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Purpose: Mechanical thrombectomy (MT) for distal medium vessel occlusions (DMVOs) in the middle cerebral artery (MCA) is less established than for large vessel occlusions. This study evaluates the safety and efficacy of MT in DMVOs, comparing it with M1-segment occlusions., Materials and Methods: This retrospective study analyzed 218 patients who underwent MT for isolated M1 (n=123) or distal M2+M3 (n=35) occlusions between January 2020 and August 2023. Outcomes included procedural complications, hemorrhagic events, reperfusion rates, and clinical severity and disability at admission and discharge. Multivariate logistic regression identified predictors of favorable outcomes (modified Rankin Scale≤2) at discharge., Results: Median admission National Institutes of Health Stroke Scale (NIHSS) scores were higher in the M1 group (13, interquartile range [IQR]: 8) compared to the distal M2+M3 group (8, IQR: 7; P<0.001), with significant improvements at discharge in both groups (6 [IQR: 8] for M1 and 2.5 [IQR: 5] for M2+M3; P=0.025). Favorable outcomes were more frequent in the M2+M3 group (50.0%) compared to M1 (28.1%; P=0.023). Recanalization rates (modified Thrombolysis in Cerebral Infarction≥2b) were excellent (>90% in both groups; P=0.300). Procedural complications were rare, with vessel perforations occurring infrequently (M1: 1.6%; M2+M3: 2.9%; P=0.531). Symptomatic intracranial hemorrhage rates were similarly low (2.4% vs. 2.9%; P=0.889). Multivariate analysis identified younger age (P=0.045) and lower NIHSS (P=0.061) as predictors of favorable outcomes in distal occlusions., Conclusion: MT is safe and effective for DMVOs of the MCA, demonstrating significant improvements in clinical outcomes and comparable complication rates to MT for M1-segment occlusions. Given the typically less severe presentations in DMVO and similar risk profiles, careful patient selection and individualized treatment remain critical.

Background Symptomatic acute occlusions of the internal carotid artery (ICA) below the circle of Willis can cause a variety of stroke symptoms, even if the major intracranial cerebral arteries remain patent; however, outcome and safety data are limited. Purpose To compare treatment effects and procedural safety of endovascular treatment (EVT) and best medical treatment (BMT) in patients with symptomatic acute occlusions of the ICA below the circle of Willis. Materials and Methods This retrospective, multicenter cohort study from 22 comprehensive stroke centers in Europe and Asia includes patients treated between January 1, 2008, and December 31, 2022. Functional (modified Rankin Scale [mRS]) and clinical (National Institutes of Health Stroke Scale [NIHSS]) outcomes, safety measures (symptomatic intracerebral hemorrhage), mortality, and procedural complications were assessed. Results A total 354 patients met the inclusion criteria (median age, 72 years [IQR, 60-81 years]; median NIHSS, 13 [IQR, 7-19]). Most frequent occlusions were in the C1 segment (243 of 354; 68.6%). Of 354 patients, 82.2% (291 patients) were administered EVT. In the overall population, favorable outcomes (mRS 0-2), mortality, and symptomatic intracerebral hemorrhage occurred in 40.6% (108 of 266 patients), 25.2% (67 of 266 patients), and 7.1% (25 of 350 patients), respectively. After adjustment, no statistically significant difference in functional outcome was observed (adjusted odds ratio [AOR], 0.82 [95% CI: 0.31, 2.12]; average treatment effect, -12.7%; P = .19) in the EVT compared with BMT group. Symptomatic intracerebral hemorrhage (average treatment effect, -0.28%; P = .95) and mortality did not differ between both groups (average treatment effect, -17.1%; P = .07). EVT resulted in complete recanalization of the occlusion in 80.9% (229 of 283) of cases. Periprocedural distal embolization occurred in 27.8% (81 of 291 patients) and was associated with poor outcomes (AOR, 0.41; 95% CI: 0.18, 0.93; P = .03). Conclusion EVT did not reveal a favorable treatment effect over BMT, and both therapies were safe. EVT had a risk for periprocedural distal embolization associated with poor outcomes. © RSNA, 2025 Supplemental material is available for this article. See also the editorial by Daou and Chaudhary in this issue.

