Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease.

BACKGROUND: Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. METHODS: Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. RESULTS: The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. CONCLUSIONS: A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Multicentre study of abdominal aortic aneurysm measurement and enlargement.

BACKGROUND: No effective treatment is currently available to prevent progression of small and medium-sized abdominal aortic aneurysms (AAAs). Identification of drugs with sufficient promise to justify large expensive randomized trials remains challenging. One potentially useful strategy is to look for associations between commonly used drugs and AAA enlargement in appropriately adjusted observational studies. METHODS: Potential AAA measurements were identified from abdominal imaging reports in the electronic data files of three medical centres from 1995 to 2010. AAA measurements were extracted manually and patients with an aneurysm of 3 cm or larger, who had at least two measurements over an interval of at least 6 months, were identified. Other data were obtained from the electronic data files (demographics, co-morbidities, smoking status, drug use) to conduct a propensity analysis of the associations of drugs and other factors with AAA enlargement. RESULTS: From 52,962 abdominal imaging studies, 5362 patients with an AAA of 3 cm or more were identified, of whom 2428 had at least two measurements over at least 6 months. Mean AAA follow-up was 3.4 years and the mean AAA enlargement rate was 2.0 mm per year. Propensity analysis demonstrated no significant association of AAA enlargement with statins, beta-blockers, angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Diabetes was associated with a reduction in AAA enlargement of 1.2 mm per year (P = 0.008), and chronic obstructive pulmonary disease was associated with increased enlargement (0.5 mm per year; P = 0.050). Moderate AAA measurement variation and substantial terminal digit preference were also observed, but the digit preference became less pronounced after 2000. CONCLUSION: This study confirms the negative association of diabetes with AAA progression. There was no evidence that commonly used cardiovascular drugs affect AAA enlargement.

Reliability of self-reported willingness-to-pay and annual income in patients treated for toenail onychomycosis.

BACKGROUND: Willingness-to-pay (WTP) is a health economics measure that has recently been used for skin diseases to evaluate patients' quality of life. However, the reliability of this measure has not been investigated in the dermatology literature and is essential in validating its use in health services research. OBJECTIVES: This study evaluated the test-retest reliability of self-reported annual income and WTP, a health economics measure of disease impact, in patients with toenail onychomycosis. METHODS: Forty-six patients enrolled in a randomized clinical trial comparing two different dosing regimens of terbinafine completed a self-administered questionnaire at baseline and 1 month later. The questionnaire asked: (i) how much patients would be willing to pay for a theoretical treatment with a cure rate of 85% for their current onychomycosis (10 categories: $0-50, $51-100, to > $800); and (ii) annual income (10 categories: $0-10,000 to > $200,000). RESULTS: Forty-four patients reported WTP at both visits, and 55% reported the same WTP. The quadratic-weighted (Fleiss-Cohen) kappa statistic indicated moderate agreement (kappa = 0.50, 95% confidence interval, CI 0.24-0.75, P < 0.01) as did the Spearman rank-order correlation coefficient (r(s) = 0.57, P < 0.01; median difference = 0, P = 0.50). Strong agreement was shown among the 42 patients who reported income at both visits; 71% reported the same annual income category (kappa = 0.72, 95% CI 0.47-0.96, P < 0.01; r(s) = 0.68, P < 0.01; median difference = 0, P = 0.77). Age, disease severity and duration, previous therapy, self-reported annual income, and medication side-effects were not statistically associated with the reliability of WTP. CONCLUSIONS: WTP and annual income demonstrated moderate and strong test-retest reliability, respectively. Self-reported WTP can serve as a reliable measure for future health economics research on onychomycosis.

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions.

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.

Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697.

AIMS: To characterize the beta-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial beta-fructofuranosidases. METHODS AND RESULTS: The beta-fructofuranosidase of B. infantis ATCC 15697 was purified 46.8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme was a monomeric protein (Mr 70 kDa) with beta-fructofuranosidase and invertase activities. The isoelectric point was pH 4.3, the optimum pH 6.0 and pKas (4.5 and 7.2) of two active groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37 degrees C and activities were unstable at 55 degrees C. beta-fructofuranosidase activity was more efficient than that of invertase with Vm/Km ratios of 0.65 and 0.025 x 10-3 l min(-1) mg(-1), respectively. The enzyme catalyses the hydrolysis of fructo-oligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. CONCLUSIONS: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has beta-fructofuranosidase and invertase activities. This protein was different from the beta-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of bacterial beta-fructofuranosidases, especially from bifidobacteria, has been gained.

