Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures.

We have developed a cryogenic fluorescence microscope system, the core of which is a reflecting objective that consists of spherical and aspherical mirrors. The use of an aspherical mirror allows the reflecting objective to have a numerical aperture (NA) of up to 0.99, which is close to the maximum possible NA of 1.03 in superfluid helium. The performance of the system at a temperature of 1.7 K was tested by recording a three-dimensional fluorescence image of individual quantum dots using excitation wavelengths (λex) of 532 nm and 635 nm. At 1.7 K, the microscope worked with achromatic and nearly diffraction-limited performance. The 1/e(2) radius (Γ) of the point spread function of the reflecting objective in the lateral (xy) direction was 0.212 ± 0.008 µm at λex = 532 nm and was less than 1.2 times the simulated value for a perfectly polished objective. The radius Γ in the axial (z) direction was 0.91 ± 0.04 µm at λex = 532 nm and was less than 1.4 times the simulated value of Γ. The chromatic aberrations between the two wavelengths were one order of magnitude smaller than Γ in each direction.

Specific messenger RNA expression for signal transduction molecules by lipopolysaccharide in intestinal macrophages.

Intestinal macrophages are known to display profound inflammatory anergy in response to lipopolysacchraide (LPS). To study the mechanisms of unresponsiveness of intestinal macrophages to LPS, we compared the mRNA expression of molecules associated with signal transduction of intestinal macrophages with those of other tissue macrophages. Also cellular localization of CD14 protein was examined. Intestinal, alveolar and peritoneal macrophages were isolated from rats or mice. The expression of mRNA was assessed by real-time PCR, and cellular localization of CD14 protein was examined by flow cytometry. Cellular responses to LPS were examined by production of TNF and NO. The expression of CD14 mRNA in intestinal macrophages was lower than for peritoneal macrophages but higher than for alveolar macrophages. The mRNA expression of other molecules corresponding to intracellular signal transduction in intestinal macrophages was similar with alveolar and peritoneal macrophages. Despite the presence of CD14 mRNA, proteins of CD14 were not detected on cell surfaces of intestinal macrophages, and induction of TNF or NO responding to LPS were not detected. Flow cytometric analysis demonstrated that CD14 protein was not expressed on the cell surface but was expressed inside intestinal macrophages. The unresponsiveness of intestinal macrophages after LPS exposure is considered to be largely attributed to the lack of CD14 protein on their cell surfaces. However, CD14 protein was expressed inside of the cells, suggesting that post-transcriptional regulation rather than transcriptional suppression may play a dominant role in determining the phenotype of the intestinal macrophages.

Bacteriological study of autologous cryoprecipitate-derived fibrin glue as the operative sealant.

Fibrin glue (FG) is frequently used to seal and cover the anastomoses in many operations such as cardiovascular surgery or orthopaedic surgery. However, in case of gastrointestinal surgery, anastomoses are potentially contaminated, and FG may promote bacterial growth, increasing the risk of leakage. The purpose of this study was to examine the effect of cryoprecipitate-derived FG (CryoFG) on bacterial growth. Bacterial growth on the CryoFG and on the commercial FG (Beriplast P) was evaluated and compared with that on control medium. In addition, the complement activities were evaluated by heat inactivation or addition of guinea-pig complement to the experimental settings. The CryoFG inhibited the growth of Escherichia coli, whereas the commercial FG had no effect. Heat inactivation of the CryoFG inhibited the bactericidal effect of CryoFG. Addition of guinea-pig complement to the heat-inactivated CryoFG could almost restore the bactericidal activity, suggesting the important role of complement. This study showed that the CryoFG preserved the complement activity, which inhibited the in vitro growth of E. coli. Therefore, we concluded that the application of the CryoFG for gastrointestinal surgical anastomoses not only would be safe but also has the advantage of reducing bacterial infection.

Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss.

