Cathepsin K gene disruption does not affect murine aneurysm formation.

Cathepsin K (catK), a lysosomal cysteine protease, exerts strong elastinolytic and collagenolytic activity and is implicated in a range of pathological disorders including cardiovascular disease. CatK expression was found to be elevated in human aortic aneurysm pointing to a role in this vasculopathy. In the angiotensin II (Ang II)-induced mouse model for aneurysm formation, catK, S and C expression was strongly upregulated. Therefore, we investigated the effect of catK deficiency on Ang II-induced aneurysm formation in the abdominal aorta of apoE-/- mice. Contrary to our expectations, catK deficiency did not protect against aneurysm formation, nor did it affect medial elastin breaks. Proteolytic activity in abdominal aortic lysates were comparable between apoE-/- and catK-//-apoE-/- mice. Adventitial presence of catS- and catC-expressing cells was significantly increased in catK-/-//apoE-/- versus apoE-/- mice, which might have compensated for the deficiency of catK-derived proteolysis in the aneurysm tissue of catK deficient apoE-/- mice. Circulating granulocytes and activated T cell numbers were significantly increased in Ang II-infused catK-/-//apoE-/- mice, which is consistent with the borderline significant increase in adventitial leukocyte content in catK-/-//apoE-/- compared to apoE-/- mice. Strikingly, despite unchanged proteolytic activity in AAA lesions, collagen content in the aneurysm was significantly increased in catK-//-apoE-/- mice. In conclusion, while catK deficiency has major impact on various vasculopathies, it did not affect murine aneurysm formation.

Molecular MRI of early thrombus formation using a bimodal alpha2-antiplasmin-based contrast agent.

OBJECTIVES: We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal alpha(2)-antiplasmin-based contrast agent (CA). BACKGROUND: Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links alpha(2)-antiplasmin to fibrin, indicating the potential of alpha(2)-antiplasmin-based CAs in the detection of early thrombus formation. METHODS: A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an alpha(2)-antiplasmin-based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of alpha(2)-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. RESULTS: In vitro-generated thrombi exposed to the alpha(2)-antiplasmin-based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the alpha(2)-antiplasmin-based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the alpha(2)-antiplasmin-based CA bound to fibrin. Immunohistochemistry demonstrated substantial alpha(2)-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 +/- 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs -0.14 +/- 0.55, p = 0.003, n = 6) and alpha(2)-antiplasmin-based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 +/- 0.23, p = 0.006, n = 6). CONCLUSIONS: A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.

Macrophage-specific expression of mannose-binding lectin controls atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: With consideration of the central role of the innate immune system in atherogenesis and mannose-binding lectin (MBL) as an innate regulator of immunity, the role of MBL in experimental and human atherosclerosis was assessed. METHODS AND RESULTS: With the use of immunohistochemistry and polymerase chain reaction, deposition and gene expression of MBL-A and -C were assessed in murine atherosclerosis from mice deficient for the low-density lipoprotein receptor (LDLR(-/-)) after 10 or 18 weeks of high-fat feeding. MBL was present and was produced in 10-week-old lesions, whereas deposition and gene expression were minimal after 18 weeks of high-fat feeding and absent in healthy vasculature. Interestingly, deposition of MBL-A and -C differed: MBL-A predominantly localized in upper medial layers, whereas MBL-C was found in and around intimal macrophages. To further study the role of local MBL production by monocytic cells in atherosclerosis, LDLR(-/-) mice with MBL-A and -C(-/-) monocytic cells were construed by bone marrow transplantation. Mice carrying MBL-A and -C double deficient macrophages had increased (30%) atherosclerotic lesions compared with wild-type controls (P=0.015) after 10 weeks of high-fat diet. Subsequently, analysis of MBL deposition and gene expression in advanced human atherosclerotic lesions revealed the presence of MBL protein in ruptured but not stable atherosclerotic lesions. Putatively in agreement with murine data, no MBL gene expression could be detected in advanced human atherosclerotic lesions. CONCLUSIONS: These results are the first to show that MBL is abundantly present and locally produced during early atherogenesis. Local MBL expression, by myeloid cells, is shown to critically control development of atherosclerotic lesions.

Effects of surfactant protein D on growth, adhesion and epithelial invasion of intestinal Gram-negative bacteria.

