Synergistic cranberry juice combinations with natural-borne antimicrobials for the eradication of uropathogenic Escherichia coli biofilm within a short time.

Urinary tract infections (UTI), one of the most common diseases in humans, are caused primarily by uropathogenic Escherichia coli (UPEC). Cranberry juice (CB) is a widely known prophylaxis for UTI, but the treatment of CB alone could not effectively eradicate preformed UPEC biofilms. The aim of this study was to develop enforced CB composites within a short time by adding a small quantity of natural borne antimicrobials. UPEC biofilms (initial: 6·0 log CFU per cm2 ), formed on silicone coupons in artificial urine medium, were exposed to CB (4-8%), caprylic acid (CAR; 0·025-0·05%) and thymol (TM; 0·025-0·05%) at 37°C for 1 min. Individual treatment of each compound did not show the significant antibacterial effect on UPEC biofilms (P > 0·05). Otherwise, the survivor counts of biofilms were synergistically reduced with CB containing any of the antimicrobials. For example combined treatment with CB (8%) + CAR (0·05%) + TM (0·05%) resulted in a 6 log reduction in UPEC populations in the biofilm (no detectable bacteria remained) with 4·6 log of synergistic bactericidal effect. The confocal laser scanning microscope images indicated that any composites including TM might result in biofilm detachment from the surface. The present method is cost-effective and more acceptable to consumers as it is based on the synergistic interaction of natural borne antimicrobials. The results of this study could be widely applicable in the functional food, medical and healthcare field. SIGNIFICANCE AND IMPACT OF THE STUDY: Anti-biofilm effect of cranberry juice (CB) has been focused mainly on inhibiting biofilm formation of uropathogenic Escherichia coli (UPEC); however, combined treatment with natural borne antimicrobials derived from coconut oil (caprylic acid) and oregano essential oil (thymol) could synergistically enhance its eradicating activity against biofilms. This study developed novel CB composites showing marked anti-biofilm effects (complete eradication of UPEC biofilms within just 1 min).

Concomitant ALK translocation and EGFR mutation in lung cancer: a comparison of direct sequencing and sensitive assays and the impact on responsiveness to tyrosine kinase inhibitor.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation are considered mutually exclusive in nonsmall-cell lung cancer (NSCLC). However, sporadic cases having concomitant EGFR and ALK alterations have been reported. The present study aimed to assess the prevalence of NSCLCs with concomitant EGFR and ALK alterations using mutation detection methods with different sensitivity and to propose an effective diagnostic and therapeutic strategy. PATIENTS AND METHODS: A total of 1458 cases of lung cancer were screened for EGFR and ALK alterations by direct sequencing and flourescence in situ hybridization (FISH), respectively. For the 91 patients identified as having an ALK translocation, peptide nucleic acid (PNA)-clamping real-time PCR, targeted next-generation sequencing (NGS), and mutant-enriched NGS assays were carried out to detect EGFR mutation. RESULTS: EGFR mutations and ALK translocations were observed in 42.4% (612/1445) and 6.3% (91/1445) of NSCLCs by direct sequencing and FISH, respectively. Concomitant EGFR and ALK alterations were detected in four cases, which accounted for 4.4% (4/91) of ALK-translocated NSCLCs. Additional analyses for EGFR using PNA real-time PCR and ultra-deep sequencing by NGS, mutant-enriched NGS increased the detection rate of concomitant EGFR and ALK alterations to 8.8% (8/91), 12.1% (11/91), and 15.4% (14/91) of ALK-translocated NSCLCs, respectively. Of the 14 patients, 3 who were treated with gefitinib showed poor response to gefitinib with stable disease in one and progressive disease in two patients. However, eight patients who received ALK inhibitor (crizotinib or ceritinib) showed good response, with response rate of 87.5% (7/8 with partial response) and durable progression-free survival. CONCLUSIONS: A portion of NSCLC patients have concomitant EGFR and ALK alterations and the frequency of co-alteration detection increases when sensitive detection methods for EGFR mutation are applied. ALK inhibitors appear to be effective for patients with co-alterations.

A Fas-associated via death domain promoter polymorphism (rs10898853, -16C/T) as a risk factor for papillary thyroid cancer.

