Effective wound bed preparation using maggot debridement therapy for patients with critical limb ischaemia.

OBJECTIVE: Patients with critical limb ischaemia (CLI) lack sufficient blood flow in to the limb, which leads to difficulties in the normal wound healing process. Therefore, maggot debridement therapy (MDT) has not generally been recommended for CLI patients. We evaluated the effectiveness of wound bed preparation by MDT in CLI patients who had undergone mid-foot amputation. METHODS: Patients who underwent mid-foot amputation after angioplasty between April 2014 and October 2016 were retrospectively investigated by classifying them into an MDT group or a conventional treatment group. The primary outcome was defined as achievement of wound healing. Secondary outcomes were the proportions of amputation-free survival (AFS) and successful ambulatory improvement. Propensity scores were used to evaluate treatment outcomes based on five factors: ankle-brachial index, skin perfusion pressure of the foot, nutritional status, experience with dialysis and age. RESULTS: A total of 39 patients (39 legs) were included, seven within the MDT group and 32 in the conventional treatment group. Clinical backgrounds of the two groups showed no significant differences except for higher albumin levels for the MDT group (3.5±0.4g/dl; p=0.014). The wound healing proportion was significantly higher in the MDT group (86%) than in the control group (38%) (p=0.035). At 6 months after amputation, no significant differences were found between the two groups for AFS (71% versus 47%; p=0.41) or ambulatory capability (43% versus 28%; p=0.65). This result was also similar to the propensity score adjustment analysis. CONCLUSIONS: The efficacy of MDT with favourable wound bed preparation was shown in our CLI patients based on effective debridement and granulation formation by maggots, avoiding the loss of their heels. Wound-healing rates after MDT were higher for patients than for those receiving conventional treatment. MDT is considered a valid adjuvant treatment strategy for patients with CLI after revascularisation treatment is conducted. More favourable wound bed preparation and successful graft take were achieved in the MDT group, suggesting the effectiveness of MDT for wound healing in CLI patients.

Clinical significance of ERG rearrangement subtype and its association with increased p53 expression in Japanese and German prostate cancer.

UNLABELLED: This study investigated differences in prevalence of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) and ETS transcription factor family member, v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion gene (TMPRSS2-ERG fusions) in clinically localized prostate cancer Japanese and German patients. A total of 105 specimens, including 69 Japanese and 36 German patients, were collected. The status of TMPRSS2-ERG fusion was determined by fluorescence in situ hybridization, and correlations of the TMPRSS2-ERG fusion with clinicopathological characteristics and immunohistochemistry were studied. Gene fusions were identified in 20% (14/69) of Japanese and 53% (19/36) of German patients (P < 0.001). The difference in the type of gene fusion between the two ethnic groups was statistically significant (P=0.024). Overexpression of ERG protein was significantly associated with gene fusion. Biochemical recurrence was significantly higher in patients with ERG overexpression than in those without, and not related to TMPRSS2-ERG fusion status. Interestingly, two types of gene fusions (deletion and increase of copy number) were significantly associated with increased p53 expression (P = 0.005). Association of specific gene fusions harboring higher genomic alterations with p53 expression levels suggests that p53 mutation might drive more aggressive arrangements of TMPRSS2-ERG fusion in prostate cancer. KEYWORDS: ERG, p53, prostate cancer, TMPRSS2-ERG fusion.

[A preliminary study on dihydropyrimidine dehydrogenase activity in breast cancer].

We studied dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS), key enzymes in regulating the pharmacokinetics and chemosensitivity to 5-FU, in 36 breast cancer patients as a control group and 18 patients as a 5-FU group, in which 5-FU was given orally for 2 weeks before surgery at a dose of 200 mg/day. Cancer tissues with adjacent normal tissue were sampled and stored until the assay. The DPD activity and TS amount were determined according to the radio-enzymatic assay and radiobinding assay, respectively. The DPD activity was significantly higher in breast cancer than in the adjacent breast tissue. This finding was observed in T1 and T2, node negative and ER positive breast cancers in the control group as well as in the 5-FU group. The DPD activity in T3 was significantly lower than in T1, and that of the adjacent breast tissue in T3 was significantly higher than in T1; therefore, the tumoral/non-tumoral ratio of DPD activity was significantly lower in T3 than T1. TS was significantly elevated in both groups, without significant differences. The clinical implication of elevated DPD activity in T1, T2, node negative or ER positive breast cancer compared to the respective normal breast tissue activity remains to be studied, because it is still unclear whether or not the tumoral DPD activity regulates the local concentration of 5-FU within the tumor. The amount of TS and DPD activity was not influenced by the oral administration of 5-FU for 2 weeks at the dose of 200 mg/day.