A second TNF gene (TNF2) has been cloned and sequenced in rainbow trout. In common with the first TNF gene isolated (TNF1), this gene is more TNF alpha-like than TNF beta-like. The full length cDNA is 1519bp, containing a 765bp open reading frame. The gene has four exons, of 380, 49, 60 and 1030bp, respectively. Analysis of the 5' flanking regions of TNF1 and TNF2 reveals several interesting differences in identified transcriptional regulatory elements, with a CATAAA box present 26bp upstream of the transcription start in both genes. Expression analysis in LPS stimulated macrophages has shown a much stronger expression of TNF2 relative to TNF1, with expression being detected earlier and lasting longer.

[Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (1999)].

From October 1999 to September 2000, we collected the specimen from 430 patients with lower respiratory tract infections in 17 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and antibiotics and patients' characteristics. Of 515 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in inflammation, 506 strains were investigated. The breakdown of the isolated bacteria were: Staphylococcus aureus 78, Streptococcus pneumoniae 101, Haemophilus influenzae 104, Pseudomonas aeruginosa (non-mucoid) 58, P. aeruginosa (mucoid) 11, Moraxella subgenus Branhamella catarrhalis 41, Klebsiella pneumoniae 18, etc. Of 78 S. aureus strains, those with 4 micrograms/ml or above of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) occupied 57.7%. Vancomycin and arbekacin showed the most potent activities against MRSA without detection of ABK-resistant strain (MIC: 64 micrograms/ml) and decrease of VCM-sensitive strains those were found in 1998. The frequency of S. pneumoniae exhibiting low sensitivity to penicillin (penicillin-intermediate S. pneumoniae: PISP + penicillin-resistant S. pneumoniae: PRSP) decreased to 34.7% from 46.0% in 1998. The frequency of PRSP was 3.0%, being the least number after 1991. Carbapenems showed strong activities against S. pneumoniae. Especially, panipenem inhibited the growth of all 101 strains with MIC of 0.063 microgram/ml. Generally, all drugs showed strong activities against H. influenzae with MIC80s of 4 micrograms/ml or below. MICs of ofloxacin ranged between 0.063 microgram/ml and 4 micrograms/ml in 1998, however, those were 0.125 microgram/ml or below in all H. influenzae in 1999 showing the strongest activity. Tobramycin and ciprofloxacin showed strong activities against P. aeruginosa (both mucoid and non-mucoid) with MIC80s of 1 microgram/ml. Number of isolated P. aeruginosa (mucoid) was little as 11, however, the susceptibilities to all drugs were better than P. aeruginosa (non-mucoid). K. pneumoniae showed good susceptibilities to all drugs except for ampicillin with decreasing of low-sensitive strains compared to those detected in 1998. Also, all drugs generally showed strong activities against M. (B.) catarrhalis. MIC80s of all drugs were 2 micrograms/ml or below. The drug which showed the strongest activity was imipenem inhibiting all 41 strains with MIC of 0.063 microgram/ml. On the patients' characteristics, the number of patients aged 80 years or older who had been increased was decreased in 1999 in the distribution by age. The percentage of the elderly patients aged 70 years or older was 47.0%, which occupied almost a half number of the total patients as in the last year. As for the incidence by disease, bacterial pneumonia and chronic bronchitis were the highest. They were noted in 37.9% and 30.5% of the patients, respectively. In 1999, bronchial asthma was frequently observed as compared in recent years. It was noted in about 10% of the patients which is the same % as in bronchiectasis. We examined the number of strains from these patients with infections before and after administration of antibiotics. In patients with bacterial pneumonia, the number of isolated strains was almost the same between those before and after administration. However, in patients with chronic bronchitis, the number of strains remarkably decreased to less than the half of the total after administration of antibiotics in the last year, but it decreased to 2/3 of the total in 1999. On the administration of antibiotics and isolated bacteria by the day of administration, the bacteria which were isolated more before administration were H. influenzae in 28.4%, S. pneumoniae in 25.7%, M. (B.) catarrhalis in 12.0% and S. aureus in 10.6%. The frequency of S. aureus after administration over 15 days was almost the same as that before administration, but the frequency of P. aeruginosa (both mucoid and non-mucoid) was 36.8% which was higher than that before administration. The frequency of isolated S. pneumoniae was decreased after administration and none of them was isolated after completion of administration. However, that of H. influenzae was decreased to 7.1% after administration within 3 days, and many H. influenzae were isolated after completion of administration as 21.4%.

Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss).

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.

Systemic fusariosis after a preparative regimen including thiotepa, VP-16 and busulfan used for blood stem cell transplantation in Hodgkin's disease.

Fusarium infection is rare but important infection after bone marrow transplantation (BMT). A 27-year-old man developed systemic fusarial infection following severe skin damage probably caused by high-dose thiotepa administration. Systemic fusariosis rapidly progressed to a variety of organs despite antifungal treatment, and he finally died of this infection on day 75. Considering that this organism usually invades via damaged skin and that the penile lesion was the first manifestation of systemic fusariosis in this patient, careful examination of the skin might be helpful for early diagnosis of fusarial infection. His clinical course provided us with an important clue for diagnosis of fusarial infection.

A new method of antitumor therapy with a high dose of TNF perfusion for unresectable liver tumors.

There are primary and secondary malignant liver tumors for which principal treatment is surgical resection. There is no established treatment for unresectable malignant liver tumors, however, and the prognosis for these is quite poor. An effective treatment for malignant liver tumors is thus urgently needed. Recent advances in molecular biology have uncovered the structures and/or functions of many cytokines thought to have a strong relation with the mechanisms of the antitumor effect of biological therapies. Availability of those cytokines in large amounts and homogeneously owing to advances in recombinant technology makes it possible to use them clinically. Among cytokines demonstrating antitumor activities, tumor necrosis factor-alpha (TNF-alpha) is one of the strongest. However, severe toxicity such as hypotension, abnormalities in liver function, leukopenia, chill and thrombus formation makes TNF-alpha difficult to use systemically as an antitumor drug. To enhance cytotoxicity while decreasing the side effects, especially hypotension, we developed a mutein called TNF-SAM2 by protein-engineering. The biological activity of TNF-SAM2 was more beneficial than TNF-alpha for antitumor therapy, since its side effects were milder. In contrast, using the isolated limb perfusion (ILP) method against malignant melanoma and soft tissue sarcoma of the extremities in combination with TNF-alpha and melphalan, a high response rate of 70-100% was observed. These observations led to the re-evaluation of TNF as an antitumor drug. A preliminary clinical trial was done using TNF-alpha combined with the formation of a closed circuit (isolated hepatic perfusion method) targeting the liver and a response rate of over 75% was achieved against malignant liver tumors. To isolate the liver from the systemic circulation, however, required a laparotomy, so that patients were subjected to excessive surgical stress. Isolated hypoxic hepatic perfusion (IHHP) using balloon catheters is a treatment developed to overcome such stress and we are planning to do clinical trials of IHHP with TNF-SAM2 in combination with a chemotherapeutic agent against malignant liver tumor patients. IHHP combined with TNF-SAM2 and a chemotherapeutic agent might be more beneficial in antitumor effects as well as in maintaining good quality of life (QOL) for the patient.

[Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (1998)].