PURPOSE: To determine whether a Fas-associated via death domain (FADD) promoter single-nucleotide polymorphism (SNP) is associated with susceptibility to papillary thyroid cancer (PTC) and clinicopathological features of PTC. METHODS: To identify a possible association with PTC, 94 patients with PTC and 346 healthy controls were recruited. One promoter SNP (rs10898853, -16C/T) was analyzed by direct sequencing. Multiple logistic regression models (co-dominant, dominant, recessive, and log-additive models) were applied, and odds ratios (ORs), 95% confidence intervals (CIs), and p values were calculated. RESULTS: The genotype of the promoter SNP (rs10898853) of FADD was found to be significantly associated with PTC in the co-dominant model 2 (T/T vs. C/C; p = 0.002, OR = 2.80, 95% CI = 1.39-5.65), the recessive model (p = 0.003, OR = 2.21, 95% CI = 1.31-3.71), and the log-additive model (p = 0.002, OR = 1.71, 95% CI = 1.20-2.44). Allele frequency analysis showed that the C allele of rs10898853 was significantly associated with an increased risk of PTC (p = 0.002, OR = 1.67, 95% CI = 1.21-2.32). CONCLUSIONS: Our results suggest that the FADD promoter polymorphism is associated with susceptibility to PTC.

Primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer harboring TKI-sensitive EGFR mutations: an exploratory study.

BACKGROUND: The mechanism of primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small-cell lung cancer (NSCLC) has not been clearly understood. PATIENTS AND METHODS: Eleven patients exhibiting primary resistance (disease progression <3 months) were identified among 197 consecutive NSCLC patients with TKI-sensitive EGFR mutations who received EGFR TKIs at Seoul National University Hospital. Treatment-naïve tumors were examined for concurrent genetic alterations using fluorescence in situ hybridization and targeted deep sequencing of cancer-related genes. Deletion polymorphism of Bcl-2-interacting mediator of cell death (BIM) gene was examined to validate its predictive role for TKI outcome. RESULTS: The median progression-free survival (PFS) for patients receiving EGFR TKIs was 11.9 months, and the response rate 78.8%. Among the 11 patients exhibiting primary resistance, a de novo T790M mutation was identified in one patient, and two exhibited mesenchymal-epithelial transition amplification and anaplastic lymphoma kinase fusion. Targeted deep sequencing identified no recurrent, coexistent drivers of NSCLC. Survival analysis revealed that patients with recurrent disease after surgery had a longer PFS than those with initial stage IV disease. However, BIM deletion polymorphism, line of treatment, EGFR genotype, and smoking were not predictive of PFS for EGFR TKIs. CONCLUSIONS: We identified coexistent genetic alterations of cancer-related genes that could explain primary resistance in a small proportion of patients. Our result suggests that the mechanism of primary resistance might be heterogeneous.

IL-9-producing invariant NKT cells protect against DSS-induced colitis in an IL-4-dependent manner.

Although the T-helper type 9 (Th9) subset has recently been revisited, interleukin (IL)-9-producing invariant natural killer T (iNKT) cells remain poorly characterized. Moreover, whether IL-9-producing iNKT cells regulate colitis is unknown. Here, we investigated functions of IL-9-producing iNKT cells in dextran sulfate sodium (DSS)-induced colitis. Wild-type (WT) mice attenuated colitis compared to Jα18(-/-) mice, which were restored by the adoptive transfer of WT, but not IL-4-deficient iNKT cells. IL-4-deficient iNKT cells failed to produce IL-9, which was reversed by recombinant IL-4. Furthermore, iNKT cells, pre-incubated with anti-CD3+CD28 monoclonal antibodies and IL-4+tumor growth factor (TGF)-ß (IL-9(+) iNKT), suppressed colitis in Jα18(-/-) mice, whereas pre-incubated IL-4-deficient iNKT cells did not. IL-9 blockade reversed IL-9(+) iNKT cell-mediated colitis by increasing colonic IL-17A and interferon (IFN)-γ transcripts, but decreasing IL-9, IL-10, TGF-ß, PU.1, IFN regulatory factor 4, and signal transducer and activator of transcription 5 in Jα18(-/-) mice. In conclusion, IL-9-producing iNKT cells protect against DSS-induced colitis through IFN-γ and IL-17A suppression, but IL-10 and TGF-ß enhancement, depending on the IL-4 production by iNKT cells.