Inhibitory effect of a synthetic protease inhibitor (gabexate mesilate) on the respiratory burst oxidase in human neutrophils.

We investigated the mechanism by which the synthetic protease inhibitor gabexate mesilate inhibits the production of the superoxide anion by human neutrophils. We found that gabexate mesilate suppressed SOD-inhibitable cytochrome c reduction in a dose-dependent manner in intact neutrophils activated with phorbol ester. Gabexate mesilate slightly scavenged the superoxide anion in the pyrogallol assay. The reagent also inhibited superoxide anion production in a dose-dependent manner in a cell-free oxidase-activating system. Translocation of the cytosolic respiratory burst oxidase components, the 47- and 65-kDa proteins, to membranes was suppressed by the reagent in intact cells stimulated with phorbol ester. Gabexate mesilate also reduced arachidonic acid-induced translocation of the components to the membrane fraction in the cell-free system. These results demonstrate that gabexate mesilate suppresses superoxide anion production by reducing the translocation of the 47- and 65-kDa proteins to the plasma membrane.

Concurrent chemotherapy (5-fluorouracil and cisplatin) and radiation therapy for inoperable squamous cell carcinoma of the esophagus potentially followed by surgery.

Twenty-four previously untreated patients with primary inoperable squamous cell carcinoma of the esophagus showing no evidence of hematogenous metastasis were treated with concurrent chemotherapy and radiation therapy (CRT) followed by surgical resection if possible. The chemotherapy regimen consisted of 5-fluorouracil 750 mg/m2 on days 1-4 and 21-24, and cisplatin 70 mg/m2 on days 1 and 21. Radiation therapy was administered over days 1-26 (200 cGy/day five times per week with an initial planned dose of 40 Gy). Five patients (8%) showed complete response (CR), 14 patients (58%) had partial response (PR), and 19 had good local control (CR 2, PR 17). Eleven cases (48%) underwent esophageal resection with no operative mortality. Curative resection was accomplished in eight cases (35%). Toxicities observed in CRT were leukopenia (grades 3 and 4) 38%, nausea and vomiting (grades 2 and 3) 67%, esophagitis 42%, and fever 42%. The median survival time (MST) for 11 neoadjuvant cases was 349 days (P < 0.05) compared to 212 days for palliative treatment (six cases) and 126 days for no treatment (six cases) after CRT. The MST of eight patients who received curative resection had not been reached after a 17-month median follow-up time. Concurrent chemotherapy with 5-fluorouracil plus cisplatin and radiation proved to be a safe regimen yielding a satisfactory response and minimal toxicity in this particular group of patients. Extensive surgery was thus determined to be feasible after CRT and to contribute to prolonging survival.

Elevation of circulating monitor peptide/pancreatic secretory trypsin inhibitor-I (PSTI-61) after turpentine-induced inflammation in rats: hepatocytes produce it as an acute phase reactant.

