Nariaki Matsuura

Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus.

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including squamous cell carcinomas (SCCs) of the esophagus, but little is known about COX-2 expression in premalignant esophageal squamous dysplasia. To elucidate the role of COX-2 in esophageal carcinogenesis, we examined the expression of this enzyme in normal squamous epithelium (n = 42), squamous dysplasia [high-grade dysplasia (HGD, n = 41; low-grade dysplasia (LGD, n = 33)]; carcinoma in situ (n = 16), mucosal invasive carcinoma (n = 18), and advanced SCC (n = 45). Immunohistochemistry showed a significantly high COX-2 expression in HGD compared with other lesions. The COX-2 score, an index determined by intensity and positivity of COX-2 staining (maximum 3.0), was 0.29 +/- 0.04 in normal esophagus, 1.75 +/- 0.11 in LGD, 2.89 +/- 0.05 in HGD, 2.17 +/-0.18 in CIS, 1.95 +/- 0.22 in mucosal invasive carcinoma, and 1.81 +/- 0.08 in advanced SCC. Results of reverse transcription-PCR assays confirmed those obtained by immunohistochemistry. COX-2 expression correlated with proliferation activity assessed by the proliferating cell nuclear antigen index in dysplastic lesions (P = 0.001) but not in SCCs. COX-2 expression in SCC did not correlate with various clinicopathological parameters including prognosis. Our results indicate that COX-2 is a sensitive marker for HGD and suggest that COX-2 may be involved in early stages of squamous carcinogenesis of the esophagus.

Spontaneous breathing during lung-protective ventilation in an experimental acute lung injury model: high transpulmonary pressure associated with strong spontaneous breathing effort may worsen lung injury.

Objective: We investigated whether potentially injurious transpulmonary pressure could be generated by strong spontaneous breathing and exacerbate lung injury even when plateau pressure is limited to <30 cm H2O. Design: Prospective, randomized, animal study. Setting: University animal research laboratory. Subjects: Thirty-two New Zealand White rabbits. Interventions: Lavage-injured rabbits were randomly allocated to four groups to receive low or moderate tidal volume ventilation, each combined with weak or strong spontaneous breathing effort. Inspiratory pressure for low tidal volume ventilation was set at 10 cm H2O and tidal volume at 6 mL/kg. For moderate tidal volume ventilation, the values were 20 cm H2O and 7–9 mL/kg. The groups were: low tidal volume ventilation + spontaneous breathingweak, low tidal volume ventilation + spontaneous breathingstrong, moderate tidal volume ventilation + spontaneous breathingweak, and moderate tidal volume ventilation + spontaneous breathingstrong. Each group had the same settings for positive end-expiratory pressure of 8 cm H2O. Measurements and Results: Respiratory variables were measured every 60 mins. Distribution of lung aeration and alveolar collapse were histologically evaluated. Low tidal volume ventilation + spontaneous breathingstrong showed the most favorable oxygenation and compliance of respiratory system, and the best lung aeration. By contrast, in moderate tidal volume ventilation + spontaneous breathingstrong, the greatest atelectasis with numerous neutrophils was observed. While we applied settings to maintain plateau pressure at <30 cm H2O in all groups, in moderate tidal volume ventilation + spontaneous breathingstrong, transpulmonary pressure rose >33 cm H2O. Both minute ventilation and respiratory rate were higher in the strong spontaneous breathing groups. Conclusions: Even when plateau pressure is limited to <30 cm H2O, combined with increased respiratory rate and tidal volume, high transpulmonary pressure generated by strong spontaneous breathing effort can worsen lung injury. When spontaneous breathing is preserved during mechanical ventilation, transpulmonary pressure and tidal volume should be strictly controlled to prevent further lung injury.

Interleukin 10 reduces mortality from severe peritonitis in mice.

Interleukin 10 (IL-10) is known to suppress the induction of proinflammatory cytokines such as tumor necrosis factor (TNF) and IL-1 and is itself induced by monocytes and macrophages during sepsis. We studied the therapeutic efficacy of IL-10 by testing its effect on the survival rate in the murine cecal ligation-and-puncture (CLP) model. Administration of 1 microgram or more of recombinant murine IL-10 6 h after induction of sepsis decreased lethality in septic mice significantly and also suppressed the elevation of circulating TNF after sepsis. However, treatment with the same dose of IL-10 simultaneously or 6 h before induction of CLP had no effect on survival, and treatment with anti-TNF antibody after induction of CLP had no effect on the survival rate. These data suggest that cytokine modulation with IL-10 is a potential candidate for the treatment of sepsis and sepsis-related multiple organ failure.