Background: Numerous questions regarding procedural details of distal stroke thrombectomy remain unanswered. This study assesses the effect of anesthetic strategies on procedural, clinical and safety outcomes following thrombectomy for distal medium vessel occlusions (DMVOs)., Methods: Patients with isolated DMVO stroke from the TOPMOST registry were analyzed with regard to anesthetic strategies (ie, conscious sedation (CS), local (LA) or general anesthesia (GA)). Occlusions were in the P2/P3 or A2-A4 segments of the posterior and anterior cerebral arteries (PCA and ACA), respectively. The primary endpoint was the rate of complete reperfusion (modified Thrombolysis in Cerebral Infarction score 3) and the secondary endpoint was the rate of modified Rankin Scale score 0-1. Safety endpoints were the occurrence of symptomatic intracranial hemorrhage and mortality., Results: Overall, 233 patients were included. The median age was 75 years (range 64-82), 50.6% (n=118) were female, and the baseline National Institutes of Health Stroke Scale score was 8 (IQR 4-12). DMVOs were in the PCA in 59.7% (n=139) and in the ACA in 40.3% (n=94). Thrombectomy was performed under LA±CS (51.1%, n=119) and GA (48.9%, n=114). Complete reperfusion was reached in 73.9% (n=88) and 71.9% (n=82) in the LA±CS and GA groups, respectively (P=0.729). In subgroup analysis, thrombectomy for ACA DMVO favored GA over LA±CS (aOR 3.07, 95% CI 1.24 to 7.57, P=0.015). Rates of secondary and safety outcomes were similar in the LA±CS and GA groups., Conclusion: LA±CS compared with GA resulted in similar reperfusion rates after thrombectomy for DMVO stroke of the ACA and PCA. GA may facilitate achieving complete reperfusion in DMVO stroke of the ACA. Safety and functional long-term outcomes were comparable in both groups., Competing Interests: Competing interests: JF: Consulting fees from Cerenovus, Medtronic, Phenox, Penumbra, Roche, Tonbridge; participation on a Data Safety Monitoring Board of Stryker and Phenox; stock holdings for Tegus and Vastrax, Associate Editor for JNIS. RC: Consultant and/or proctor for BALT, Stryker, Microvention, Rapid Medical, Siemens Medical Systems. MM: Institutional grants: Balt, Medtronic, MicroVention, Stryker. AG: Proctor/consultant/speaker for Medtronic, Stryker and Penumbra. MM-G: Consultant for Medtronic, Stryker and Balt, Associate Editor for JNIS. FD: Grant from Cerenovus/ Johnson&Johnson, consulting fees from Cerus Endovascular, Balt, Cerenovus/Johnson&Johnson, honoraria for lectures Asahi, Cerenovus/Johnson&Johnson, Acandis, Stryker, Advisory Board Cerenovus Johnson&Johsno, Associate Editor for JNIS. JK: Grants from SAMW/Bangerter, grants from Swiss Stroke Society, and grants from Clinical Trial Unit Bern outside the submitted work. LLLY: Consultant for Stryker, SeeMode, and See-mode, Cerenovus honoraria, Jakarta Neuroupdate honorarium, Research Support from National Medical research Council (NMRC) Singapore and Ministry of Health (MOH). Stock holdings for Cereflo, SNVIS vice president. BT: Grants from ExxonMobil-NUS Research Fellowship for Clinicians. PN: Consultant/Proctor for Balt, Cerenovus, Medtronic, Penumbra, Stryker. AG: Honoraria for lectures from Stryker Neurovascular, Medtronic, Penumbra. BG: grants from the French Ministry of Health and is the primary investigator of the TITAN, DIRECT ANGIO, and IA-RESCUE trial; consulting fees from Air Liquide, MIVI, Medtronic, Microvention, and Penumbra. LM: Compensation as a speaker for Balt Prime. GB: Compensation as a speaker for Balt Prime., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background Evidence supporting a potential benefit of thrombectomy for distal medium vessel occlusions (DMVOs) of the anterior cerebral artery (ACA) is, to the knowledge of the authors, unknown. Purpose To compare the clinical and safety outcomes between mechanical thrombectomy (MT) and best medical treatment (BMT) with or without intravenous thrombolysis for primary isolated ACA DMVOs. Materials and Methods Treatment for Primary Medium Vessel Occlusion Stroke, or TOPMOST, is an international, retrospective, multicenter, observational registry of patients treated for DMVO in daily practice. Patients treated with thrombectomy or BMT alone for primary ACA DMVO distal to the A1 segment between January 2013 and October 2021 were analyzed and compared by one-to-one propensity score matching (PSM). Early outcome was measured by the median improvement of National Institutes of Health Stroke Scale (NIHSS) scores at 24 hours. Favorable functional outcome was defined as modified Rankin scale scores of 0-2 at 90 days. Safety was assessed by the occurrence of symptomatic intracerebral hemorrhage and mortality. Results Of 154 patients (median age, 77 years; quartile 1 [Q1] to quartile 3 [Q3], 66-84 years; 80 men; 94 patients with MT; 60 patients with BMT) who met the inclusion criteria, 110 patients (median age, 76 years; Q1-Q3, 67-83 years; 50 men; 55 patients with MT; 55 patients with BMT) were matched. DMVOs were in A2 (82 patients; 53%), A3 (69 patients; 45%), and A3 (three patients; 2%). After PSM, the median 24-hour NIHSS point decrease was -2 (Q1-Q3, -4 to 0) in the thrombectomy and -1 (Q1-Q3, -4 to 1.25) in the BMT cohort ( P = .52). Favorable functional outcome (MT vs BMT, 18 of 37 [49%] vs 19 of 39 [49%], respectively; P = .99) and mortality (MT vs BMT, eight of 37 [22%] vs 12 of 39 [31%], respectively; P = .36) were similar in both groups. Symptomatic intracranial hemorrhage occurred in three (2%) of 154 patients. Conclusion Thrombectomy appears to be a safe and technically feasible treatment option for primary isolated anterior cerebral artery occlusions in the A2 and A3 segment with clinical outcomes similar to best medical treatment with and without intravenous thrombolysis. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Zhu and Wang in this issue.