Monitor peptide (MP) is a trypsin-sensitive cholecystokinin (CCK)-releasing peptide purified from rat pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion and has been reported to exhibit cell growth-stimulating activity. Pancreatic secretory trypsin inhibitor (PSTI) prevents premature activation of trypsinogen in the pancreatic duct. There are two PSTIs (PSTI-61 and -56) purified from rat pancreatic juice on the basis of trypsin inhibitory activity as reported previously. Fushiki et al. (1989, FASEB J. 3, 121) showed that MP is structurally the same peptide as PSTI-61. We measured the serial changes of circulating MP/PSTI-61 in rat and those in the level of PSTI-61 mRNA in the rat liver to investigate another novel role of this peptide in the turpentine-induced acute inflammation model. The elevation of serum MP/PSTI-61 as well as the alpha 2-globulin fraction, which is known to include several acute phase reactants such as alpha 2-macroglobulin and haptoglobin, was observed after induction of the turpentine inflammation. The serum alpha 2-globulin fraction had increased approximately 3-fold over the initial level at 48 hr after the injection. In contrast, serum MP/PSTI-61 had increased approximately 17-fold over the initial level at 48 hr after the injection. The elevation of circulating MP/PSTI-61 was significantly related with that of the alpha 2-globulin fraction (r = 0.91, P < 0.01). Immunoreactive MP/PSTI-61 was detected in the liver after induction of the inflammation (152.5 +/- 16.5 ng/g wet weight), but in the normal rat liver there was no immunoreactive MP/PSTI-61.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced attachment and elastase-releasing capacity of neutrophils after surgery.

Perioperative changes in neutrophil attachment level and neutrophil elastase-releasing capacity were examined in patients who underwent surgery for either esophageal or gastric cancer. The neutrophil attachment level was significantly increased in both groups to approximately three times that of the preoperative value. The elastase-releasing capacity of nonstimulated neutrophils was not significantly changed, but it was significantly increased in neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). Moreover, both neutrophil attachment and elastase-releasing capacity of FMLP-stimulated neutrophils were more enhanced in patients with postoperative complications than in patients without complications. Peak levels of serum interleukin 6, serum alpha 1 proteinase inhibitor, and plasma neutrophil elastase were also significantly higher in patients with postoperative complications than in patients without. These results suggest that during the postoperative period, neutrophils may be primed and activated in response to inflammatory mediators such as cytokines, and that such an alteration of neutrophil functions may reflect the extent of inflammation and may, at times, be implicated in postoperative complications.

The mechanism of hepatic cellular injury in sepsis: an in vitro study of the implications of cytokines and neutrophils in its pathogenesis.

Neutrophil-mediated injury to hepatocytes was evaluated in vitro. A new in vitro coculture system of neutrophils and a human hepatoblastoma cell line (HuH-6), instead of normal hepatocytes, was established. Recombinant human tumor necrosis factor (TNF) activated neutrophils to release neutrophil elastase and showed the significant cytotoxicity for HuH-6 cells, which was determined by measuring the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), from HuH-6 cells. The concentration of neutrophil elastase from zymosan-primed/TNF (1.0 ng/ml)-stimulated neutrophils cocultured with HuH-6 cells reached to the level of 1.59 +/- 0.18 micrograms/10(6) cells in 24 hr. The release of LDH from HuH-6 cells in this coculture system was 84.8 +/- 17.8 units/liter after 24 hr incubation. Purified human neutrophil elastase also increased LDH release from HuH-6 cells. When HuH-6 cells were cocultured with zymosan-primed/TNF (1.0 ng/ml)-stimulated neutrophils, the secretion of the negative acute phase reactant (APR), alpha-fetoprotein, from HuH-6 cells was significantly decreased, and the production of the positive APR, pancreatic secretory trypsin inhibitor, was decreased in response to the stimulation of interleukin 6. Urinary trypsin inhibitor, the inhibitor of neutrophil elastase, decreased the release of LDH from HuH-6 cells cocultured with stimulated neutrophils, while superoxide scavenger did not. These results show that human neutrophils activated by TNF injure hepatocytes, thus causing hepatic dysfunction, through the release of neutrophil elastase.

Differential activation of polymorphonuclear leukocytes from peripheral blood and exudate in surgical patients.

We examined the effects of surgical trauma on polymorphonuclear leukocyte (PMN) chemotaxis, production of phagocytosis-dependent intracellular chemiluminescence (CL) and production of phagocytosis-independent total CL in two groups: group A with esophageal cancer, and group B with gastric cancer. The scale of surgical trauma was quantified by measuring interleukin-6 in plasma and exudate from drainage tubes. We found a significant augmentation of chemotaxis, total CL and intracellular CL in both groups during the post-operative week. In group A, the increments in both chemotaxis and total CL of circulating cells were smaller than those in group B, but there was no significant difference in intracellular CL between the two groups. Exudate PMNs were more chemotactic, but produced smaller amounts of total CL than circulating cells. No significant difference in intracellular CL between exudate and circulating PMNs was detected, indicating that phagocytic activity was not affected. We conclude that severe surgical trauma causes circulating PMNs with high chemotactic activity to migrate to sites of injury, but that preactivated PMNs in exudate examined in vitro produce lower amounts of reactive oxygen metabolites in response to soluble chemoattractants in vitro than cells circulating in plasma.