Effects of oxidative stress on intestinal type I insulin-like growth factor receptor expression.

INTRODUCTION: Oxidative stress activates multiple signaling transduction pathways, including the phosphatidylinositol 3-kinase (PI3-K), in an injured intestine as occurs in necrotizing enterocolitis (NEC). We have previously shown that hydrogen peroxide (H2O2)-induced PI3-K activation is significantly enhanced with exogenous insulin-like growth factor (IGF)-1 in intestinal epithelial cells. However, the effects of oxidative stress on IGF receptor type I (IGF-IR) activation and expression in the neonatal intestine during NEC are unknown. MATERIAL AND METHODS: Intestinal sections from neonates undergoing bowel resections (control = 3, NEC = 20) were analyzed for IGF-IR expression. NEC was induced in newborn mouse pups using hypoxia and hyperosmolar feeds, and distal small bowel segments were analyzed for IGF-IR expression (control = 3, NEC = 7). H2O2 was used to induce oxidative stress in rat (RIE-1) and fetal human (FHs74 Int) intestinal epithelial cells. Phosphorylation of IGF-IR, Akt, a downstream effector of PI3-K, and IGF-IR levels were determined by Western blotting. Flow cytometry, immunofluorescence, immunohistochemistry, IGF-IR tyrosine phosphorylation array, cell death enzyme-linked immunosorbent assay, and Western blotting were used to determine the IGF-IR expression. RESULTS: An increased IGF-IR expression was noted in intestinal sections from NEC as well as murine model of NEC. H2O2 treatment rapidly activated IGF-IR and increased the expression in RIE-1 and FHs74 Int cells. Inhibition of IGF-IR resulted in significant RIE-1 cell apoptosis during oxidative stress. IGF-IR tyrosine phosphorylation array showed the recruitment of several key SH2 domain-containing proteins and oncogenes to the IGF-IR tyrosine kinase domain in H2O2-treated RIE-1 cells. CONCLUSION: IGF-IR-mediated activation of intracellular signaling may play a critical role during oxidative stress-induced apoptosis in NEC.

Proteins involved in DNA damage response pathways and survival of stage I non-small-cell lung cancer patients.

BACKGROUND: Biological complexity leads to significant variation in the survival of patients with stage I non-small-cell lung cancer (NSCLC). DNA damage response (DDR) pathways play a critical role in maintaining genomic stability and in the progression of NSCLC. Therefore, the development of a prognostic biomarker focusing on DDR pathways is an intriguing issue. PATIENTS AND METHODS: Expression of several proteins (ATM, ATMpS1981, γH2AX, 53BP1, 53BP1pS25, Chk2, Chk2pT68, MDC1, MDC1pS964, BRCA1pS1423, and ERCC1) and overall survival were investigated in 889 pathological stage I NSCLC patients. RESULTS: Low expression of BRCA1pS1423 or ERCC1 was significantly associated with worse survival in the whole cohort of patients. Analysis performed based on histology revealed that low expression of γH2AX, Chk2pT68, or ERCC1 was a poor prognostic factor in squamous cell carcinoma patients [adjusted hazard ratio (aHR), Cox P: 1.544, 0.012 for γH2AX; 1.624, 0.010 for Chk2pT68; 1.569, 0.011 for ERCC1]. The analysis of the interaction between two proteins showed that this effect was more pronounced in squamous cell carcinoma patients. However, these effects were not detected in adenocarcinoma patients. CONCLUSIONS: The proteins involved in DDR pathways exhibited differential expression between squamous cell carcinoma and adenocarcinoma and were important determinants of survival in stage I squamous cell carcinoma patients.

Tankyrase-1 function at telomeres and during mitosis is regulated by Polo-like kinase-1-mediated phosphorylation.