Background: The optimal endovascular strategy for reperfusing distal medium-vessel occlusions (DMVO) remains unknown. This study evaluates angiographic and clinical outcomes of thrombectomy strategies in DMVO stroke of the posterior circulation., Methods: TOPMOST (Treatment for Primary Medium Vessel Occlusion Stroke) is an international, retrospective, multicenter, observational registry of patients treated for DMVO between January 2014 and June 2020. This study analyzed endovascularly treated isolated primary DMVO of the posterior cerebral artery in the P2 and P3 segment. Technical feasibility was evaluated with the first-pass effect defined as a modified Thrombolysis in Cerebral Infarction Scale score of 3. Rates of early neurological improvement and functional modified Rankin Scale scores at 90 days were compared. Safety was assessed by the occurrence of symptomatic intracranial hemorrhage and intervention-related serious adverse events., Results: A total of 141 patients met the inclusion criteria and were treated endovascularly for primary isolated DMVO in the P2 (84.4%, 119) or P3 segment (15.6%, 22) of the posterior cerebral artery. The median age was 75 (IQR, 62-81), and 45.4% (64) were female. The initial reperfusion strategy was aspiration only in 29% (41) and stent retriever in 71% (100), both achieving similar first-pass effect rates of 53.7% (22) and 44% (44; P =0.297), respectively. There were no significant differences in early neurological improvement (aspiration: 64.7% versus stent retriever: 52.2%; P =0.933) and modified Rankin Scale rates (modified Rankin Scale score 0-1, aspiration: 60.5% versus stent retriever 68.6%; P =0.4). In multivariable logistic regression analysis, the time from groin puncture to recanalization was associated with the first-pass effect (adjusted odds ratio, 0.97 [95% CI, 0.95-0.99]; P <0.001) that in turn was associated with early neurological improvement (aOR, 3.27 [95% CI, 1.16-9.21]; P <0.025). Symptomatic intracranial hemorrhage occurred in 2.8% (4) of all cases., Conclusions: Both first-pass aspiration and stent retriever thrombectomy for primary isolated posterior circulation DMVO seem to be safe and technically feasible leading to similar favorable rates of angiographic and clinical outcome.

