Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.     

Reconstruction of the anterior cruciate ligament with a patella-tendon-bone graft may lead to a permanent loss of bone mineral content due to decreased patellar tendon stiffness

Immobilisation induces bone loss. Evidence from studies in animals and healthy humans that were immobilised for a limited time indicates that, in general, bone mass may be restored even in adults. Following conservative management of partial tears of the anterior cruciate ligament (ACL), bone loss is often negligible (2-3%). After surgical reconstruction, however, there is greater bone loss (15-20%), with little or no recovery. Bones adapt to the stresses they experience. Also, the largest forces in the musculoskeletal system arise from muscle pull. Tendons transmit these forces. Many surgical techniques for ACL reconstruction use autologous tendon grafts. We hypothesise that tissue harvesting causes weakening of the formerly intact tendon, which, in turn, leads to reduced muscle pull and subsequent bone loss in those parts of the bone that are loaded by the tendon. If our hypothesis holds true, it may change patients' and surgeons' choice of management. Clinical follow-up should assess the functional result with greater scrutiny, possibly including the assessment of bone mineral content. This may be particularly important since there is accumulating evidence that a decrease in bone mineral density (BMD) preceedes, and hence may be a cause of, osteoarthritis.     

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.     

Papillary carcinoma of the thyroid: hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells

Tissue distribution of dendritic cells was investigated in eight cases of papillary carcinoma of the thyroid using immunohistochemistry. Most dendritic cells had an immature phenotype (CD1a++, CD11c+, CD40+, CD86-, HLA-DR-) and were located at the invasion edge of the tumor. This pattern of distribution was profoundly different from that of CD68+ macrophages, which were evenly distributed throughout the tumor. The ability of tumor cells to release chemotactic factors active on dendritic cells was investigated in primary cultures of the same cases of papillary carcinoma, and was compared to that of the corresponding normal thyroid cells obtained from the tumor-free contralateral lobe. Chemotactic activity of culture supernatants was tested against dendritic cells in a chemotaxis chamber. It was found that papillary carcinoma cells were active in releasing chemotactic activity, that hepatocyte growth factor (HGF; 100 ng/ml) or interleukin (IL)-1beta (10(3) U/ml) induced a fourfold increase in the amount of chemotactic activity released, and that normal thyroid cells obtained from the same patients were as effective as tumor cells. Characterization of chemokines at RNA level revealed that unstimulated cells contain large amounts of IL-8 and monocyte chemotactic protein (MCP)-1 RNAs, and that stimulation with HGF or IL-1beta induced RNAs for regulated upon activation normal T expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-3alpha, interferon-gamma-inducible protein 10 (IP-10), and, to a lesser extent, MIP-1alpha and MIP-1beta. The possibility that HGF/Met interaction has a biological role in vivo was investigated in serial sections of six tumors immunostained for CD1a+, Met protein, and HGF. It was found that all six tumors were intensely and diffusely positive for Met protein, that HGF staining was present in tumor cells of the advancing edge, and that HGF+/Met+ tumor cell nests were infiltrated by CD1a+ dendritic cells. The foregoing observations are consistent with the possibility that HGF stimulation of Met+ tumor cells is one of the molecular mechanisms involved in the recruitment of dendritic cells.     

Seroprevalence of Ehrlichia canis and of Canine Granulocytic Ehrlichia Infection in Dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

Recruiting and retaining GPs and patients in intervention studies: the DEPS-GP project as a case study

Background  

Recruiting and retaining GPs for research can prove difficult, and may result in sub-optimal patient participation where GPs are required to recruit patients. Low participation rates may affect the validity of research.     

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.     

Combined skin prick and patch testing enhances identification of peanut-allergic patients with atopic dermatitis

BACKGROUND: Food atopy patch tests (APTs) are considered a useful tool for the diagnosis of food allergy. Hypersensitivity to peanuts has not been investigated by means of APTs so far. METHODS: APTs and skin prick tests (SPTs) with peanuts were performed in 136 atopic dermatitis (AD) patients. Relevance of positive and negative responses to these tests was assessed by repeated open challenges with peanuts. RESULTS: Nine percent of our AD patients reacted to the challenge. Positive responses to APTs were recorded in 19% of the patients, whereas in 12% positive SPTs were observed. APTs were more frequently positive in subjects with eczematous responses after challenge with respect to those with urticarial reactions. SPT reactivity proved to be higher in patients above 12 years of age, whereas APT positivity was more frequent in children under 6 years. APT sensitivity proved significantly higher than SPT sensitivity, in particular in children under 12 years of age. On the contrary, SPT specificity and positive predictive value were significantly higher with respect to those of APT in the age group of subjects under 6 years of age. CONCLUSIONS: Our data suggest that APTs with peanuts may represent a useful integration to standard testing modalities employed for the diagnosis of peanut allergy in AD patients.     

Cytotoxicity on tumour cells of human mononuclear phagocytes: defective tumoricidal capacity of alveolar macrophages.

Human cells of the monocyte-macrophage lineage were isolated by adherence from peripheral blood, peritoneal exudate, non-neoplastic ascites, benign ovarian cystic fluid and bronchoalveolar lavages. Cytolytic activity was measured as 3H-thymidine release from prelabelled mKSA TU5 tumour cells over 48-72 hr and cytostasis was evaluated in a 72-hr spectrophotometric assay. Mononuclear phagocytes from the various anatomical sites examined, except lung alveolar spaces, were significantly cytolytic and cytostatic on target cells. Unlike other cells of the monocyte-macrophage lineage, alveolar macrophages were not cytocidal, but significantly inhibited tumour cell proliferative capacity. Peripheral blood monocytes and peritoneal macrophages showed enhanced cytotoxicity in the presence of partially purified human fibroblast interferon or of lymphokine supernatants from mitogen-stimulated lymphocytes. In contrast, interferon did not affect the cytotoxic potential of alveolar macrophages, whereas lymphokines augmented their cytostatic activity and rendered them weakly cytolytic.