The bacteria isolated from the patients with lower respiratory tract infections were collected by institutions located throughout Japan, since 1981. Ikemoto et al. have been investigating susceptibilities of these isolates to various antibacterial agents and antibiotics, and analyzed some characteristics of the patients and isolates from them each year. Results obtained from these investigations are discussed. In these 18 institutions around the entire Japan, 532 strains of presumably etiological bacteria were isolated mainly from the sputa of 438 patients with lower respiratory tract infections during the period from October in 1998 to September in 1999. MICs of various antibacterial agents and antibiotics were determined against 85 strains of Staphylococcus aureus, 100 strains of Streptococcus pneumoniae, 96 strains of Haemophilus influenzae, 75 strains of Pseudomonas aeruginosa (non-mucoid strains), 6 strains of Pseudomonas aeruginosa (mucoid strains), 38 strains of Moraxella subgenus Branhamella catarrhalis, 26 strains of Klebsiella pneumoniae etc., and the susceptibilities of 517 strains were assessed except for those strains that died during transportation. S. aureus strains for which MICs of oxacillin (MPIPC) were higher than 4 micrograms/ml (methicillin-resistant S. aureus: MRSA) accounted for 60.0%. Vancomycin (VCM) and arbekacin (ABK) showed the most potent activities against MRSA. But one of MRSA showed resistance to ABK with the MIC of 64 micrograms/ml. The sensitive strains of MRSA to VCM have decreased. The frequency of penicillin (PC)-intermediate S. pneumoniae (PISP) + PC-resistant S. pneumoniae (PRSP) have increased in 46.0% for 1998 comparatively from 30.9% of 1997's. But PRSP decreased, and PISP increased into 39.0% of 1998 years from 19.8% of 1997's. Panipenem (PAPM), imipenem (IPM) and faropenem (FRPM) showed the most potent activities against S. pneumoniae with MIC80s of 0.125 microgram/ml or below. Against H. influenzae and M. (B.) catarrhalis, almost all the drugs showed good activities. The sensitive strains of them against ceftazidime (CAZ) decreased in 1997, but those have increased in 1998. Inversely, the susceptibility of them against cefotiam (CTM) had been higher in 1997, but those have been lower in 1998. Tobramycin (TOB) showed the most potent activity against P. aeruginosa (both mucoid and nonmucoid strains). All drugs except ampicillin (ABPC) were active against K. pneumoniae. A quite few of K. pneumoniae showed low susceptibilities. Also, we investigated year to year changes in the characteristics of patients, their respiratory infectious diseases, and the etiology. The examination of age distribution indicated that the proportion of patients with ages over 70 years was 48.6% of all the patients showing a slight increase in every year. About the proportion of diagnosed diseases as follows: Bacterial pneumonia was the most frequent with 40.2%. The ratio of it has increased slightly, and the increased rate was 10% in patients with ages over 70 years compared with the results in 1997. Chronic bronchitis have decreased slightly with 27.6% in 1998. Number of strains isolated from patients before administration of antibiotics were more than those after administration of them in chronic bronchitis, but these were almost same number in bacterial pneumonia. Administration of antibiotics has changed the results of the frequency of isolation of bacterial species. Bacterial isolations before administration of antibiotics were as follows: S. pneumoniae 26.7%, H. influenzae 23.8%, S. aureus 13.3% and M. (B.) catarrhalis 10.8%. The frequencies of S. aureus decreased after antibiotics administration over 15 days, but the frequencies of P. aeruginosa (both mucoid and non-mucoid) was not affected. The frequencies of P. aeruginosa was 45.5% after administration over 15 days. The frequencies of S. pneumoniae decreased upon administration of antibiotics, these were only 4.5% over 15 days. The frequencies of H. (

Pharmacokinetics of isolated hepatic perfusion with high dose tumor necrosis factor in rat model.

BACKGROUND: Although the application of an isolation procedure with tumor necrosis factor (TNF) to the liver is quite attractive, an animal model is not yet available to evaluate antitumor effects by TNF in isolated hepatic perfusion (IHP). To establish the rat model in IHP, the pharmacokinetics of TNF, both in a perfusate and in a systemic circuit should be examined. METHODS: All rats underwent IHP with TNF. After a 10 min perfusion, a washout procedure was performed for 5 min, after which isolation was terminated. Throughout the procedure and afterward, blood samples were obtained from the systemic blood and concentrations of TNF were assayed by L-929 cytotoxicity. RESULTS: After the administration of 240 micrograms of TNF in the circuit, TNF reached a plateau at about 12.7 micrograms/ml of perfusion fluid, lasting until the end of IHP. As a result of the washout procedure, regional TNF concentrations declined from 12.7 micrograms/ml to 1.5 micrograms/ml. At the beginning of the IHP, all rats exhibited no detectable level of TNF activity in the systemic circulation (< 100 pg/ml). With time, TNF plasma levels quickly increased to reach a plateau of about 0.2 microgram/ml at 15 min. Systemic leakage of TNF is calculated as less than 2% of the total TNF in perfusate during perfusion. CONCLUSION: Rat IHP models with TNF showed that systemic leakage of TNF was higher than that of pig models, although a large enough amount of TNF in perfusate was achieved without death. Rat models might be feasible to evaluate antitumor effect of IHP against liver metastatic tumors.

