Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells

Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficile toxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC). Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)- or tumor necrosis factor- (TNF )-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-B activation (NF- B). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F₁ (6k-PGF₁). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF₁ liberation when TcdB-10643 was added 10 h after LPS or TNF stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF- B-dependent LPS- and TNF-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.     

Interleukin-1β induces expression of cyclooxygenase-2 mRNA in human gingival fibroblasts

The effect of interleukin-1 (IL-1) on the expression of cyclooxygenase-1 and –2 (COX-1 and COX-2) mRNA and its relation to prostaglandin E₂ (PGE₂) biosynthesis in human gingival fibroblasts was studied. IL-1 increased levels of mRNA for COX-2 whereas the COX-1 mRNA level was unaffected. The increased COX-2 mRNA levels were accompanied by enhanced PGE₂ formation. The phorbol, 12-myristate 13-acetate (PMA), known to stimulate protein kinase C (PKC), also induced expression of COX-2 mRNA. When gingival fibroblasts were treated simultaneously with IL-1 and PMA, the cytokine IL-1 synergistically increased levels of COX-2 mRNA, accompanied by a corresponding increase in PGE₂ biosynthesis. The anti-inflammatory steroid, dexamethasone (DEX) abolished the enhanced expression of COX-2 mRNA as well as PGE₂ formation induced by IL-1, PMA or the combination of IL-1 and PMA. The study indicates that the IL-1 induced PGE₂ formation is mediated by an enhanced gene expression of COX-2 in gingival fibroblasts suggesting that the enzyme COX-2 may play an important role in the regulation of prostanoid formation at inflammatory lesions in gingival tissue.     

In vitro activity of saperconazole (R66 905) compared with amphotericin B and itraconazole againstAspergillus species

Twenty isolates of aspergillus were tested against saperconazole and 16 of these against itraconazole and amphotericin B using a macrodilution broth method. For 18 (90 %) of 20isolates tested against saperconazole, MICs were 3.1 mg/l and for 15 (75 %) of 20 isolates MFCs were 3.1 mg/l. For 9 (56 %) of 16 isolates tested against itraconazole, MICs were 3.1 mg/l, and for 4 (33 %) of 12 isolates MFCs were 3.1 mg/l. For all 16 isolates tested against amphotericin B MICs and MFCs were 4.0 mg/l; for 11 of 16 isolates MICs were 2.0 mg/l. Saperconazole appears to be highly active againstAspergillus spp. in vitro, with a bimodal distribution of MICs and MFCs.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.     

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGCTTC (cysteinephenylalanine) and the other codon 175; CGCCAC (argininehistidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.     

IL-4 and TNF-α induce changes in integrin expression and adhesive properties and decrease the lung-colonizing potential of HT-29 colon carcinoma cells

The colon carcinoma cell line HT-29 was used to explore the potential of interleukin-4 (IL-4) and tumor necrosis factor (TNF-) to modify integrin expression and adhesive functions of tumor cells in vitro and to examine corresponding metastatic effects in vivo. Preincubation of HT-29 cells with 100 U/ml of IL-4 for 48 h downregulated the surface expression of the integrin subunits 2, 3, l and 4 after 48 h, whereas the 1 subunit was upregulated. In contrast, 100 U/ml of TNF-a selectively upmodulated the expression of av. Attachment to fibronectin of cells treated with IL-4 increased twofold (63.5% vs 32.4%). Adhesion to fibronectin (54.0% vs 32.4%) and vitronectin (37.9% vs 16.4%) was elevated in the case of TNF-a stimulation. Using an experimental metastasis model, HT-29 cells showed a significant reduction of their lung-colonizing potential in nude mice when preincubated with IL-4 for 48 h before intravenous injection. The decrease also observed for TNF--treated cells was less pronounced. The data indicate that the cytokines IL-4 and TNF- can act as direct regulators of adhesive mechanisms of tumor cells bearing adequate receptors, thus influencing lung-colony formation.     

Action of a serum protein on muscular contraction

Summary The mechanism of action of a serum protein isolated from human serum was assessed in several experimental preparations including glycerol-treated muscle fibers, rat heart papillary muscle and isolatedin vitro perfused rat heart. The action of the serum protein was studied also on canine and human heart papillary muscles which were made to respond to electrical stimulation with ultrasonication modified epinephrine. In addition the action of the protein on adenosine 5 triphosphate generated precipitation of purified human actomyosin was investigated.The serum protein enhanced and intensified the generation of ATP induced tension in glycerol-extracted muscle fibers. It intensified the developed tension (DT) and increased the rate of development of tension (dT/dt) without influencing the time peak tension (TPT) of capillary muscles from rat, canine and human hearts in response to electrical stimulation. The serum protein increased the force of contraction of the isolatedin vitro perfused rat heart, and accelerated the adenosine 5 triphosphate generated precipitation of purified human heart actomyosin.     