Fermentation of chicory fructo-oligosaccharides in mixtures of different degrees of polymerization by three strains of bifidobacteria.

We estimated and compared the action of three selected strains of bifidobacteria in a semi-synthetic medium for different degrees of polymerization of fructo-oligosaccharides contained in three commercial products derived from chicory inulin: Fibrulose F97 (shorter chains), Fibruline Instant (native chains), Fibruline LC (longer chains). Biomass and production of lactate and acetate were greater when the substrate contained mostly shorter chain fructo-oligosaccharides. Shorter chains were first to be consumed, and one strain could use longer chains. As the degree of polymerization increased, residual fructo-oligosaccharides increased after growth of the strains, and the rate of consumption of fructo-oligosaccharides decreased.

Obesity is associated with premature occurrence of acute myocardial infarction.

BACKGROUND: The American Heart Association has classified obesity as a major modifiable risk factor for coronary artery disease, but its relationship with age at presentation with acute myocardial infarction (AMI) is poorly documented. HYPOTHESIS: The study was undertaken to evaluate the impact of obesity on age at presentation, and on in-hospital morbidity and mortality in patients with AMI. METHODS: Our analysis includes a consecutive series of 906 Olmsted County patients (mean age 67.7 years, 51% male) admitted with AMI to the Mayo Clinic Coronary Care Unit (CCU). The patients were entered into the Mayo CCU Database, a prospective registry of data pertaining to patients admitted to the Mayo Clinic CCU with AMI. Age at AMI occurrence and in-hospital morbidity and mortality were noted. RESULTS: Obese patients (body mass index [BMI] >30) with AMI were significantly younger than patients with AMI in the overweight (BMI 25-30) and normal-weight (BMI < 30) groups (62.3+/-13.1 vs. 66.9+/-13.2 and 72.9+/-13.4, respectively. p < 0.001). Obesity and overweight status were associated with male gender, diabetes mellitus, hypercholesterolemia, and smoking history; however, after multivariate adjustment for these risk factors, excess weight and premature AMI remained significantly associated. Compared with normal-weight patients, overweight patients presenting with AMI were 3.6 years younger (p < 0.001, confidence interval [CI] 1.9-5.4) and obese patients 8.2 years younger (p < 0.001, Cl 6.2-10.1). No significant increase in in-hospital morbidity and mortality was seen. CONCLUSION: In this population-based study, overweight and obese status are independently associated with the premature occurrence of AMI, but not with an increased incidence of in-hospital complications.

Length of stay in myocardial infarction.

OBJECTIVE: We evaluated the association between length of hospital stay (LOS) and clinical factors, treatment intensity, and use of percutaneous coronary revascularization from 1988 to 1997. BACKGROUND: Multiple factors contribute to the observed reduction in LOS for patients with myocardial infarction. METHODS: We studied a series of 849 consecutive patients admitted with acute myocardial infarction to the Mayo Clinic Coronary Care Unit within three time periods: period I (1988-1990), period II (1991-1993), and period III (1994-1997). RESULTS: Median LOS decreased significantly between 1988 and 1997 (9 days to 5 days, 36% reduction, p < 0.0001), with significant reductions (p < 0.001) associated with certain therapies: primary reperfusion (6 days vs 7 days), b-blockers (6 days vs 8 days), and aspirin (6 days vs 8 days). Hospitalizations were lengthened by coronary artery bypass grafting (12 vs 6 days) and by serious complications (10 vs 6 days). The era of the admission (period I vs II vs III) is a significant, powerful predictor of LOS, even after adjustment for other key variables. CONCLUSION: The 36% reduction in LOS for acute myocardial infarction between 1988 and 1997 is related both to therapeutic modalities and temporal trends. Further study is needed to clarify whether the trend for decreasing LOS persists and influences outcome and health care quality variables.