Reduction of hepatotoxicity of tumor necrosis factor in isolated hepatic perfusion by administration of glucocorticoid as well as lipopolysaccharide.

BACKGROUND: The application of an isolation procedure with tumor necrosis factor (TNF) to the liver is quit attractive, however, one of problems to overcome is reducing the toxicity to the liver caused by high doses of TNF. MATERIALS AND METHODS: Rats underwent isolated hepatic perfusion (IHP) with TNF and pre-treatment of subcutaneous administration of dexamethasone (4 mg/kg) and/or intradermal administration of LPS (50 micrograms/rat). After a 10 min perfusion, a washout procedure was performed for 5 min, after which isolation was terminated. RESULTS: SD or Wister rats and F344 rats tolerated up to 120 mg/rat or 4 micrograms/rat, respectively. Dexamethasone and/or LPS was tolerated at 40 micrograms/rat of TNF in F344 rat and showed a significant reduction of hepatotoxicity, and indicated histologically the suppression of ballooning and of necrosis during and after perfusion by TNF. CONCLUSION: We propose new a protocol for IHP as follows: 1. the intradermal administration of LPS for protection against toxicity of TNF, 2. IHP with TNF-SAM2, a mutain of TNF-alpha, having less toxicity than conventional TNF-alpha, and 3. simultaneous perfusion with chemotherapeutic agents such as 5-fluorouracil (5-FU).

Significance of anaerobic preincubation of Helicobacter pylori for measuring metronidazole susceptibility by the Etest.

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration>8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.

Enteral administration of glutamine in purulent peritonitis.

The intestinal hypomotility associated with purulent peritonitis is generally regarded as a contraindication to enteral nutrition. However, enteral nutrition may be feasible in suppurative peritonitis if administered with great caution, i.e., assuring the appropriate amount, delivery speed, and osmolality of the enteral formulation. Glutamine (Gln) increases muscle protein synthesis and decreases muscle protein degradation in sepsis, regardless of the route of administration. Therefore, administering small amounts of supplemental Gln via the enteral route to peritonitis patients may be beneficial. Two purulent peritonitis patients received L-Gln through a jejunostomy tube. The average amount of supplemental Gln was 16 g/d. Systemic inflammatory responses, i.e., high temperature and a high serum C-reactive protein level, persisted throughout the treatment period. Femoral arterial and venous blood samples were drawn simultaneously for determination of amino acid levels before and after 7 d of Gln supplementation. Enterally administered Gln was well-tolerated by both patients. There was an increase in plasma Gln levels after Gln supplementation. Moreover, the release of Gln, alanine, and phenylalanine from the lower extremities was lower after as compared to before Gln supplementation. Enteral administration of Gln may be feasible even in purulent peritonitis.

Mechanisms by which chemotherapeutic agents augment the antitumor effects of tumor necrosis factor: involvement of the pattern shift of cytokines from Th2 to Th1 in tumor lesions.

BACKGROUND: The antitumor effect exerted by tumor necrosis factor (TNF) is characteristic in that it causes central necrosis of the tumor mass. Viable tumor cells surrounding the tumor mass remain, however, even after most of the mass is necrotized, and these cells gradually regrow and form tumors. To overcome this, we analyzed the combined effects of chemotherapeutic agents used with TNF. Alkylating agents such as cyclophosphamide altered the antitumor effect qualitatively, leading to complete regression which TNF alone could not achieve. The mechanism, behind the enhancement of endogenous TNF production and expression of mRNA of various cytokines by the combination of chemotherapeutic agents with TNF inducer was investigated in Meth A fibrosarcoma. METHODS: Seven days after the inoculation of Meth A fibrosarcoma into BALB/c mice, cyclophosphamide (CY, 100-150 mg/kg) was injected intraperitoneally, and 7 days later endogenous TNF was induced by the intradermal administration of lipopolysaccharide (LPS, 400 micrograms/kg) or intravenous injection of ONO-4007, a synthetic lipid A derivative (30 mg/kg). RESULTS: A combination therapy of LPS or ONO-4007 with CY showed the effect of complete regression in 50-100% of tested mice, while CY, LPSp or ONO-4007 alone did not cause complete regression. The amount of endogenous TNF induced by LPSp or ONO-4007 around a tumor lesion with CY was 4-5 fold higher than that without CY. The expression of mRNA of transforming growth factor-beta was suppressed by CY seven days after the injection, and expressions of mRNA of IL-1 beta and TNF-alpha were augmented by the administration of CY 1 to 3 hours after the administration of ONO-4007. CONCLUSION: Some chemotherapeutic agents thus appear to augment the antitumor effect of TNF around tumor lesions, leading to tumor regression through a mechanism in which the agent changes the host's immune status, especially around a tumor lesion and pattern shift of cytokines from Th2 to Th1.