Expression and ligand binding of α2β1 integrin on breast carcinoma cells

We examined the expression and ligand specificity of the 21 integrin on human mammary epithelial cells (HMEC) and a panel of breast carcinoma cell lines in vitro. We found that the 21 integrin was universally, but quite variably expressed on these cells by FACS analysis. No significant correlation was observed between its expression and other known cellular phenotypes. Substrate attachment assays using blocking antibodies demonstrated that 21 integrin served as a receptor for collagen on HMEC and almost all breast carcinoma cells. However, its contribution to laminin binding of these cells appeared to be related to cellular differentiation as evaluated by sex steroid receptor status and by markers of epithelial-mesenchymal transition, i.e. loss of E-cadherin and expression of vimentin. Two different populations of non-malignant immortalized HMEC (184A1N4 and MCF-10A) contained cells capable of using 21 integrin as a laminin receptor. Breast cancer cell lines positive for estrogen receptor (ER) and E-cadherin (MCF-7, T47D, ZR75-1) could also use 21 integrin as a laminin receptor. Conversely, 21 integrin appeared to be incapable of binding to laminin or to be a very minor receptor for laminin on metastatic ER-negative breast carcinoma cells that expressed vimentin (MDA-MB 231, MDA-MB 435, and MDA-MB 436). These findings suggest that the ligand specificity of 21 integrin, i.e. its function as a laminin receptor, may be regulated during the malignant progression of breast carcinoma cells. A reduced contribution of 21 integrin to the cellular laminin binding appears to be associated with an increased malignant phenotype and with an epithelial-mesenchymal transition of breast carcinoma cells.     

The Pulmonary Inflammatory Response to Experimental Fecal Peritonitis: Relative Roles of Tumor Necrosis Factor-α and Endotoxin

The roles of endotoxin (LPS) and tumor necrosis factor- (TNF-) in the causation of organ injury during sepsis are unclear. To study LPS and TNF- in the genesis of lung inflammation after cecal ligation and puncture (CLP), we used endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive mice (C3H/HeOuJ). We examined lung neutrophil sequestration, interleukin 1 (IL-1) mRNA expression, IL-1 protein expression, and injury. We also determined the expression of two C-X-C chemokine mRNAs, macrophage inflammatory protein-2 (MIP-2) and KC, in the lung to determine whether in vivo, endotoxin, or TNF- are significant modulators of MIP-2 and KC mRNA expression. After CLP, increased neutrophils sequestrated in the lungs of both strains of mice and coincided with an increase in expression of IL-1, MIP-2 and KC mRNAs, and IL-1 protein. Lung and serum TNF- were significantly increased in the C3H/HeOuJ strain but not in the C3H/HeJ strain. Histologic studies of the lung revealed similar injury in both strains. Our results suggest that bacterial factors other than endotoxin cause lung neutrophil sequestration and injury after CLP and further, that TNF- production is not a prerequisite. Our findings also suggest a potential role for local pulmonary chemokine production in the control of neutrophil sequestration after CLP.     

Measurement precision of a portable instrument to assess vibrotactile perception threshold

Summary The objective of the present study was to evaluate the reliability of a modified version of the commercially available Biothesiometer, and to examine vibrotactile perception thresholds with respect to age and gender. A standardized protocol for measuring vibrotactile perception threshold was administered to 80 subjects, once a week over 4 weeks. Inter-session variability was stable (analysis of variance for repeated measures; P>0.05) and correlations were high (Pearson's: 0.87r0.90; P0.001). For sites on both hands and feet, there was a significant increase with age (0.19r ²0.52; P0.001). Five factor analysis of variance model showed that vibrotactile perception threshold was significantly different with stimulus site, age category and gender; no differences were observed with alcohol consumption or smoking status. The findings indicate that the measurements from this device are highly reproducible and sensitive to expected threshold differences with age and gender. The authors attribute this to technical improvements of the original apparatus, rigid adherence to test protocol and maintenance of standard conditions. This type of instrument would be useful in assessing vibrotactile perception loss in occupational health studies.     

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1) and tumor necrosis factor-alpha (TNF-) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 and TNF- were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 and TNF- were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)₂-vitamin D₃, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF- stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 and TNF- failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 and l nM TNF-) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.     