Fermentations of fructo-oligosaccharides and their components by Bifidobacterium infantis ATCC 15697 on batch culture in semi-synthetic medium.

AIMS: To compare the physiological behaviour of Bifidobacterium infantis ATCC 15697 growing on synthetic oligofructose or its components. METHODS AND RESULTS: The studies were carried out in regulated or non-regulated batch cultures on semi-synthetic media. Differences between the carbohydrate utilization patterns with glucose, fructose, sucrose and fructo-oligosaccharides (FOS) were determined. Glucose was the preferred substrate for growth and biomass production, whereas fructose was the best for lactate and acetate production. With sucrose, biomass production reached the level obtained with glucose, whereas with FOS, more metabolites were produced, as with fructose. In a mixture of FOS, the shorter saccharides were used first and fructose was released in the medium. Fructofuranosidase, an enzyme necessary to hydrolyse FOS, was inducible by fructose. CONCLUSION: Glucose contained in FOS and sucrose might sustain growth and cell production, while fructose might enable the production of major metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the bifidogenic nature of oligofructose has been gained.

Bile salt toxicity to some bifidobacteria strains: role of conjugated bile salt hydrolase and pH.

The purpose of this work was to study some aspects of bile salt toxicity towards bifidobacteria. A strain (Bifidobacterium coryneforme ATCC 25911) was selected for its lack of conjugated bile salt hydrolase activity (CBSH-), and was used with three deconjugating strains (CBSH+), for study of their growth and viability in the presence of two dihydroxylated conjugated bile salts (tauro- and glyco-deoxycholic acids). The presence of the glycoconjugate induced a more significant growth inhibition for the four strains than the tauroconjugate. The viability of the strains was measured at several pH levels. Glycodeoxycholic acid, but not taurodeoxycholic acid, exerted a lethal effect, which increased at low pH. This phenomenon was more pronounced for the CBSH- strain. We explain some of these results using an hypothesis based on the consequence of dissociation of conjugated and deconjugated bile salts, and the value of their pKa.

Improved survival after acute myocardial infarction in patients with advanced Killip class.

BACKGROUND: The continuing applicability of the Killip classification system to the effective stratification of long-term and short-term outcome in patients with acute myocardial infarction (MI) and its influence on treatment strategy calls for reanalysis in the setting of today's primary reperfusion treatments. HYPOTHESIS: Our study sought to test the hypothesis that Killip classification, established on admission in patients with acute MI, is an effective tool for early prediction of in-hospital mortality and long-term survival. METHODS: A series of 909 consecutive Olmsted County patients admitted with acute MI to St. Marys Hospital, Mayo Clinic, between January 1988 and March 1998 was analyzed. Killip classification was the primary variable. Endpoints were in-hospital death, major in-hospital complications, and post-hospital death. RESULTS: Patients analyzed included 714 classified as Killip I, 170 classified as Killip II/III, and 25 classified as Killip IV. Increases in in-hospital mortality and prevalence of in-hospital complications correspond significantly with advanced Killip class (p < 0.01), with in-hospital mortality 7% in class I, 17.6% in classes II/III, and 36% in class IV patients (p < 0.001). Killip classification was strongly associated with mode of therapy administered within 24 h of admission (p < 0.01). Killip IV patients underwent primary angioplasty most commonly and were less likely to receive medical therapy. CONCLUSIONS: Killip classification remains a strong independent predictor of in-hospital mortality and complications, and of long-term survival. Early primary angioplasty has contributed to a decrease in mortality in Killip IV patients, but effective adjunctive medical therapy is underutilized.

Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance.

Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance.

Effects of Lactobacillus amylovorus and Bifidobacterium breve on cholesterol.

