproducibility of computer-assisted measurements were assessed and compared with manual measurements. METHODS: CT scans of 24 consecutive lung cancer patients who were referred to our hospital over a period of 6 months were analyzed. The tumor sizes were measured manually by 3 independent radiologists, according to World Health Organization (WHO) and the Revised Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. At least 10 months later, measurements were repeated semi-automatically on the same scans by the same radiologists. The inter-observer reproducibility of all measurements was assessed and compared between manual and semi-automated measurements. RESULTS: Manual measurements of the tumor longest diameter were significantly (p < 0.05) smaller compared with the semi-automated measurements. The intra-rater correlations coefficients were significantly higher for measurements of longest diameter (intra-class correlation coefficients: 0.998 vs. 0.986; p < 0.001) and area (0.995 vs. 0.988; p = 0.032) using semi-automated compared with manual method. The variation coefficient for manual measurement of the tumor area (WHO guideline, 15.7% vs. 7.3%) and the longest diameter (RECIST guideline, 7.7% vs. 2.7%) was 2-3 times that of semi-automated measurement. CONCLUSIONS: By using computer-assisted size assessment in primary lung tumor, interobserver-variability can be reduced to about half to one-third compared to standard manual measurements. This indicates a high potential value for therapy monitoring in lung cancer patients.

First record of Aedes japonicus japonicus in Mississippi.

In July 2011, 7 late-stage larvae of Aedes japonicus japonicus were collected from a 5-gal bucket located behind a house in Fulton, MS. Three of the larvae were reared to the adult stage, with the remaining retained in 70% ethanol. Fifteen subsequent attempts over the next month to collect specimens by larval dipping in artificial containers at the property and surrounding towns in 3 adjacent counties all failed to produce any additional Ae. j. japonicus.

Evidence for immunosurveillance in intestinal premalignant lesions.

The immunosurveillance theory argues that the immune system recognizes tumour-specific antigens expressed by transformed cells, which results in the destruction of cancer precursors before they become clinically manifest. As a model for the development of cancer, we set out to study premalignant lesions and immune responses in sentinel lymph nodes from patients with long-standing ulcerative colitis and progression of mucosal dysplasia. Mesenteric lymph nodes draining dysplastic and normal intestinal segments were identified by sentinel node technique during surgery in 13 patients with ulcerative colitis who were subjected to colectomy because of intestinal dysplasia. T cells were extracted from the lymph nodes and analysed by flow cytometry, and lymphocyte proliferation assays were set up in the presence of extracts from dysplastic and normal intestinal mucosa. Increase in CD4/CD8 ratio was observed in sentinel lymph nodes draining dysplastic epithelium compared to normal mucosa. The increase in CD4(+) T cells in relation to CD8(+) T cells correlated with the degree of dysplasia reflected by a significant increase in the ratio against low-grade dysplasia compared to indefinite dysplastic lesions. The T-cell response was specific to antigens from dysplastic epithelial lining as seen in proliferation assays. The observation suggests an important surveillance role for the immune system against premalignant intestinal lesions in patients with long-standing ulcerative colitis.

A device for noninvasive assessment of vascular impairment risk in the lower extremity.

The repeatability and resolution of the clinical gold standard of vascular assessment, the ankle-brachial index (ABI), was compared to that of a new device that dynamically assesses tissue perfusion during external loading utilizing laser Doppler flowmetry. Eight subjects of varying levels of vascular impairment were tested in successive weeks using two different sites on the subject's posterior calf. These new measures included the perfusion decrease as well as the unloading delay during cyclic loading. Some new dynamic tissue perfusion measures demonstrated comparable levels of reproducibility with the ABI (e.g., 10%-20%). Only the unloading delay showed potentially enhanced resolution over ABI measures. The perfusion decrease showed little resolution, and the remaining parameters exhibited too great variability (25%-90%). The unloading delay associated with the reperfusion response during cyclic loading displayed the greatest combination of reproducibility and differentiation between subject groups of varying levels of vascular impairment. The preliminary results of this pilot study were also used to estimate sample sizes necessary to detect possible significant (P<0.05) differences between subject groups for all measured perfusion parameters. From these calculations, at least 30 subjects are needed for future study in each of the five subject groups.

Branching patterns and drainage territories of the middle hepatic vein in computer-simulated right living-donor hepatectomies.