Surfactant protein D (SP-D) interacts with various different microorganisms and plays an important role in pulmonary innate immunity. SP-D expression has also been detected in extrapulmonary tissues, including the gastro-intestinal tract. However, its function in the intestine is unknown and may differ considerably from SP-D functions in the lung. Therefore, the effects of porcine SP-D (pSP-D) on several strains of intestinal bacteria were studied by means of bacterial growth assays, colony-count assays, radial diffusion assays and differential fluorescent staining. Furthermore, the effect of pSP-D on the adhesion- and invasion-characteristics was investigated. All bacterial strains tested in this study were aggregated by pSP-D, but only Escherichia coli K12 was susceptible to pSP-D-mediated growth inhibition. Bacterial membrane integrity of E. coli K12 was affected by pSP-D, but this did not lead to a reduced bacterial viability. Therefore, it is unlikely that pSP-D has a direct antimicrobial effect, and the observed effects are most likely due to pSP-D-mediated bacterial aggregation. The effects of pSP-D on bacterial adhesion and invasion were studied with the porcine intestinal epithelial cell line IPI-2I. Preincubation with pSP-D results in a several-fold increase in adhesion (E. coli and Salmonella) and invasion (Salmonella), but did not affect the IL-8 production induced by the bacteria. Results obtained in this study suggest that pSP-D promotes uptake of pathogenic bacteria by epithelial cells. This may reflect a scavenger function for pSP-D in the intestine, which enables the host to generate a more rapid response to infectious bacteria.

Expression sites of the collectin SP-D suggest its importance in first line host defence: power of combining in situ hybridisation, RT-PCR and immunohistochemistry.

Surfactant proteins A and D are pattern recognition molecules that play a role in pulmonary host defence. In this paper, we describe for the first time the expression and localisation of both collectins in various porcine tissues using a combination of in situ hybridisation (ISH), RT-PCR and immunohistochemistry (IHC). SP-D was expressed in several tissues including lung, tongue, intestinal tract, thymus, skin, gall bladder and lacrimal gland. Focal SP-D expression was detected in oesophagus, stomach, kidney, liver, prostate and spleen with both histological techniques. These tissues tested negative with RT-PCR. In contrast, SP-A expression was limited to the lung as measured by ISH and IHC. Interestingly, analysis by RT-PCR showed that thymus, trachea, jejunum and duodenum are positive for the presence of SP-A mRNA. We conclude that the combination of different methods can be advantageous if tissue-specific expression is studied. The importance of SP-D in innate immune defence of the pig is underlined by its expression at the potential ports of entry of pathogens.

Probiotic effects of Lactobacillus casei on DSS-induced ulcerative colitis in mice.

We tested the effect of Lactobacillus casei strain Shirota (LcS) on the murine model of ulcerative colitis induced by dextran sodium sulphate. The effect of LcS was tested either as a prophylactic 10 days before the onset of the disease, simultaneously with ulcerative colitis induction or continued 10 days after the disease was induced. LcS was not able to prevent the disease induction in any of the experiments. However, important clinical parameters including blood anemia indicators, body weight, and organ weight were improved in the animals receiving LcS as compared with the ulcerative colitis-induced controls. Increased colonic epithelial regeneration in the LcS treated animals was observed in the chronic stage. The results seemed better for the simultaneous short LcS treatment where some parameters remained similar to the PBS controls, including disease activity scores measured in the acute stage. We can conclude that although LcS alone cannot prevent the induction of ulcerative colitis by dextran sodium sulphate, it can improve the clinical condition of the mice. This could imply important biological consequences for the human situation. Further studies including LcS or other probiotic bacteria together with the available treatment are encouraged.

Immunomodulatory effects of Lactobacillus plantarum colonizing the intestine of gnotobiotic rats.

We have studied the effect of the probiotic strain Lactobacillus plantarum 299v on the immune functions of gnotobiotic rats. One group of germ-free rats was colonized with the type 1-fimbriated Escherichia coli O6:K13:H1 and another group with the same E. coli strain together with L. plantarum 299v. One and 5 weeks after colonization, bacterial numbers were determined in the contents of the small intestine, caecum and mesenteric lymph nodes. Small intestinal sections were examined for CD8+, CD4+, CD25+ (IL-2R alpha-chain), IgA+ and MHC class II+ cells and mitogen-induced spleen cell proliferation was determined. Immunoglobulin levels and E. coli-specific antibodies were measured in serum. Rats given L. plantarum in addition to E. coli showed lower counts of E. coli in the small intestine and caecum 1 week after colonization compared with the group colonized with E. coli alone, but similar levels after 5 weeks. Rats colonized with L. plantarum + E. coli had significantly higher total serum IgA levels and marginally higher IgM and IgA antibody levels against E. coli than those colonized with E. coli alone. They also showed a significantly increased density of CD25+ cells in the lamina propria and displayed a decreased proliferative spleen cell response after stimulation with concanavalin A or E. coli 1 week after colonization. The results indicate that L. plantarum colonization competes with E. coli for intestinal colonization and can influence intestinal and systemic immunity.