To determine the validity of the hypothesis of assimilation and/or precipitation of cholesterol by Lactobacillus and Bifidobacterium species, culture were undertaken in TPY medium containing oxgall or taurocholic acid. In the case of growing cells, both strains were able to remove cholesterol in the presence of bile salts. Nevertheless, the behaviour was different according to the kind of bile salt. In the presence of taurocholic acid, the removal of cholesterol was due to both bacterial uptake and precipitation. In the presence of Oxgall, bacterial uptake and precipitation were observed for Lactobacillus but only precipitation occurred for Bifidobacterium.

Effects of fructooligosaccharides and their monomeric components on bile salt resistance in three species of bifidobacteria.

The influence of fructooligosaccharides (FOS) and their monomeric components on bile salt resistance of Bifidobacterium breve ATCC 15700, Bif. longum ATCC 15707 and Bif. animalis ATCC 25527 was examined. The neosugars induced fructofuranosidase activities for the degradation of these saccharides. For the three strains tested the growth was identical and bile salts had the same inhibitory effect on growth whatever the carbohydrate used. The survival of Bif. breve and Bif. longum, in the presence of glycodeoxycholic acid depended, however, on carbohydrates: the toxic effects of the bile salt could be partly alleviated by the addition of a metabolizable C-source. For Bif. animalis, the presence of any carbohydrate in the incubation medium did not enhance the viability of the strain. But in the three deconjugating strains of bifidobacteria studied, the presence of neosugar during the growth led to improved resistance to the bactericidal effect of the bile salt compared with the monomeric components of these neosugars (glucose and fructose).

A randomized phase II trial of two schedules of topotecan for the treatment of advanced stage non-small cell lung cancer.

We conducted a randomized phase II trial of two different schedules of topotecan in patients with advanced-stage non small lung cancer (NSCLC) without prior cytotoxic chemotherapy. All patients had histologic or cytologic confirmation of stage IV (M1) or III-B NSCLC. Patients were stratified by performance status, stage and weight loss. Patients were randomized to receive topotecan at intravenous doses of 1.5 mg/m(2)/day over 30 min for 5 days every 3 weeks (Arm A) or 1.3 mg/m(2)grade 3 in both arms included leukopenia, thrombocytopenia, malaise, constipation, diarrhea, lethargy, pulmonary, vomiting, infection and myalgia. Severe (> or = grade 3) thrombocytopenia occurred in 15.8% of Arm A patients and 37.8% of Arm B patients and this difference was statistically significant (P=0.03). The median times to progression are 101 and 63 days (P=0. 75) and the median survival times are 257 and 179 days (P=0.83) for Arms A and B, respectively. These differences in time to progression and overall survival are not statistically significant. Topotecan has limited, single agent activity in advanced NSCLC when given as 1. 5 mg/m(2)/day over 30 min for 5 days every 3 weeks. We do not intend to pursue further investigations with topotecan in patients with NSCLC.

Paclitaxel and G-CSF in previously untreated patients with extensive stage small-cell lung cancer: a phase II study of the North Central Cancer Treatment Group.

Paclitaxel is an antimicrotubule agent that interferes with cell division. It has demonstrated promising single-agent activity against non-small-cell lung cancer. The purpose of this study was to evaluate the therapeutic effectiveness of paclitaxel in previously untreated patients with extensive stage small-cell lung cancer (SCLC). The study was designed as a two-stage phase II trial. All patients who entered received paclitaxel by intravenous infusion at a dose of 250 mg/m2 during 24 hours. Granulocyte colony stimulating factor was also provided to ameliorate neutropenia. Cycles were repeated at 21-day intervals. Patients who achieved a complete response received a maximum of 10 cycles of treatment, whereas those who achieved a partial response/regression continued treatment until progression or undue toxicity developed. Patients who progressed or maintained stable disease for six cycles were crossed over to cisplatin and etoposide. Forty-three patients entered the study and all were evaluable for analysis. Responses were observed in 23 (53%) of the patients. There was no significant difference in the response rates in patients with measurable or evaluable disease (13/23 versus 10/20, p = 0.76). At the time of analysis, 39 patients had progressed with a median time to progression of 95 days, and 39 patients had died with a median survival of 278 days. The 1-year achieved survival rate was 24%. Significant neutropenia (absolute neutrophil count <1,000/microl) occurred in 24 (56%) of the patients, but only 2 patients experienced severe infection (grade > or = 3), and there were no septic deaths. The results indicate that paclitaxel is active against SCLC. Myelosuppression was the main side effect in this patient population. Response duration was short (median = 3.4 months), which suggests that paclitaxel is not sufficient as a single agent. Further studies of paclitaxel in combination with other agents against SCLC are currently in progress within the North Central Cancer Treatment Group and other cancer treatment groups. Key Words: Paclitaxel-G-CSF-Small-cell lung cancer-North Central Cancer Treatment Group.