Preservation of metastatic ability of colorectal tumor cells stratified by inducibility of endogenous tumor necrosis factor after orthotopic transplantation in nude mice.

BACKGROUND: Inducibility of endogenous tumor necrosis factor (TNF) by colorectal tumor cells can be regarded as a novel prognostic factor in terms of distant metastasis or local recurrence following curative operation, especially at Duke's stage C. In this study, the metastatic ability of human colorectal tumor cells stratified by inducibility of endogenous TNF was analyzed by orthotopic transplantation in nude mouse. METHODS: Fifty three cases of freshly resected colorectal tumor specimens cut into about 50mg pieces were inoculated into the cecum wall of nude mice. Two to nine months after transplantation, tumor growth on this wall and metastases to the peritoneal wall as well as to the liver were assessed. RESULTS: Of forty one evaluable cases, successful transplantation was observed in twenty nine (70%), and metastases to the peritoneal wall or to the liver was found in thirteen (32%), or eight cases (20%), respectively. In the twenty nine cases with local tumor growth, incidence of the liver metastases in nude mice when tumor specimens from patients with liver metastases (4/8) were used was significantly higher than that from patients without liver metastases (4/21) (P = 0.096). Inducibility of endogenous TNF was separately analyzed in fourteen of the evaluable twenty nine cases. Seven cases belonged to the high group and seven to the low group in terms of the amount of endogenous TNF secreted by tumor cells. Incidence of metastases in mice was 1/7 in the high group and 6/7 in the low group, and there was a statistically significant difference between liver metastases in mice and inducibility of endogenous TNF by colorectal tumor cells (p = 0.0057). CONCLUSION: From these results, it is strongly suggested that inducibility of endogenous TNF by colorectal tumor cells can affect a patient's prognosis since it regulates metastatic ability to the liver.

[Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (1996)].