Interleukin-1 receptor antagonist modifies the changes in vital organs induced by acute necrotizing pancreatitis in a rat experimental model

OBJECTIVE Interleukin-1 (IL-1) is a mediator in some critical conditions such as septic shock and multiple organ failure. Acute pancreatitis is one of the noted causes of multiple organ failure but the mechanism by which local inflammation progresses to systemic disease is unknown. In this study, we used an IL-1 receptor antagonist (IL-1ra) to investigate whether multiple organ failure due to acute pancreatitis is mediated by IL-1, as in other causes such as severe infection, trauma, and major surgery. DESIGN Prospective, randomized, controlled trial. SETTING Research laboratory of a university medical school. SUBJECTS Specific pathogen-free male Wistar rats weighing 200 to 250 g. INTERVENTIONS Necrotizing pancreatitis was induced by retrograde injection of deoxycholate solution into the biliopancreatic duct. IL-1ra was injected intravenously at a dose of 10 mg/kg 15 mins before induction of acute pancreatitis and then infused continuously at a rate of 5 mg/kg/hr for the following 24 hrs. MEASUREMENTS AND MAIN RESULTS Although treatment with recombinant human IL-1ra did not affect the degree of local pancreatic insult, it significantly reduced mortality, improved urine output as an indicator of the state of shock, and ameliorated the accumulation of neutrophils into the lung in a rat experimental pancreatitis model. CONCLUSIONS We concluded that multiple organ failure in severe pancreatitis is mediated, at least in part, by IL-1 through the activation of neutrophils. Furthermore, we concluded that circulatory collapse may also be important in the mechanism of the lethal effect of pancreatitis.

The Comparison of Spontaneous Breathing and Muscle Paralysis in Two Different Severities of Experimental Lung Injury.

Objectives:The benefits of spontaneous breathing over muscle paralysis have been proven mainly in mild lung injury; no one has yet evaluated the effects of spontaneous breathing in severe lung injury. We investigated the effects of spontaneous breathing in two different severities of lung injury compared with muscle paralysis. Design:Prospective, randomized, animal study. Setting:University animal research laboratory. Subjects:Twenty-eight New Zealand white rabbits. Interventions:Rabbits were randomly divided into the mild lung injury (surfactant depletion) group or severe lung injury (surfactant depletion followed by injurious mechanical ventilation) group and ventilated with 4-hr low tidal volume ventilation with spontaneous breathing or without spontaneous breathing (prevented by a neuromuscular blocking agent). Inspiratory pressure was adjusted to control tidal volume to 5–7 mL/kg, maintaining a plateau pressure less than 30 cm H2O. Dynamic CT was used to evaluate changes in lung aeration and the regional distribution of tidal volume. Measurements and Results:In mild lung injury, spontaneous breathing improved oxygenation and lung aeration by redistribution of tidal volume to dependent lung regions. However, in severe lung injury, spontaneous breathing caused a significant increase in atelectasis with cyclic collapse. Because of the severity of lung injury, this group had higher plateau pressure and more excessive spontaneous breathing effort, resulting in the highest transpulmonary pressure and the highest driving pressure. Although no improvements in lung aeration were observed, muscle paralysis with severe lung injury resulted in better oxygenation, more even tidal ventilation, and less histological lung injury. Conclusions:In animals with mild lung injury, spontaneous breathing was beneficial to lung recruitment; however, in animals with severe lung injury, spontaneous breathing could worsen lung injury, and muscle paralysis might be more protective for injured lungs by preventing injuriously high transpulmonary pressure and high driving pressure.

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome

Abstract Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279–281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing.