Telomere length is critical for chromosome stability that affects cell proliferation and survival. Telomere elongation by telomerase is inhibited by the telomeric protein, TRF1. Tankyrase-1 (TNKS1) poly(ADP-ribosyl)ates TRF1 and releases TRF1 from telomeres, thereby allowing access of telomerase to the telomeres. TNKS1-mediated poly(ADP-ribosyl)ation also appears to be crucial for regulating the mitotic cell cycle. In searching for proteins that interact with polo-like kinase-1 (Plk1) by using complex proteomics, we identified TNKS1 as a novel Plk1-binding protein. Here, we report that Plk1 forms a complex with TNKS1 in vitro and in vivo, and phosphorylates TNKS1. Phosphorylation of TNKS1 by Plk1 appears to increase TNKS1 stability and telomeric poly(ADP-ribose) polymerase (PARP) activity. By contrast, targeted inhibition of Plk1 or mutation of phosphorylation sites decreased the stability and PARP activity of TNKS1, leading to distort mitotic spindle-pole assembly and telomeric ends. Taken together, our results provide evidence of a novel molecular mechanism in which phosphorylation of TNKS1 by Plk1 may help regulate mitotic spindle assembly and promote telomeric chromatin maintenance.

Natural killer T cells are dispensable in the development of allergen-induced airway hyperresponsiveness, inflammation and remodelling in a mouse model of chronic asthma.

Natural killer T (NK T) cells have been shown to play an essential role in the development of allergen-induced airway hyperresponsiveness (AHR) and/or airway inflammation in mouse models of acute asthma. Recently, NK T cells have been reported to be required for the development of AHR in a virus induced chronic asthma model. We investigated whether NK T cells were required for the development of allergen-induced AHR, airway inflammation and airway remodelling in a mouse model of chronic asthma. CD1d-/- mice that lack NK T cells were used for the experiments. In the chronic model, AHR, eosinophilic inflammation, remodelling characteristics including mucus metaplasia, subepithelial fibrosis and increased mass of the airway smooth muscle, T helper type 2 (Th2) immune response and immunoglobulin (Ig)E production were equally increased in both CD1d-/- mice and wild-type mice. However, in the acute model, AHR, eosinophilic inflammation, Th2 immune response and IgE production were significantly decreased in the CD1d-/- mice compared to wild-type. CD1d-dependent NK T cells may not be required for the development of allergen-induced AHR, eosinophilic airway inflammation and airway remodelling in chronic asthma model, although they play a role in the development of AHR and eosinophilic inflammation in acute asthma model.

Decreased phosphorylation of STAT-1, STAT-4 and cytokine release in MDR-TB patients with primary resistance.

SETTING: We recently showed that treatment failure rate was higher among multidrug-resistant tuberculosis (MDR-TB) patients without a previous history of tuberculosis (TB) treatment, or so-called 'primary resistance'. OBJECTIVE: To investigate the phosphorylation levels of signal transducers and activators of transcription-1 (STAT-1) and STAT-4 and the subsequent cytokine release as a possible cause of a poor prognosis in MDR-TB patients with primary resistance. DESIGN: Ten patients with successfully treated pulmonary TB without resistance, 12 MDR-TB patients with acquired resistance and 10 MDR-TB patients with primary resistance were enrolled. After 24 h stimulation of peripheral blood mononuclear cells (PBMC) with interferon-gamma (IFN-gamma), interleukin-12 (IL-12), purified protein derivative (PPD), or lysate of Mycobacterium tuberculosis, flow cytometric analysis of intracellular pSTAT-1 and pSTAT-4 were performed and secretion of IFN-gamma, IL-12p40 and tumour necrosis factor-alpha (TNF-alpha) was measured in culture supernatant. RESULTS: The mean fluorescent intensities of pSTAT-1 and pSTAT-4 in PBMC of MDR-TB patients with primary resistance decreased on stimulation of IFN-gamma, PPD or lysate of M. tuberculosis when compared with patients with acquired resistance. In addition, secretion of IFN-gamma, IL-12p40 and TNF-alpha in these patients decreased on various stimuli. CONCLUSION: Decreased phosphorylation of STAT-1, STAT-4, and of subsequent cytokine release, might be associated with a poor prognosis in MDR-TB patients with primary resistance.

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals.

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15 +/- 0.02 microg l(-1) and an IC50 value of 1.13 +/- 0.16 microg l(-1). Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0-909.8 microg kg(-1). Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant zearalenone kit (r2 = 0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.

High tumour islet macrophage infiltration correlates with improved patient survival but not with EGFR mutations, gene copy number or protein expression in resected non-small cell lung cancer.