Background: Whether to approach distal occlusions endovascularly or not in medium-sized vessels secondary to proximal large vessel occlusion stroke remains unanswered., Objective: To investigates the technical feasibility and safety of thrombectomy for secondary posterior circulation distal, medium vessel occlusions (DMVO)., Methods: TOPMOST (Treatment fOr Primary Medium vessel Occlusion STroke) is an international, retrospective, multicenter, observational registry of patients treated for distal cerebral artery occlusions. This study subanalysis endovascularly treated occlusions of the posterior cerebral artery in the P2 and P3 segment secondary preprocedural or periprocedural thrombus migration between January 2014 and June 2020. Technical feasibility was evaluated with the modified Thrombolysis in Cerebral Infarction (mTICI) scale. Procedural safety was assessed by the occurrence of symptomatic intracranial hemorrhage (sICH) and intervention-related serious adverse events., Results: Among 71 patients with secondary posterior circulation DMVO who met the inclusion criteria, occlusions were present in 80.3% (57/71) located in the P2 segment and in 19.7% (14/71) in the P3 segment. Periprocedural migration occurred in 54.9% (39/71) and preprocedural migration in 45.1% (32/71) of cases. The first reperfusion attempt led in 38% (27/71) of all cases to mTICI 3. On multivariable logistic regression analysis, increased numbers of reperfusion attempts (adjusted odds ratio (aOR)=0.39, 95% CI 0.29 to 0.88, p=0.009) and preprocedural migration (aOR=4.70, 95% CI,1.35 to 16.35, p=0.015) were significantly associated with mTICI 3. sICH occurred in 2.8% (2/71)., Conclusion: Thrombectomy for secondary posterior circulation DMVO seems to be safe and technically feasible. Even though thrombi that have migrated preprocedurally may be easier to retract, successful reperfusion can be achieved in the majority of patients with secondary DMVO of the P2 and P3 segment., Competing Interests: Competing interests: JF: research support from the German Ministry of Science and Education (BMBF), German Ministry of Economy and Innovation (BMWi), German Research Foundation (DFG), European Union (EU), Hamburgische Investitions-/Förderbank (IFB), Medtronic, Microvention, Philips, Stryker; consultancy appointments; Acandis, Bayer, Boehringer Ingelheim, Cerenovus, Covidien, Evasc Neurovascular, MD Clinicals, Medtronic, Medina, Microvention, Penumbra, Route92, Stryker, Transverse Medical; stock holdings for Tegus. PP: consultant for Penumbra and Ab Medica. AG: has served as proctor/consultant/speaker for Medtronic, Stryker, and Penumbra M. MM-G: consultant of Medtronic, Stryker, and Balt. FD: research support from Cerenovus; consultant for Cerus Endovascular, AB Medica, and Phenox; speaker honorary from Acandis, Cerenovus. JK reports grants from SAMW/Bangerter, grants from Swiss Stroke Society, and grants from Clinical Trial Unit Bern outside the submitted work. LLLY: consultant for Stryker and SeeMode; research support from National Medical Research Council (NMRC) Singapore and Ministry of Health (MOH); stock holdings for Cereflo. PBS: consultant/proctor for Balt, Acandis, Microvention. RC: consultant and/or proctor for BALT, Stryker, Microvention, Rapid Medical, Siemens Medical Systems. PN: consultant/proctor for Balt, Stryker, and Penumbra. KZ: support under the Operational Programme Integrated Infrastructure for the project: TENSION – complementary project, IMTS: 313011W875, co-financed by the European Regional Development Fund., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Importance: Clinical evidence of the potential treatment benefit of mechanical thrombectomy for posterior circulation distal, medium vessel occlusion (DMVO) is sparse., Objective: To investigate the frequency as well as the clinical and safety outcomes of mechanical thrombectomy for isolated posterior circulation DMVO stroke and to compare them with the outcomes of standard medical treatment with or without intravenous thrombolysis (IVT) in daily clinical practice., Design, Setting, and Participants: This multicenter case-control study analyzed patients who were treated for primary distal occlusion of the posterior cerebral artery (PCA) of the P2 or P3 segment. These patients received mechanical thrombectomy or standard medical treatment (with or without IVT) at 1 of 23 comprehensive stroke centers in Europe, the United States, and Asia between January 1, 2010, and June 30, 2020. All patients who met the inclusion criteria were matched using 1:1 propensity score matching., Interventions: Mechanical thrombectomy or standard medical treatment with or without IVT., Main Outcomes and Measures: Clinical end point was the improvement of National Institutes of Health Stroke Scale (NIHSS) scores at discharge from baseline. Safety end point was the occurrence of symptomatic intracranial hemorrhage and hemorrhagic complications were classified based on the Second European-Australasian Acute Stroke Study (ECASSII). Functional outcome was evaluated with the modified Rankin Scale (mRS) score at 90-day follow-up., Results: Of 243 patients from all participating centers who met the inclusion criteria, 184 patients were matched. Among these patients, the median (interquartile range [IQR]) age was 74 (62-81) years and 95 (51.6%) were female individuals. Posterior circulation DMVOs were located in the P2 segment of the PCA in 149 patients (81.0%) and in the P3 segment in 35 patients (19.0%). At discharge, the mean NIHSS score decrease was -2.4 points (95% CI, -3.2 to -1.6) in the standard medical treatment cohort and -3.9 points (95% CI, -5.4 to -2.5) in the mechanical thrombectomy cohort, with a mean difference of -1.5 points (95% CI, 3.2 to -0.8; P = .06). Significant treatment effects of mechanical thrombectomy were observed in the subgroup of patients who had higher NIHSS scores on admission of 10 points or higher (mean difference, -5.6; 95% CI, -10.9 to -0.2; P = .04) and in the subgroup of patients without IVT (mean difference, -3.0; 95% CI, -5.0 to -0.9; P = .005). Symptomatic intracranial hemorrhage occurred in 4 of 92 patients (4.3%) in each treatment cohort., Conclusions and Relevance: This study suggested that, although rarely performed at comprehensive stroke centers, mechanical thrombectomy for posterior circulation DMVO is a safe, and technically feasible treatment option for occlusions of the P2 or P3 segment of the PCA compared with standard medical treatment with or without IVT.