The status of Fas and Fas ligand expression can predict recurrence of hepatocellular carcinoma

The status of Fas and Fas ligand (Fas L) expression was investigated in this study for 103 hepatocellular carcinomas (HCC). We studied the expression of the following three factors, Fas and Fas L expression in carcinoma cells and Fas L expression in stromal mononuclear cells (defined as stromal Fas L index). Fas expression in HCC cells was significantly decreased in cases with poor differentiation (P< 0.0001) and of larger size (P = 0.0058). Fas L expression in carcinoma cells was observed exclusively in moderately or poorly differentiated cases. Furthermore, each factor had prognostic significance for disease-free survival (DFS) (P< 0.0001, P = 0.0222 and 0.0027 respectively). We then scored the results of each factor and defined the total score as ‘Fas-Fas L risk score’. The P -value of the score for DFS was even lower than that of the clinical stage by multivariate analysis. These results suggest that the evaluation of Fas and Fas ligand expression potentially has a significant prognostic value for DFS of HCC patients, in addition to the clinical stage, and can be regarded as a new prognostic marker.

Both cell proliferation and apoptosis significantly predict shortened disease-free survival in hepatocellular carcinoma

In this study, we investigated the proliferating cell index by the percentage of Ki-67 expressing cells (Ki-67 LI) and the apoptotic index (AI) by the number of morphologically apoptotic cells per 1000 carcinoma cells in haematoxylin and eosin sections of 76 hepatocellular carcinomas (HCC). Both indices showed excellent correlation with each other (P < 0.0001) and were significantly higher in cases of poor differentiation, of advanced stages, with portal invasion and with intrahepatic metastasis. Furthermore, cases with higher Ki-67 LI or higher AI displayed poor outcomes for disease-free survival (P = 0.0001 and P = 0.0005) by univariate analysis. By multivariate analysis, both indices could be regarded as independent prognostic factors. These results strongly suggest that Ki-67 LI and AI have very similar clinical significance, reflecting the existence of biologically aggressive phenotypes and poor disease-free survival rate in HCC.

Expression and Prognostic Role of Cyclin-Dependent Kinase 1 (cdc2) in Hepatocellular Carcinoma

The expression of cyclin-dependent kinase 1 (cdc2), cyclin A and cyclin B1 was immunohistochemically studied in 101 hepatocellular carcinomas (HCC). cdc2 overexpression was directly related to advanced stage, portal invasion, intrahepatic metastasis, poor differentiation, high α-fetoprotein level, large size, high Ki-67 labeling index and poor prognosis. Cyclin A and B1 overexpression showed similar tendency to that of cdc2, but they were not recognized as independent prognostic factors by multivariate analysis. These findings suggest that cdc2 plays the most crucial role of the G2/M modulators in cell cycle progression and cell proliferation of HCC and significantly predicts the recurrence of this carcinoma.

Expression of Tumor Suppressor Gene p16INK4 Products in Primary Gastric Cancer

Recent studies have shown that the cyclin-dependent kinase (CDK) inhibitor p27Kip1 represents an indicator for patients’ outcome in several human malignancies including gastric cancer. However, the clinicopathologic value of another class of CDK inhibitor, p16INK4, has not been determined. In a retrospective study, we examined the expression of p16INK4 by immunohistochemical assay of 80 samples of primary gastric cancers and their adjacent nonneoplastic mucosas. Less than 10% of non-tumor gastric mucosal cells were p16INK4 positive, whereas the expression of p16INK4 in gastric cancer cells varied widely from 0 to 100% (mean, 24.5%). The expression of p16INK4 was not seen in 11.3% (9/80) of the cancer cases, but in 65% (52/80) this protein was even overexpressed when compared with the nonneoplastic mucosa. A clinicopathologic survey indicated that a low or no expression of p16INK4 was associated with poorly differentiated carcinoma (p = 0.0133), but the level of expression did not correlate with other parameters including patients’ prognosis or with the expression of the pRb protein. In an effort to explore the underlying mechanism for the p16INK4-negative cases, a prospective study was also performed on 20 cases of gastric cancer to compare the level of the p16INK4 protein with the methylation status of the p16INK4 promoter. Gastric cancer tissues with methylation expressed significantly lower levels of the p16INK4 protein (p = 0.0013) and two of them lacked p16INK4 expression altogether, whereas all the cancer tissues without methylation expressed it. These findings suggest that the p16INK4 protein may be associated with differentiation of gastric cancer tissues and that methylation of the p16INK4 promoter may, in part, account for the loss of p16INK4 expression.