Full right hepatic grafts are most frequently used for adult-to-adult living donor liver transplantation (LDLT). One of the major problems is venous drainage of segments 5 and 8. Thus, this study was designed to provide information on venous drainage of right liver lobes for operation-planning. Fifty-six CT data sets from routine clinical imaging were evaluated retrospectively using a liver operation-planning system. We defined and analyzed venous drainage segments and the impact of anatomic variations of the middle hepatic vein (MHV) on venous outflow from segments 5 and 8. MHV variations led to significant shifts of segment 5 drainage between the middle and right hepatic vein. In cases with the most frequent MHV branching pattern (n = 33), a virtual hepatectomy closely right to the MHV intersected drainage vessels that provided drainage for 30% of the potential graft, not taking into account potential veno-venous shunts. In individuals with inferior MHV branches that extend far into segments 5 and 6 (n = 10), the overall graft volume at risk of impaired venous drainage increased by 5% (p < 0.001). If this is confirmed in clinical trials and correlated with intraoperative findings, the use of liver operation-planning systems would be beneficial to improve overall outcome after right lobe LDLT.

Sentinel node lymphocytes: tumour reactive lymphocytes identified intraoperatively for the use in immunotherapy of colon cancer.

The sentinel node is the first lymph node to receive lymphatic drainage from a tumour and is usually the first site of metastases. Today, the sentinel node is used for tumour staging. Here, we focus on its immunological role and investigate lymphocytic function in sentinel nodes, identified intraoperatively by peritumoural dye injection, from 15 patients with colon cancer. Tumour infiltrating lymphocytes, sentinel and nonsentinel lymph node cells and peripheral blood leukocytes were studied by flow cytometry, proliferation assays and interferon-gamma secretion after activation with autologous tumour homogenate. Whereas tumour-infiltrating lymphocytes were nonresponsive in the proliferation assays, lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon-gamma upon stimulation with tumour homogenate. The responses were of varying magnitude and tended to be weaker in metastatic sentinel nodes. Sentinel node lymphocytes represents an enriched source of tumour reactive lymphocytes, and may be useful in future trials of adoptive immunotherapy.

[The role of 3-D imaging and computer-based postprocessing for surgery of the liver and pancreas]. / Bedeutung der 3-D-Bildgebung und computerbasierten Nachverarbeitung für die Chirurgie der Leber und des Pankreas.

Cross-sectional imaging based on navigation and virtual reality planning tools are well-established in the surgical routine in orthopedic surgery and neurosurgery. In various procedures, they have achieved a significant clinical relevance and efficacy and have enhanced the discipline's resection capabilities. In abdominal surgery, however, these tools have gained little attraction so far. Even with the advantage of fast and high resolution cross-sectional liver and pancreas imaging, it remains unclear whether 3D planning and interactive planning tools might increase precision and safety of liver and pancreas surgery. The inability to simply transfer the methodology from orthopedic or neurosurgery is mainly a result of intraoperative organ movements and shifting and corresponding technical difficulties in the on-line applicability of presurgical cross sectional imaging data. For the interactive planning of liver surgery, three systems partly exist in daily routine: HepaVision2 (MeVis GmbH, Bremen), LiverLive (Navidez Ltd, Slovenia) and OrgaNicer (German Cancer Research Center, Heidelberg). All these systems have realized a half- or full-automatic liver-segmentation procedure to visualize liver segments, vessel trees, resected volumes or critical residual organ volumes, either for preoperative planning or intraoperative visualization. Acquisition of data is mainly based on computed tomography. Three-dimensional navigation for intraoperative surgical guidance with ultrasound is part of the clinical testing. There are only few reports about the transfer of the visualization of the pancreas, probably caused by the difficulties with the segmentation routine due to inflammation or organ-exceeding tumor growth. With this paper, we like to evaluate and demonstrate the present status of software planning tools and pathways for future pre- and intraoperative resection planning in liver and pancreas surgery.

Identification of sentinel nodes in patients with colon cancer.