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semi-quantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222 and miR-938) had decreased expression in the stool of patients with colon cancer, which was also more pronounced from early to later TNM stages. Results from colon mucosal tissues were similar to those from stool samples, although with more apparent changes in expression. Cytological studies on purified stool colonocytes that employed Giemsa staining showed 80% sensitivity for detecting tumor cells in stool smears. The performance characteristics of the test confirmed that stool is a medium well-suited for colon cancer screening, and that the quantitative changes in the expression of few mature miRNA molecules in stool associated with colon cancer progression provided for more sensitive and specific non-invasive diagnostic markers than tests currently available on the market. Thus, a larger prospective and properly randomized validation study of control individuals and patients exhibiting various stages of colon cancer progression (TNM stages 0-IV) is now needed in order to standardize test conditions, and provide a means for determining the true sensitivity and specificity of a miRNA screening approach in stool for the non-invasive detection of colon cancer, particularly at an early stage (0-I). Eventually, we will develop a chip to enhance molecular screening for colon cancer, as has been accomplished for the detection of genetically-modified organisms (GMOs) in foods.

We carried out this study to present proof-of-principal application, showing that by using a global microarray expression analysis, followed by quantitative stem-loop reverse transcriptase in conjunction with TaqMan® polymerase chain reaction (PCR) analysis of micro(mi)RNA genes, on limited number of plasma and tissue samples obtained from 20 individuals (five healthy, five TNM stage 0-1 colon cancer, five stage 2 and five stage 3), we were able to quantitatively monitor miRNA changes at the various TNM stages of colon cancer progression, particularly at the early, pre-malignant adenoma stage (e.g. polyps ≥ 1 cm with high grade dysplasia). The expression of some of the tested miRNAs showed less variability in tissue than in plasma. Nevertheless, our limited preliminary data on the plasma by itself show that plasma is well-suited for screening, and that the quantitative changes in the expression of a few cell-free circulatory mature miRNA molecules in plasma, that are associated with colon cancer progression, would provide for more sensitive and specific markers than those tests currently available on the market. In addition, analysis of miRNA molecules offers a quantitative and cost-effective non-invasive diagnostic approach for screening, than currently employed methods in a prevalent cancer that can be cured if it is detected at the early TNM stages, and that becomes deadly if not diagnosed before metastasis. Thus, a larger prospective and properly randomized clinical study using plasma derived from many control individuals and at various stages of colon cancer (TNM stages 0-IV) from patients, in order to corroborate the initial results, is now urgently needed in order to allow for a statistically valid analysis, standardizing test conditions which will provide a means for determining the true sensitivity and specificity of a miRNA-screening approach. This approach, when combined with bioinformatics analysis to correlate miRNA seed data with mRNA target data, would allow for a mechanistic understanding of how miRNAs regulate mRNA gene expression, and would offer a better comprehensive diagnostic screening test for early-detection of colon cancer non-invasively.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase followed by TaqMan PCR expression analysis on stool and tissue samples using fifteen human (Homo sapiens, hsa) micro(mi)RNA genes selected by careful analysis of the peer-reviewed literature, we were able to monitor changes at various stages of CRC, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages, and for difficult-to-treat active ulcerative colitis (UC). Although the expression of some of the miRNA genes tested in tissue showed less variability in CRC or UC patients than in stool, the stool by itself appears well-suited to screening. A miRNA approach using stool samples promises to offer more sensitivity and specificity than currently used screening genomic, methylomic or proteomic methods for colon cancer. Larger prospective clinical studies utilizing stool derived from many control, colon cancer or UC patients, to allow for a statistically valid analysis, are now urgently required to standardize test performance and determine the true sensitivity and specificity of the miRNA screening approach, and to provide a numerical underpinning for these diseases as a function of total RNA. Moreover, when a miRNA screening test is combined with analysis of a messenger(m)RNA expression test, which has also been considered in earlier studies to be a highly sensitive and a very specific and reliable transcriptomic approach, as outlined in this article, bioinformatics can be used to correlate microRNA seed data with mRNA target data in order to gain a mechanistic understanding of how miRNAs regulate gene expression, enabling understanding of why these miRNA genes should be informative in a screening test.