[Establishment of EGF, EGF-R and FN-R positive human adenocarcinoma cell line (GAC-1)].

A human adenocarcinoma cell line designated as GAC-1, was established from ascites of the 56-year old male patient with rapidly progressive gastric cancer. The doubling time was about 18.5 hours in vitro, and cell cycle analysis using flow cytometry showed marked increase of S phase (46.1%). Immunohistochemical demonstration of GAC-1 cells revealed positive staining of TGF-alpha, EGF, EGF-R, FN-R, laminin and negative staining of fibronectin. Histogram of them indicated aneuploidy with modal number 57 and they formed tumors in nude mice.

Protease inhibitors (gebexate mesylate and ulinastatin) stimulate intracellular chemiluminescence in human neutrophils.

The effect of protease inhibitors on the intracellular production of free radicals was investigated by measuring chemiluminescence (CL) elicited from phagocytosed luminol-bound microspheres (Lumispheres) in human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), phorbol 12-myristate 13-acetate, or diacylglycerol. Both gabexate mesylate (Foy) and ulinastatin (Miraclid), urinary trypsin inhibitor, increased intracellular CL in a dose dependent manner. Compared to control buffer without protease inhibitor, gabexate mesylate (322 micrograms/ml) caused about a 10-fold increase in intracellular CL in stimulated neutrophils, and ulinastatin (3100 U/ml) a twofold increase in neutrophils stimulated with fMLP or IL-8. When the protease inhibitors were added to the cell suspension after the phagocytosis of lumispheres, CL responses rapidly increased again to the level which was observed when both protease inhibitors and neutrophil stimulants were incubated simultaneously. In contrast, extracellular release of oxygen metabolites from stimulated neutrophils, assayed by a conventional measurement of luminol-dependent CL, was reduced by the protease inhibitors in a dose dependent fashion. When luminol-unbound microspheres were incubated with neutrophils stimulated by fMLP in luminol solution, extracellular CL was almost completely inhibited by gabexate mesylate. These results indicate that the protease inhibitors enhance the generation of intracellular CL and suppress the extracellular release of free radicals.

Circulating interleukin 6 as a useful marker for predicting postoperative complications.

We examined postoperative serial changes in the levels of serum interleukin 6 (IL-6), serum acute phase reactants (APRs) and plasma neutrophil elastase (NE) in patients with various cancers and reviewed these changes in patients who did, and did not, show postoperative complications. Serum IL-6 level was elevated after surgery, peaking on the first postoperative day. Elevation of serum APRs and plasma NE levels also followed. There was a significant correlation between the serum peak level of IL-6 and those of APRs and NE (P less than 0.01). Moreover, there was a significant difference in the serum IL-6 level in patients with and without complications. The relationship between the serum IL-6 greater than 400 pg/ml and the incidence of postoperative complications was also marked. These results suggest that circulating IL-6 is a clinically useful marker for the earliest detection and prediction of postoperative complications.

Development of a radioimmunoassay for human group-II phospholipase A2 and demonstration of postoperative elevation.

A radioimmunoassay (RIA) for the determination of human group-II phospholipase A2 (M-PLA2) has been developed. M-PLA2 was purified from human spleen. Monoclonal antibody (IgG) was prepared by fusion of splenic cells from immunized mice with M-PLA2 and the mouse myeloma cell line NS-1. The RIA was carried out by a single antibody method. The assay is sensitive (0.78 micrograms/l), reproducible and specific. In healthy individuals, the serum M-PLA2 concentration ranges from 1.4 to 4.2 micrograms/l, the average being 2.2 +/- 0.1 micrograms/l (mean +/- SE). Using the RIA, we found increased serum M-PLA2 in patients with various infections and malignant tumors. We also showed the postoperative transient elevation of serum M-PLA2 in cases without any infectious complications. The elevation was independent of the surgical procedure or site. The maximum serum M-PLA2 level was seen on the 2nd to 4th postoperative day. In these patients, the serum M-PLA2 and C-reactive protein levels were significantly correlated. The present study indicated that serum M-PLA2 is an acute phase reactant.