AIMS: Lymphatic mapping and sentinel node biopsy entails better staging in malignant melanoma and breast cancer and we used this technique in patients with colon cancer to possibly improve detection of lymphatic spread. METHODS: Thirty patients with invasive adenocarcinomas were investigated. The tumour status in identified sentinel node(s) was compared with the status in all other harvested regional nodes for each patient. Patients were followed at regular visits for more than 30 months. RESULTS: Sentinel nodes were identified in all cases, either per-operatively (28 cases) or at dissection of the formalin-fixed specimen (2 cases). The sentinel nodes were diagnostic for the entire lymphatic field in 28 patients and the false negative rate was 2/12. In four cases, the sentinel nodes were the only metastatic nodes. After at minimum 30 months, three patients had died of colon cancer metastases. CONCLUSION: This method improved the identification of patients with lymph-node metastases, especially those with only few metastatic nodes. Patients dying from metastatic disease had lymph-node metastases at diagnosis.

Phenotypic and functional characterization of clinical grade dendritic cells generated from patients with advanced breast cancer for therapeutic vaccination.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.

Nonlinear viscoelastic material property estimation of lower extremity residual limb tissues.

Axisymmetric nonlinear finite-element analysis was used to simulate force-relaxation and creep data obtained during in vivo indentation of the residual limb soft tissues of six individuals with trans-tibial amputation [1]. The finite-element models facilitated estimation of an appropriate set of nonlinear viscoelastic material coefficients of extended James-Green-Simpson material formulation for bulk soft tissue at discrete, clinically relevant test locations. The results indicate that over 90% of the experimental data can be simulated using the two-term viscoelastic Prony series extension of James-Green-Simpson material formulation. This phenomenological material formulation could not, however, predict the creep response from relaxation experiments, nor the relaxation response from creep experiments [2-5]. The estimated material coefficients varied with test location and subject indicating that these coefficients cannot be readily extrapolated to other sites or individuals.

[Bladder cancer and the sentinel node concept]. / Sentinel-Node-Diagnostik beim invasiven Blasenkarzinom.

PURPOSE: Lymph node status is one of the most important prognostic factors in muscle-invasive bladder cancer. The extent of lymphadenectomy performed in conjunction with cystectomy and the question as to whether this is a staging or therapeutic intervention are matters of discussion. The aim of this study was to evaluate the sentinel node (SN) concept and to correlate findings with tumour status in excised regional lymph nodes. MATERIAL AND METHOD: 26 patients scheduled for cystectomy were investigated with preoperative lymphoscintigraphy, peroperative dye detection (Patent Blue) and dynamic lymphoscintigraphy (Nanocoll or Albures 50 MBq/ml). The substances were injected adjacent to the tumour in the detrusor muscle. RESULTS: Sentinel nodes were detected in 21 of the 26 of the investigated patients. 7/21 SN were located outside the obturator fossa. Of the eight patients with lymph node metastasis, five displayed metastasis in lymph nodes outside the obturator fossa. There was one false negative SN in a patient with multifocal tumour, while in the other seven patients with lymph node metastasis, these were detected in the SN. CONCLUSION: Sentinel node detection is possible in most cases of bladder cancer scheduled for cystectomy. The significance of utilizing this method to detect lymph node metastasis outside the obturator fossa warrants further investigation.

Immunobiological effects of glucosamine in vitro.

Glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) were assayed in vitro for their effects on proliferation, cytotoxicity and cytokine secretion in primary and secondary mixed lymphocyte cultures (MLCs). In addition, we studied the effect of GlcN and GlcNAc on the proliferation of purified CD4+ T cells exposed to immobilized anti-CD3 antibody. The present data show that GlcN, but not GlcNAc, inhibits CD4+ T-cell proliferation, the generation of alloreactive cytotoxic T lymphocytes (CTLs) and the secretion of interferon-gamma (IFN-gamma) and interleukin-5 (IL-5) in primary MLC. In secondary T helper-2 (Th2)-polarized MLC, GlcN, but not GlcNAc, inhibits IL-4 and IL-5 secretion, whereas no effect was found on IFN-gamma secretion in Th1-polarized MLC. Dendritic cells treated with GlcN showed a 75-80% decreased capacity for antigen cross-presentation and allostimulation. In cellular bioassays, GlcN was shown to inhibit the stimulatory activity of IL-4 and IL-2, as well as the cytotoxic activity of tumour necrosis factor-alpha (TNF-alpha). In conclusion, GlcN suppresses unprimed T-cell responses by interfering with antigen-presenting cell functions and by a direct inhibitory effect on T-cell proliferation. In addition, GlcN inhibits the secretion of cytokines in antigen-stimulated unprimed T cells and primed Th2-polarized cells.