Cerebral toxicity in patients treated for small cell carcinoma of the lung.

OBJECTIVE: To present the clinical characteristics of patients enrolled in a trial of treatment of small cell carcinoma (SCC) of the lung and to describe the central nervous system toxicity associated with the chemotherapy and prophylactic cranial irradiation (PCI). MATERIAL AND METHODS: We performed a retrospective analysis of 60 patients with SCC who received chemotherapy and thoracic radiation therapy. PCI was administered to patients who had limited disease or who had extensive disease that was subsequently down-staged to only residual chest disease after initial treatment. The total PCI dose was 3,200 cGy administered in 16 fractions of 200 cGy, given concurrently with systemic chemotherapy. Diagnostic criteria for leukoencephalopathy were based on previously published guidelines. RESULTS: Of the 60 eligible and enrolled patients, 35 received PCI and 25 did not. Leukoencephalopathy developed in 5 of the 35 patients (14%) who received PCI. The median age of the patients in whom leukoencephalopathy developed was 64 years (range, 57 to 69), and the median follow-up time was 59 months. The most common signs and symptoms of leukoencephalopathy were intellectual changes, memory alterations, and motor abnormalities. The mean time to onset of symptoms after termination of irradiation was 357 days (range, 30 to 524). Of all 60 patients, 6 were still alive 4 years after enrollment, and 3 of them (50%) already had leukoencephalopathy. CONCLUSION: Small dosage fractions of PCI may still result in leukoencephalopathy. The routine use of PCI in the management of SCC should be reassessed because of increasing evidence of the toxicity associated with it.

Phase II study of high-dose somatostatin analogue in patients either previously treated or untreated who have extensive-stage small cell lung cancer.

The authors conducted a phase II study of somatostatin analogue in 18 patients with extensive stage small cell lung cancer (four with previous treatment, 14 without previous treatment). Patients received 2,000 mg subcutaneously thrice daily. They were required to have an Eastern Cooperative Oncology Group performance score of 0-2 and acceptable pretreatment biochemical parameters. No patient responded to treatment. The median time to progression was 44 days. The median survival was 106 days. Toxicity related to treatment consisted of mild diarrhea and anorexia. Somatostatin analogue is not active as a single agent in the treatment of extensive-stage small cell lung cancer.

Laryngeal chondrosarcomas: the Mayo Clinic experience.

BACKGROUND: Laryngeal chondrosarcomas occur infrequently. Their management is often guided by inferences made from the management of sarcomas arising from more commonly afflicted organs. METHOD: A retrospective analysis of patients with laryngeal chondrosarcomas treated at the Mayo Clinic between 1959 and 1992 was performed to assess prognostic factors and outcomes after various treatments. RESULTS: A total of 20 patients received treatment during this time period. All chondrosarcomas were low grade; 19 involved the cricoid cartilage and one arose in the supraglottic larynx. Initial treatment consisted of local excision (often subtotal removal) alone in 12 patients (60%), hemilaryngectomy in 2 (10%), near total laryngectomy in 2 (10%), and total laryngectomy in 4 (20%). Six patients (30%) had local recurrence: five initially had local excision and one had hemilaryngectomy. All local recurrences or tumor progression developed >3 years after initial treatment. Salvage surgery was performed in five of the six patients who had local recurrence, and the other patient was observed. Of the five patients who had salvage surgery, three required another resection because of a second recurrence. CONCLUSIONS: These results suggest that initial conservative subtotal laryngectomy should be explored further because this treatment may provide long-term voice preservation in most patients, and patients who experience a recurrence after local excision often have been given several years of voice preservation.