The purpose of this study was to investigate the prognostic value of tumour-associated macrophages with a focus on micro-anatomical localisation and determine whether molecular changes of the epidermal growth factor receptor (EGFR) are related to macrophage infiltration in resected non-small cell lung cancer (NSCLC). One hundred and forty-four patients were included in this study. Immunohistochemistry was used to identify CD68+ macrophages in the tumour islet and surrounding stroma. Epidermal growth factor receptor mutations were studied by direct sequencing. The EGFR gene copy number and protein expression were analysed by fluorescence in situ hybridisation and immunohistochemistry. Patients with a high tumour islet macrophage density survived longer than did the patient with a low tumour islet macrophage density (5-year overall survival rate was 63.9 vs 38.9%, P=0.0002). A multivariate Cox proportional hazard analysis revealed that the tumour islet macrophage count was an independent prognostic factor for survival (hazard ratio 0.471, 95% confidence interval 0.300-0.740). However, EGFR mutations, gene copy number, and protein expression were not related to the macrophage infiltration. In conclusion, tumour islet macrophage infiltration was identified as a strong favourable independent prognostic marker for survival but not correlated with the molecular changes of the EGFR in patients with resected NSCLC.

N-myc is a novel regulator of PI3K-mediated VEGF expression in neuroblastoma.

Angiogenesis in neuroblastoma (NB) correlates with increased expression of vascular endothelial growth factor (VEGF) and a worse clinical outcome. Other cellular markers, such as Akt activation and MYCN amplification, are also associated with poor prognosis in NB; therefore, we sought to determine the role of N-myc in the regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt/VEGF pathway. PI3K inhibition, using small-molecule inhibitors or phosphatase and tensin homolog adenovirus, led to decreased levels of VEGF mRNA and/or protein by reducing phosphorylation of Akt and mammalian target of rapamycin (mTOR), and attenuating hypoxia-inducible factor 1alpha expression. Moreover, PI3K inhibition decreased levels of N-myc expression in MYCN-amplified cells. To further clarify the importance of N-myc as a target of PI3K in VEGF regulation, we inhibited N-myc expression by siRNA transfection. MYCN siRNA significantly blocked VEGF secretion, irrespective of serum conditions, in MYCN-amplified NB cells; this effect was enhanced when combined with rapamycin, an mTOR inhibitor. Interestingly, in cells with low-N-myc expression, MYCN siRNA reduction of VEGF secretion was only effective with MYCN overexpression or insulin-like growth factor-1 stimulation. Our results show that N-myc plays an important role in the PI3K-mediated VEGF regulation in NB cells. Targeting MYCN, as a novel effector of PI3K-mediated angiogenesis, has significant potential for the treatment of highly vascularized, malignant NB.

Microbial assessment in school foodservices and recommendations for food safety improvement.

This study evaluated microbial food safety in school foodservices. Five school foodservices were randomly selected, and samples from water, cooking utensils, tableware, foodservice surroundings, and linen were collected in summer and winter (N=420). Tap and drinking water samples were collected, samples of food contact surfaces were collected by swab-kit, and samples for foodservice workers' hands and gloves were prepared by glove juice method. Aerobic plate count (APC) and coliform bacterial populations were enumerated on plate count agar (PCA) and desoxycholate lactose agar, respectively. The presence of Escherichia coli, Salmonella, Listeria monocytogenes, and Staphylococcus aureus was also examined by biochemical identification tests. In addition, PCA agar for APCs and Baird-Parker agar for S. aureus were used to enumerate airborne microorganisms. Higher APCs (< 0 to 5.1 log CFU/mL) than acceptable level were generally observed in water samples, while low coliform counts were found in the samples. High APCs were enumerated in cooking utensils, foodservice workers, tableware, and food-service surroundings, and coliforms were also found in the samples for both seasons. The presence of Salmonella was found from only 10% of plastic glove samples (summer), and the presence of L. monocytogenes was not observed in all samples. S. aureus was detected in some of water, cooking utensils, tableware, employees, and foodservice surroundings, and E. coli was observed in cooking utensils (10% to 20%; summer). No obvious airborne bacteria were detected. These results showed that sanitation practice in school foodservices should be improved, and the results may be useful in microbial assessment of school foodservices.

Allergenicity of proteolytic hydrolysates of the soybean 11S globulin.