Establishing test performance criteria for a transcriptomic colon cancer marker approach must be carried out in a standardized fashion in order tso ensure that the test will perform the same way in any laboratory, anywhere. Condition of sample preservation and shipping prior to total RNA extraction is critical, and we recommend preserving stool samples in an appropriate preservative and shipping them in cold packs so as to keep stools at 4 degrees C. It is not necessary to isolate colonocytes to obtain adequate RNA for testing. It is, however, important to obtain samples from both mucin-rich and non-mucin rich to have a good representation of both left- and right-side colon cancers. Employing a commercial total RNA extraction kit that contains an RLT buffer from Qiagen Corporation (Valencia, CA, USA) removes bacterial RNA from stool preparations and results in a high yield of undegraded RNA of human origin. Genes selected based on the enormous resources of NCI's Cancer Genome Anatomy project give good results. Primers for PCR should span more than one exon. Use of semiquantitative PCR, preferably with several reference housekeeping genes of various copy numbers, depending on tested genes, should enhance confidence in the quantitative results. Having standardized the testing conditions in our ongoing work, it is now imperative that a larger prospective randomized clinical study utilizing stool and tissue samples derived from several control and colon cancer patients, to allow for statistically valid analyses, be conducted in order to determine the true sensitivity and specificity of the transcriptomic screening approach for this cancer whose incidence is on the rise worldwide.

There is a need for sensitive and specific diagnostic molecular markers that can be used to monitor early patterns of gene expression in non-invasive exfoliated colonocytes shed in the stool, and in situ in adenoma-carcinoma epithelium of the colon. RNA-based detection methods are more comprehensive than either DNA-, protein- or methylation-based screening methods. By routinely and systematically being able to perform quantitative gene expression studies on these samples using less than ten colon cancer genes selected by the enormous resources of the National Cancer Institute's Cancer Genome Anatomy Project, we were able to monitor changes at various stages in the neoplastic process, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages. Although the expression of some of the genes tested in tissue showed less variability in normal or cancerous patients than in stool, the stool by itself is suitable for screening. Thus, a transcriptomic approach using stool or tissue samples promises to offer more sensitivity and specificity than currently used molecular screening methods for colon cancer. A larger prospective clinical study utilizing stool and tissue samples derived from many control and colon cancer patients, to allow for a statistically valid analysis, is now urgently required to determine the true sensitivity and specificity of the transcriptomic screening approach for this preventable cancer.

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.

Pneumonia in the critically ill surgical patient often results from the bombardment of a previously normal pulmonary system with therapeutic foreign bodies, hospital pathogens, and impairment of the host defenses. Despite its long history as a significant clinical problem, a woefully inadequate amount of study has been directed toward therapy. We created an experimental model of a differential pulmonary infection using a strain of Klebsiella pneumoniae. We then compared the progressively affected pneumonic process versus the normal parenchyma. We measured neutrophil and monocyte complement antibody receptor expression and monocyte and macrophage class II major histocompatibility antigens (HLA-DR) via percent of cells and mean fluorescent intensity outcomes from flow cytometry. The main difference between infected versus noninfected tissues was monocyte DR expression, which was consistently depressed in cells from infected parenchyma. What follows is a discussion of the implications of this work as well as other work in the immunology of pneumonia and cytokine expression. Possible therapeutic modalities are included.