Provisional quality control parameters and interpretive criteria for testing susceptibility ofStreptococcus pneumoniae andHaemophilus influenzae to quinupristin/dalfopristin (RP59500)

Studies were undertaken to select tentative criteria for susceptibility testing of quinupristin/dalfopristin againstStreptococcus pneumoniae andHaemophilus influenzae. Against 612 isolates ofStreptococcus pneumoniae, MICs of quinupristin/dalfopristin were 1.0 g/ml for all but one strain. With a tentative MIC breakpoint of either 1.0 g/ml or 2.0 g/ml for susceptible, a disk diffusion zone diameter breakpoint of 19 mm embraced all but two of the susceptible pneumococci; 16 mm included all strains. ForHaemophilus influenzae, MICs of quinupristin/dalfopristin clustered near the tentative breakpoints; 91.5% of the MICs were 2.0 to 8.0 g/ml. This precluded satisfactory performance of the disk diffusion test in discriminating between resistant and susceptible isolates unless MIC breakpoints are modified for this species: clinical experience will be needed before that can be justified. Based on data from a multilaboratory study, the following quality control limits are proposed forStreptococcus pneumoniae ATCC 49619 when testing quinupristin/dalfopristin: 0.25 to 1.0 g/ml for broth microdilution tests and 19 to 24 mm for disk diffusion tests. For tests ofHaemophilus influenzae ATCC 29247, MIC limits are 2.0 to 16 g/ml; disk tests were very reproducible but are not yet recommended.     

Cognitive spatial-motor processes

Summary Fourteen human subjects performed in a modified Sternberg memory-scanning task. First, they made a series of 2–6 movements in different directions from a central point towards peripheral lights on a planar working surface (list trials). Then, after a warning signal, one of the previous list stimuli, except the last, was presented again (test trial). Subjects were instructed to move in the direction of the stimulus which was presented next in sequence in the list. The mean reaction time (RT) in the test trials increased as a linear function of the number of movements, S, in the list: Mean RT (ms)=105+205.8S (2S6). This finding suggests that the task involves memory scanning of visuomotor list items.     

Humanβ:α but not γ interferon binding site is a product of the chromosome 21 interferon action gene

The binding of human interferons to their binding site(s) was measured by the amount of radioiodinated human beta Interferon (HuIFN) displaceable by unlabeled human beta, alpha, and gamma Interferon (HuIFN, , and ). By this approach, HuIFN and HuIFN were found to interact with specific binding sites in cell membranes derived from human cells and mouse-human cell hybrids containing chromosome 21 as their only human chromosome. Specific binding was not observed with cell membranes derived from parental mouse cells or from mouse-human cell hybrids in subsequent generations that have lost human chromosome 21. Although the chromosome 21-positive mouse-human cell hybrids are sensitive to the antiviral effects of HuIFN and HuIFN, they are found to be insensitive to the antiviral effect of HuIFN and to lack specific HuIFN binding sites. These results suggest that the HuIFN and HuIFN but not HuIFN binding sites are coded for by genes located on chromosome 21. The lack of a chromosome 21 gene dosage effect on the inducibility of the antiviral state by HuIFN is consistent with this hypothesis.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.     

Characterization of human larynx carcinoma cell lines HLaC′79 and HLaC′82: a common origin but diverged malignancies

As part of a study on the relationship of tumour phenotype and behaviour, we have characterized two head and neck squamous cell carcinoma cell lines, derived from human laryngeal carcinomas and designated HLaC79 and HLaC82. Cytogenetic analysis revealed that HLaC79 and HLaC82 shared 10 major chromosome rearrangements indicating that the cell lines had a common origin. In the extremely complex chromosomal patterns, abnormalities were found in chromosomes 1, 3 (surplus 3q) and 5 (i(5p) × 2). Both cell lines displayed constitutive expression of vimentin and were capable of anchorage-independent growth in agarose gels. However, in spite of their common origin specific differences were found. Cells of HLaC79 were spindle shaped and formed tumours in athymic mice. In contrast, cells of HLaC82 had a compact morphology, contained less vimentin, were more contact inhibited and were not tumorigenic. These results indicate that malignant transformation in HLaC82 was partially reversed.     