A substantial portion of the human population has immune hypersensitivities to various food materials. Soybean is one of the most common foods involved in such hypersensitivity reactions, especially in younger children. In this study, we investigated the effect of peptic and chymotryptic hydrolysis on the allergenicity of the 11S soybean globulin, which is the primary soybean allergen. The 11S globulin is composed of both acidic and basic polypeptides, and we found that the acidic polypeptide was effectively hydrolyzed, while basic polypeptide was more resistant to hydrolysis. The 11S globulin hydrolysate was size-fractionated by gel filtration, and 9 of the fractions obtained were tested for allergenicity against sera from 6 soybean-allergenic patients. The overall allergenicity of soybean 11S globulin was reduced by peptic and chymotryptic hydrolysis, although a gel filtration fraction with a major peptide of 20 kDa was highly immunoreactive. Hydrolyzed fragments of less than about 20 kDa were not immunoreactive.

Interstitial lung disease in patients with polymyositis, dermatomyositis and amyopathic dermatomyositis.

OBJECTIVE: To assess the prevalence, characteristics and prognostic factors of interstitial lung disease (ILD) in Korean patients with polymyositis (PM), dermatomyositis (DM) and amyopathic dermatomyositis (ADM). METHODS: We reviewed the medical records of 72 consecutive PM and DM patients, including six patients with ADM, who were seen at the Rheumatology Clinic of Seoul National University Hospital between 1984 and 2003. RESULTS: Twenty-nine PM/DM patients (40.3%) developed ILD. Anti-Jo-1 antibody and arthralgia were associated with the presence of ILD (P = 0.022 and P = 0.041, respectively), whereas dysphagia was more frequently found in patients without ILD (P = 0.041). Lung biopsies revealed diffuse alveolar damage (DAD) (n = 2), usual interstitial pneumonia (UIP) with DAD (n = 2), UIP (n = 1), and non-specific interstitial pneumonia (n = 2). Of the 29 patients, 11 (37.9%) died. The mean survival time in ILD patients was significantly shorter than in those without ILD (13.8+/-1.8 vs 19.2+/-0.9 yr, P = 0.017). Poor survival in ILD patients was associated with a Hamman-Rich-like presentation (P = 0.0000), ADM features (P = 0.0001) and an initial forced vital capacity (FVC) < or =60% (P = 0.024). CONCLUSIONS: ILD was observed in 40.3% of Korean PM/DM patients and was associated with poor survival. A Hamman-Rich-like presentation, ADM features and an initial FVC < or =60% were associated with poor survival in ILD.

Autologous cardiomyocyte transplantation in an ovine myocardial infarction model.

BACKGROUND: To evaluate the possibility of autologous cardiomyocyte transplantation, we transplanted cultured autologous cells into an infarct region developed by coronary artery ligation in an ovine model. MATERIALS AND METHODS: A chronic heart failure model with a considerable portion of myocardial infarction was created in sheep using sequential ligation of the homonymous artery and its diagonal branch. Autologous cardiomyocytes were cultured and isolated from the right ventricular infundibulum. After a predetermined interval (one animal for two months and the other for three months), the two animals were reanesthetized and a suspension of cultured autologous vetricular cells in 0.3 ml of culture medium (1.2 x 10(7) cells) was injected into the center of three out of the four sites in the infarcted area using a tuberculin syringe. The same amount of culture medium was injected with an identical procedure into the center of the remaining site, as control. The animals were kept alive for a further month, and then sacrificed for postmortem heart examinations. Light microscopic analysis and immunohistochemical study for myoglobin were done. RESULTS AND CONCLUSIONS: On postmortem gross examination, well-demarcated thin-walled anteroseptal infarcts with chamber enlargement were clearly seen in specimens from the two animals. Microscopic analysis showed homogenous fibrosis throughout the infarcted areas. In both animals, one of the three sites of cardiomycyte injection showed an islet of cardiomyocytes in the mid-myocardium, while none were observed in the control site of either animal. A layer of cardiomyocytes was observed in subendocardial regions, as it was in the control areas. In conclusion, cardiomyocyte transplantation into the infarct regions developed by coronary artery ligation in an ovine model was achieved with only limited success. An understanding of why only 33% of cardiomyocyte-injection sites demonstrated viable cardiomyocytes, in the form of tiny cell islets, remains to be elucidated.