The systemic manifestations of sepsis are associated with increased cardiac output, peripheral vasodilatation, and mesenteric vasoconstriction. Our objective was to determine whether tumor necrosis factor (TNF)-alpha regulates small intestinal microcirculatory changes observed during sepsis. An intact loop of terminal ileum of an anesthetized rat was exteriorized into modified Krebs solution and then topically suffused with varying concentrations of TNF-alpha (10(-4) ng/ml to 10(2) ng/ml), norepinephrine (10(-4) M), and sodium nitroprusside (10(-5) M). Videomicroscopy was used to measure arteriolar (A1, A2, A3) and venular (V1, V2) diameter changes in response to topical TNF-alpha. First order vessel diameters did not change in response to TNF-alpha. However, second and third order arterioles dilated maximally by 35 +/- 16 and 52 +/- 12 per cent, respectively, in a dose dependent manner in response to TNF-alpha. Higher order vessels were more sensitive to TNF-alpha than lower order vessels. Norepinephrine (10(-4) M) produced vasoconstriction in all vessels tested (A2 18 +/- 3 per cent, p < 0.05; A3 6 +/- 6 per cent; V2 13 +/- 4 per cent, p < 0.05). Topical TNF-alpha caused dilation in preconstricted vessels as in the nonpreconstricted vessels. TNF-alpha induced vasodilation was prolonged and not reversed by removal of TNF-alpha. These data demonstrate statistically significant dilation in response to TNF-alpha in second and third order arterioles and venules of the small intestine. Persistent vasodilation suggests an induced mechanism of vasodilation in response to TNF-alpha that remains active even after removal of exogenous TNF-alpha. We, therefore, conclude that TNF-alpha causes persistent vasodilatation beyond the period of actual exposure to TNF-alpha in the small intestinal microcirculation. This effect is not altered by the presence of norepinephrine. These data suggest that small intestinal vasoconstriction observed during clinical conditions such as sepsis is unlikely to be mediated by TNF-alpha.

Local and systemic host immune functions are markedly altered after trauma. Activated macrophages (M phi) are the major effector cells of protective immunity, mediated in part by nitric oxide (NO). This study was undertaken to determine the effects of hemorrhage (HEM) on M phi cytotoxic function as measured by NO production. Male Sprague-Dawley 350- to 400-g rats were studied. Sham animals only had their carotid artery exposed and ligated. Hemorrhaged animals had carotid artery cannulation followed by HEM to SBP of 40 mmHg, sustained for 45 min, followed by resuscitation with shed blood and crystalloid. Recovered animals were sacrificed (n = 5 each) at 6, 12, 24, and 72 hr after HEM; blood and alveolar M phi were then isolated and examined. A monolayer of adherent M phi was examined for NO production in resting state and after in vitro LPS stimulation. (1) HEM resulted in significantly reduced alveolar M phi yield at 12 and 24 hr. (2) HEM increased NO production by circulating M phi at 24 hr (P < 0.05), while alveolar M phi had significantly increased NO production at all time points after HEM (P < 0.05). (3) LPS stimulation significantly increased NO production in both circulating and alveolar M phi in sham animals and 6 hr after HEM but not at any other times. We therefore conclude that HEM causes early and prolonged activation of NO production by alveolar M phi and delayed and brief activation of circulating M phi. Alveolar and circulating M phi are unable to significantly increase NO production in response to LPS stimulation during the later phases of HEM.(ABSTRACT TRUNCATED AT 250 WORDS)