Inhibitory effect of pentoxifylline and prostaglandin E1 on the release of neutrophil elastase from FMLP-stimulated neutrophils.

We measured the enzymatic and immunologic activities of neutrophil elastase to determine whether pentoxifylline and prostaglandin E1 are useful for inhibiting the enzymatic activity of released neutrophil elastase. Both activities in the medium decrease in the presence of pentoxifylline and prostaglandin E1. These results showed that pentoxifylline and prostaglandin E1 inhibited the release of neutrophil elastase from activated neutrophils, and that they may be useful in the improvement of neutrophil-mediated cellular injury.

Identification and characterization of receptors specific for human pancreatic secretory trypsin inhibitor.

Specific binding sites for human pancreatic secretory trypsin inhibitor (PSTI) on 3T3 Swiss albino cells were studied using radioiodinated recombinant PSTI. Some ion species, pH, and temperature significantly influenced the binding of 125I-PSTI. Kinetic studies showed that the binding of 125I-PSTI to 3T3 Swiss albino cells reached the maximum level within 120 min at 4 degrees C, with a slow dissociation rate. The half-maximal inhibition (ID50) of 125I-PSTI binding by unlabeled PSTI occurred at 1.0 x 10(-10) M. On Scatchard analysis of the competitive binding data, linear plots indicated a single class of receptors with high affinity (Kd = 5.3 x 10(-10) M) on 3T3 Swiss albino cells, the number of receptors being 5,400 per cell. Treatment of surface-bound radiolabeled PSTI with a chemical crosslinker (disuccinimidyl suberate) led to the identification of a membrane polypeptide of Mr 140,000 to which PSTI was crosslinked. The formation was inhibited by an excess amount of unlabeled PSTI in a dose-dependent manner. The binding of 125I-PSTI to 3T3 Swiss albino cells was competitively inhibited by unlabeled PSTI but not by other peptide hormones, such as epidermal growth factor (EGF), bovine fibroblast growth factor, insulin-like growth factor, transforming growth factor alpha, platelet-derived growth factor, and tumor necrosis factor, indicating the presence of receptors specific for PSTI. Various protease inhibitors had no or only a little effect, and mercaptoethanol and dithiothreitol strongly decreased the binding of 125I-PSTI. Incubation at 37 degrees C resulted in rapid internalization of cell-bound 125I-PSTI, followed by the appearance of trichloroacetic acid-soluble 125I-radioactivity in the culture medium, due to degradation of internalized PSTI. In addition, PSTI stimulated [3H]thymidine incorporation into DNA on 3T3 Swiss albino cells in a dose-dependent manner. The combined addition of PSTI and EGF stimulated [3H]thymidine incorporation to an extent greater than that seen with either agent alone. These results indicated that the biological effect of PSTI was mediated by high affinity plasma membrane receptors, which were not a cell-surface proteinase(s). Specific binding of 125I-PSTI was noted with the following cells: WI-38, 3T3 Swiss albino, HUVE, BDC-1, and H4-II-E-C3.

Serum interleukin 6, C-reactive protein and pancreatic secretory trypsin inhibitor (PSTI) as acute phase reactants after major thoraco-abdominal surgery.

We measured serum immunoreactive interleukin 6 (IL-6) levels in patients after major thoraco-abdominal surgery and compared them with changes in serum C-reactive protein (CRP) and pancreatic secretory trypsin inhibitor (PSTI) levels. Serum IL-6 levels were elevated earlier than serum CRP or PSTI. There were significant relationships between the peak levels of serum IL-6 and the operation time (p less than 0.05) or the volume of blood loss during surgery (p less than 0.05), showing that serum IL-6 can be a useful laboratory test for the evaluation of tissue injuries due to surgical intervention. Though there was a significant relationship between the peak levels of IL-6 and CRP (p less than 0.01), there was no relationship between the peak levels of IL-6 and PSTI.