The bacteria isolated from the patients with lower respiratory tract infections were collected by institutions located throughout Japan, since 1981. Ikemoto et al. have been investigating susceptibilities of these isolates to various antibacterial agents and antibiotics, and characteristics of the patients and isolates from them each year. Results obtained from these investigations are discussed. In 16 institutions around the entire Japan, 557 strains of presumably etiological bacteria were isolated mainly from the sputa of 449 patients with lower respiratory tract infections during the period from October 1996 to September 1997. MICs of various antibacterial agents and antibiotics were determined against 98 strains of Staphylococcus aureus, 93 strains of Streptococcus pneumoniae, 84 strains of Haemophilus influenzae, 84 strains of Pseudomonas aeruginosa (non-mucoid strains), 17 strains of Pseudomonas aeruginosa (mucoid strains), 31 strains of Moraxella subgenus Branhamella catarrhalis, 21 strains of Klebsiella pneumoniae etc., and the drug susceptibilities of these strains were assessed except for those strains that died during transportation. 1) S. aureus S. aureus strains for which MICs of oxacillin (MPIPC) were higher than 4 micrograms/ml (methicillin-resistant S. aureus) accounted for 67.3%. The frequency of the drug resistant bacteria increased comparing to the previous year's 52.7%. Arbekacin (ABK) and vancomycin (VCM) showed the highest activities against both S. aureus and MRSA with MIC80s of 1 microgram/ml. 2) S. pneumoniae Imipenem (IPM) and panipenem (PAPM) of carbapenems showed the most potent activities with MIC80s of 0.063 microgram/ml. Faropenem (FRPM) showed the next potent activity with MIC80 of 0.125 microgram/ml. The other drugs except erythromycin (EM), clindamycin (CLDM) and tetracycline (TC) were active against S. pneumoniae tested with MIC80s of 8 micrograms/ml or below. 3) H. influenzae The activities of all drugs were potent against H. influenzae tested with MIC80s of 4 micrograms/ml or below. Cefotiam (CTM), cefmenoxime (CMX), cefditoren (CDTR) and ofloxacin (OFLX) showed the most potent activities with MIC80s of 0.063 microgram/ml. 4) P. aeruginosa (mucoid strains) Tobramycin (TOB) showed the most potent activity against P. aeruginosa (mucoid strains) with MIC80 of 1 microgram/ml. Ceftazidime (CAZ), cefsulodin (CFS), IPM, gentamicin (GM), ABK and ciprofloxacin (CPFX) showed the next potent activities, with MIC80s of 2 micrograms/ml. The MIC80s of the other drugs ranged from 4 micrograms/ml to 16 micrograms/ml. 5) P. aeruginosa (non-mucoid strains) TOB and CPFX showed the most potent activities against P. aeruginosa (non-mucoid strains) with MIC80s of 1 microgram/ml. The MIC80s of piperacillin (PIPC) and cefoperazone (CPZ) were 16 micrograms/ml in 1995, and they were 64 micrograms/ml in 1996. 6) K. pneumoniae All drugs except ampicillin (ABPC) were active against K. pneumoniae. CMX, cefpirome (CPR), cefozopran (CZOP) and carumonam (CRMN) showed the most potent activities against K. pneumoniae with MIC80s of 0.125 microgram/ml. The MIC80s of the other drugs ranged from 0.25 microgram/ml to 2 micrograms/ml. 7) M.(B) catarrhalis Against M.(B.) catarrhalis, all drugs showed good activities with MICs of 4 micrograms/ml or below. IPM and minocycline (MINO) showed the most potent activities with MICs of 0.063 microgram/ml. Also, we investigated year to year changes in the characteristics of patients, their respiratory infectious diseases, and the etiology. Patients' backgrounds were examined for 557 isolates from 449 cases. The examination of age distribution indicated that the proportion of patients with ages over 60 years was 71.0% of all the patients showing a slight increase over that in 1994. Proportions of diagnosed diseases were as follows: Bacterial pneumonia and chronic bronchitis were the most frequent with 35.9% and 30.3% respectively. They were followed by bronchiectasis with a proportion of 10.

Effects of trauma and sepsis on soluble L-selectin and cell surface expression of L-selectin and CD11b.

OBJECTIVES: To examine (1) the effects of trauma on changes in neutrophil L-selectin and CD11b expression and on the levels of soluble L-selectin and (2) whether these alterations are different on leukocyte subpopulations in those patients who develop multiple organ dysfunction syndrome. MATERIALS AND METHODS: Twenty patients with Injury Severity Score (ISS) > or = 16 and 15 patients with ISS score < 16 were studied. Arterial blood were collected serially after injury. The staining of leukocyte surface adhesion molecules was performed with antibodies against L-selectin and CD11b. Positive cell count and mean fluorescence intensity were determined by flow cytometry. Soluble L-selectin was measured using enzyme-linked immunosorbent assay. RESULTS: In patients with ISS > or = 16, neutrophil L-selectin expression showed an immediate increase, reaching peak levels between 3 to 4 hours after injury (p < 0.05 vs. patients with ISS < 16), followed by a gradual decrease. Plasma levels of soluble L-selectin reached peak levels at 6 hours after injury. However, in patients with ISS < 16, minimal changes in L-selectin expression and soluble L-selectin were observed. Neutrophil CD11b expression showed an immediate increase for the first 3 hours followed by a gradual increase up to 24 hours after injury. In patients who developed multiple organ dysfunction syndrome, CD11b both on neutrophils and lymphocytes remained elevated for 120 hours. CONCLUSIONS: These findings suggest that acute neutrophil activation is an early event after trauma and may be implicated as "a vulnerable window" for leukocyte-mediated end organ injury.