As long as infection remains the most common cause of morbidity and mortality in severely ill patients, there exists the need for more effective anti-infective therapy. The current study was undertaken to determine whether continuous infusion (CONT) is superior to intermittent administration (INT) of an equal amount of cefazolin (CEF) in a model of surgical infection. The thigh suture model consists of the surgical placement of 1 cm of cotton suture with absorbed K. pneumoniae into the thigh muscle of mice. The experimental groups were: 1) controls (n = 20) with thigh suture inoculation and treatment with intraperitoneal (IP) sterile saline; 2) CONT infusion group that received CEF at 60 mg/kg IP 30 minutes before inoculation followed by CONT IP infusion at 180 mg/kg/day (n = 22) for 3 days; and 3) INT injection group that received CEF at 60 mg/kg IP 30 minutes before inoculation followed by INT IP injections every 8 hours at 180 mg/kg/day (n = 20) for 3 days. All CEF treated animals received identical quantities of total CEF, and all groups were followed for 10 days. The control and INT CEF groups had 20% survival, whereas the CONT CEF group had 81% survival, (P < 0.001). Continuous CEF yielded constant serum levels of 19 +/- 1 micrograms/mL, whereas INT injections resulted in peak serum level of 74 +/- 12 micrograms/mL at one minute but declined to 3.9 +/- 0.9 micrograms/mL in 2 hours. Although there was statistically significant tissue bacterial growth in the INT injection group, there was extensive tissue bacterial clearance in the CONT infusion group.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: The authors undertook a prospective study of trauma victims in the intensive care unit (ICU) to investigate the clinical course of pneumonia and the local and systemic immune responses to the pneumonia., Summary Background Data: The silent epidemic of pneumonia has been an "unappreciated killer" in terms of being overlooked in surgical ICUs for the past 5 years, and specifically, the most common major infection after severe trauma. Little is known about the immune response to an acute pulmonary infection., Methods: The authors studied 50 consecutive, critically ill trauma patients, with a mean injury severity score of 28 +/- 2, who developed pneumonia while ventilated mechanically. Patients were observed clinically, and specific immunologic parameters, including major histocompatibility antigen HLA-DR, complement receptor (CR3), and Fc receptor (FcRIII), were measured in circulating and local alveolar leukocytes for up to 30 days. Eleven patients provided unique clinical data via bronchoscopy for unilateral pneumonia, with collection of bronchoalveolar lavage (BAL) fluid from both the infected and uninfected sides., Results: Patients developed clinical pneumonia 5.3 +/- 0.4 days after admission to the ICU. At diagnosis, mean temperature was 101.4 F, white blood cell count was 16,000/mm3, arterial oxygen tension was 104 +/- 14, fraction of inspired oxygen was 0.47, and positive end-expiratory pressure was 5. Thirty patients (Group A) recovered relatively promptly; 20 patients had prolonged illnesses (Group B), 15 of whom ultimately survived, and five of whom died. Patients with poor outcomes had greater leukocytosis (p < 0.05) and temperature elevation (p < 0.05) after 5 days of pneumonia. Immunologically, peripheral leukocyte expression of HLA-DR, FcRIII, and CR3 was equivalent in both groups. However, the expression of all three antigens on local alveolar leukocytes was decreased to a greater extent in the poor outcome group compared to the good outcome group, evident before any clinical differentiation between the two outcome groups., Conclusions: Pneumonia prolonged duration of mechanical ventilation, ICU and hospital stay, and overall infectious morbidity. Although immune suppression has been recognized as a result of initial injury, the development of pneumonia coincided with the nadir of immune function. Poor outcome patients were clinically identifiable 5 days after pneumonia and immunologically identifiable within 2 days. Moreover, there was localized suppression of pulmonary leukocytes at the site of the infiltrate compared to the uninfected lobes. This same alteration was noted in experimental Klebsiella pneumoniae pneumonia. This evidence suggests that there is active immune participation within the respiratory system. It also suggests that there are predispositions to pulmonary infections, and it may allow immune modulation targeted to pulmonary leukocytes to hasten clinical recovery and minimize pulmonary dysfunction.

Intact peritoneal macrophage (M phi) function is critical to successful localization of intra-abdominal infection. Peritoneal macrophage antigen presentation capacity (APC), interleukin-1 (IL-1) expression, and immune-associated (Ia) antigen expression and abscess formation were determined following cecal ligation and puncture. APC and IL-1 expression were measured by coculture with a T-helper cell clone and by measuring subsequent proliferation. Ia expression was determined in blood, peritoneal M phi, and splenocytes using anti-Ia monoclonal antibody stain and flow cytometric analysis. Significant reductions in both Ia expression and APC were found 1 and 4 days after CLP with no change in IL-1 expression. Muramyl dipeptide, which enhances M phi phagocytosis, partially abrogated the depression in antigen presentation but did not affect Ia expression. Peritoneal M phi Ia expression and APC, but not IL-1 expression, were depressed after experimental peritonitis. The recovery of M phi function by day 14 coincides with clinical recovery and abscess formation, and restoration of early M phi depression may improve outcome.

The extent to which circulating leukocytes reflect functional capacity at the site of infection is unclear despite a century of debate. We conducted experiments designed to clarify those relationships as well as the relationship of pleural and peritoneal responses to infection. Empyema was established in inbred CBA/J mice by introducing Escherichia coli and Bacteroides fragilis into the thoracic cavity through a limited thoracotomy. Walled off abscesses appeared seven days after the introduction of infection. Cell surface expression of murine class II major histocompatibility antigen was measured in peripheral leukocytes, thoracic and peritoneal cavity exudate leukocytes and abscess cells for 60 days after the introduction of infection. The peripheral antibody response was determined by measuring immunoglobulin M (IgM) and immunoglobulin G (IgG) specific for the two organisms. The results demonstrated that, after introduction of infection into the thoracic cavity, mice mounted a specific systemic humoral immune response by producing IgM and IgG. There was a pronounced concomitant cellular response in thoracic and peritoneal cavity exudate cells. However, there was no evidence of a systemic cellular response in circulating lymphocytes or monocytes and polymorphonuclear leukocytes measured as a single group. We conclude from these experiments that measurements of peripheral immune parameters may adequately reveal the humoral response to infection, but it does not reflect the local immune cellular response. Furthermore, a special relationship exists between the peritoneal and the thoracic cavities, whereby an inflammatory process in the thoracic cavity leads to a marked activation of inflammatory process in the peritoneal cavity without affecting peripheral circulating cells to the same extent.