Nonlinear elastic material property estimation of lower extremity residual limb tissues.

The interface stresses between the residual limb and prosthetic socket have been studied to investigate prosthetic fit. Finite-element models of the residual limb-prosthetic socket interface facilitate investigation of the mechanical interface and may serve as a potential tool for future prosthetic socket design. However, the success of such residual limb models to date has been limited, in large part due to inadequate material formulations used to approximate the mechanical behavior of residual limb soft tissues. Nonlinear finite-element analysis was used to simulate force-displacement data obtained during in vivo rate-controlled (1, 5, and 10 mm/s) cyclic indentation of the residual limb soft tissues of seven individuals with transtibial amputation. The finite-element models facilitated determination of an appropriate set of nonlinear elastic material coefficients for bulk soft tissue at discrete clinically relevant test locations. Axisymmetric finite-element models of the residual limb bulk soft tissue in the vicinity of the test location, the socket wall and the indentor tip were developed incorporating contact analysis, large displacement, and large strain, and the James-Green-Simpson nonlinear elastic material formulation. Model dimensions were based on medical imaging studies of the residual limbs. The material coefficients were selected such that the normalized sum of square error (NSSE) between the experimental and finite-element model indentor tip reaction force was minimized. A total of 95% of the experimental data were simulated using the James-Green-Simpson material formulation with an NSSE less than 5%. The respective James-Green-Simpson material coefficients varied with subject, test location, and indentation rate. Therefore, these coefficients cannot be readily extrapolated to other sites or individuals, or to the same site and individual some time after testing.

A model for evaluating radiological impacts on organisms other than man for use in post-closure assessments of geological repositories for radioactive wastes.

Bioaccumulation and dosimetric models have been developed that allow the computation of dose rates to a wide variety of plants and animals in the context of the deep geological disposal of solid radioactive wastes. These dose rates can be compared with the threshold dose rates at which significant deleterious effects have been observed in field and laboratory observations. This provides a general indication of whether effects on ecosystems could be observable, but does not quantify the level of those effects. To address this latter issue, two indicator organisms were identified and exposure-response relationships were developed for endpoints of potential interest (mortality in conifers and the induction of skeletal malformations in rodents irradiated in utero). The bioaccumulation, dosimetry and exposure-response models were implemented and used to evaluate the potential significance of radionuclide releases from a proposed deep geological repository for radioactive wastes in France. This evaluation was undertaken in the context of a programme of assessment studies being performed by the Agence nationale pour la gestion des déchets radioactifs (ANDRA).

Inhibition of bacterial alpha-glucosidases by castanospermine in pure cultures and activated sludge.

Castanospermine (CAST) is a known and potent inhibitor of various alpha-glucosidases in eukaryotes. In this work, we elucidated whether CAST could also be used for determining bacterial alpha-glucosidase activity, when measured with 4-methylumbelliferyl-alpha- D-glucoside as a substrate, both in a complex bacterial community, in activated sludge and in pure cultures of bacterial isolates. We found that 140 microM CAST inhibited alpha-glucosidase activity by 30% in a pure culture of Pseudomonas stutzeri. The alpha-glucosidase activity in Chryseobacterium gleum was inhibited by 90% at a concentration of 150 microM CAST, whereas the alpha-glucosidase in Paracoccus denitrificans was resistant to the inhibitor. CAST (140 microM) reduced alpha-glucosidase activity in activated sludge by 40%, the respiration rate being reduced by only 12%. No significant inhibition of the respiration rate was observed in Ps. stutzeri or Pa. denitrificans, whereas the respiration rate in C. gleum grown in a medium containing starch was inhibited by 50% with 140 microM CAST. No effect of CAST was observed in C. gleum grown in a complex medium. This indicated that CAST, at the concentration used, did not cause a general negative effect on bacterial activity. The results suggest that the CAST assay may potentially be useful in determining whether alpha-glucosidase activity, starch, poly- and disaccharides contribute appreciably to the overall activity of a bacterial community. However, the assay should not be used for quantitative measurements of such activity.