Differential expression of basement membrane type-IV collagen α1, α2, α5 and α6 chains among the histological subtypes of adenoid cystic carcinoma

Six distinct (IV) chains in the basement membrane (BM) of adenoid cystic carcinoma (ACC) of the salivary gland were immunohistochemically examined by anti-(IV) chain-specific antibodies, and their expressions were compared with the histological subtypes and the expressions of cytokeratin 19 (CK19), cytokeratin 14 (CK14) and -smooth muscle actin (-SMA) and Ki-67. In the BM of normal salivary ducts, 1(IV), 2(IV), 5(IV) and 6(IV) chains were continuously stained, but 3(IV) and 4(IV) chains were negative. In the tubular and cribriform subtypes of ACC, tubules with continuous staining of 5(IV) and 6(IV) chains showed the biphasic-staining pattern among the expressions of CK19, CK14 and -SMA. However, in cancer-cell nests with discontinuous or negative staining of 5(IV) and 6(IV) chains, the biphasic pattern was ambiguous. In the solid subtype, the staining of 1(IV) and 2(IV) chains was discontinuous, the staining of 5(IV) and 6(IV) chains was negative and the biphasic-staining pattern was unclear. The mitotic activity of cancer cells analyzed by the Ki-67 labeling index was significantly related to the expression of 5(IV) and 6(IV) chains in the cribriform subtype. These results suggest that BM irregularity with the differential expression of (IV) chains in ACC closely relates to cell proliferation, cell differentiation and histological structure.     

In vitro susceptibility ofHaemophilus influenzae to cefaclor,cefixime, cefetamet and loracarbef

The susceptibility of 2,212Haemophilus influenzae isolates cultured in UK clinical laboratories in 1991 was determined for four orally-administered -lactam drugs. These isolates included 1,893 ampicillin-susceptible, 191 -lactamase-positive and 128 ampicillin-resistant, -lactamase-negativeHaemophilus influenzae. While 150 (6.8 %) isolates were resistant to cefaclor (MIC 16 mg/l) and 85 (3.8 %) to loracarbef, all were inhibited by 2 mg/l cefetamet and 1 mg/l cefixime and were therefore susceptible to these agents. Ranges and modes of inhibition zone diameters and MICs indicated that the susceptibility of a variable proportion of the 191 -lactamase-positive isolates to cefaclor, loracarbef and cefetamet was reduced compared with the fully susceptible population. In contrast, a major reduction in susceptibility to all four antimicrobial agents was seen among the 128 ampicillin-resistant (MIC 1–64 mg/l) -lactamase-negative isolates such that these accounted for 53 % and 67 % of the total number of organisms resistant to cefaclor and loracarbef respectively. In addition, 23 of 25 isolates inhibited only by 1 mg/l cefetamet and all eight inhibited only by 0.5 mg/l cefixime showed this type of resistance to ampicillin. Results indicate the importance of detecting non--lactamase-mediated resistance to ampicillin and any concomitant diminished susceptibility to other -lactam drugs.     

Comparative in vitro activity of cefdinir (CI-983; FK-482) against staphylococci,gram-negative bacilli and respiratory tract pathogens

The in vitro activity of cefdinir (CI-983; FK-482), a new oral cephalosporin, was compared with that of other antimicrobial agents against clinical isolates of staphylocci, gram-negative bacilli and common respiratory tract pathogens. Cefdinir (MIC90 2.0 µg/ml) was more active than cefixime (MIC90 >64 µg/ml) and equally as active as cefuroxime (MIC90 2.0 µg/ml) against oxacillin-susceptible staphylococci. Cefdinir was active againstHaemophilus influenzae, including -lactamase producers (MIC90 0.5 µg/ml),Moraxella catarrhalis (MIC90 0.12 µg/ml),Streptococcus pneumoniae (MIC90 0.06 µg/ml) andStreptococcus pyogenes (MIC90 0.06 µg/ml). The activity of cefdinir against gram-negative bacilli was variable; organisms with chromosomal cephalosporinases were often resistant.     

Expansion of CD14+CD16+Monocytes in Critically Ill Cardiac Surgery Patients

We have asked whether critically ill cardiac valve surgery patients identified by a high APACHE II score exhibit an increase in the number of proin-flammatory CD14⁺ CD16⁺ monocytes. A group of 12 patients was studied over a period of 5 days post cardiac valve surgery for changes in blood monocyte populations. Patients were selected on day 1 post surgery to either be in good clinical condition (APACHE II Score of 14; N = 9) or to be critically ill (APACHE II score of 24; N = 3). The 14 patients had an uneventful course and could leave the ICU after 2–3 days. Among the 24 patients two showed a decrease of the score to 14 within the 5 days of observation and they could leave the ICU thereafter. One 24 patient (patient #2) had a persistently high score and finally died on day 28. Analysis of blood monocytes on day 1 post surgery revealed that the 14 patients had normal values of CD14⁺CD16⁺ monocytes (44 ± 9/l). By contrast the 24 patients had increased values of these cells with 243 ± 106 cells per 1 on day 1. The numbers of CD14⁺CD16⁺ monocytes returned to the control range over the 5 days of observation in 2 of the 24 patients concomitant with the improvement of the APACHE II score. CD14⁺CD16⁺ monocytes remained, however, at a high level in patient #2, the patient with persistently high APACHE II score.