Increased antibody production against gut-colonizing Escherichia coli in the presence of the anaerobic bacterium Peptostreptococcus.

Germ-free rats were colonized with E. coli alone, or with E. coli plus Lactobacillus acidophilus and a strain of the obligate anaerobic gram-positive species, Peptostreptococcus. The presence of Peptostreptococcus reduced translocation of E. coli, but increased the serum antibody response to E. coli antigen. Whereas the immunoglobulin G (IgG) anti-E. coli antibodies largely represented cross-reactive antibodies, those of the immunoglobulin M (IgM) isotype represented true anti-E. coli antibodies because they could not be absorbed by L. acidophilus or Peptostreptococcus but could with E. coli. We suggest that peptostreptococci prime the gut immune system to other bacterial antigens and that this could be a mechanism behind the reduced translocation of facultative anaerobes in the presence of obligate anaerobes.

Antibodies to Escherichia coli and Shigella flexneri in milk from undernourished mothers: studies on sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated antigens.

Analysis of IgA, IgM, and IgG antibodies against Escherichia coli O6, its lipopolysaccharide (LPS), and Shigella flexneri were performed in the milk of moderately undernourished Guatemalan women receiving either a low or a high calorie supplement, using SDS-PAGE. As expected, the immunostaining analysis of milk antibodies showed that IgA was the predominant isotype in both groups. Concerning the other Igs, antibodies against O6 LPS were mainly of the IgM isotype, whereas IgG antibodies were more prominent than IgM against the bacterial whole cell preparations. Seven to nine distinct bands, ranging in molecular mass from 13.5 to 109 kD were selected for each antigen to compare the milk antibodies between the two groups of women. After a 20-wk supplementation period, the IgA and IgG antibodies to the E. coli, O6 LPS, and S. flexneri were not found negatively affected by a low calorie intake. A significantly lower immunostaining intensity was, however, obtained for the low calorie intake group regarding the IgM antibody activity against four high molecular mass bands of the E. coli whole cell preparation. A decreased immunostaining intensity was also found in the same group for IgM antibodies against two bands of E. coli O6 LPS when analyzing paired samples collected at d 0 and wk 20. No differences were found for IgM antibodies against any of the S. flexneri antigens. In conclusion, the results suggest that low calorie intake does not significantly affect the production of milk IgA antibodies to E. coli and S. flexneri antigens in these women. Still, IgM antibodies against certain proteins and LPS molecules of E. coli may be decreased. IgG antibodies, although also present in milk, seemed to be directed mainly against bacterial proteins and not to LPS.

Escherichia coli K5 capsule expression enhances colonization of the large intestine in the gnotobiotic rat.

The role of capsule expression in the capacity of Escherichia coli to colonize in the large intestinal environment was studied in a gnotobiotic rat model. The rats were given perorally a mixture of two mutant strains differing in K5 expression. After 2 weeks, the rats were sacrificed, and subsequently intestinal contents, intestinal mucosae, and mesenteric lymph nodes were homogenized and bacterial numbers were quantified. Two E. coli mutant pairs were used, the first pair (972-998) lacking the O-specific side chain and the second pair (973-997) carrying the O75 lipopolysaccharide. The K5+ mutants established themselves at a higher level than the K5- mutants (10(9) versus 10(6) CFU/g [P < 0.001] for the first pair and 10(9) versus 10(8) CFU/g [P < 0.01] for the second pair, respectively). The results were confirmed by serology showing a K5+ phenotype for practically all isolates. The bacterial population associated with the mucosa was similar to that in the luminal contents with respect to the proportions of the respective mutants, and translocation occurred in numbers proportional to the intestinal population densities of the respective mutants. All mutants were able to express type 1 as well as P fimbriae. After colonization, the expression of P fimbriae remained high whereas only a minority of the isolates expressed type 1 fimbriae. The results suggest that capsule expression and P fimbriae enhance intestinal colonization by E. coli and that these virulence factors, by increasing bacterial densities in the intestine, secondarily increase translocation.

Role of Escherichia coli P fimbriae in intestinal colonization in gnotobiotic rats.

Adherence via P fimbriae is associated with long-term persistence of Escherichia coli in the human large intestine, but a causal relationship has not been proven. In the present study, germfree rats were colonized with a mixture of two isogenic E. coli strains, one P fimbriated and the other type 1 fimbriated. Both types of fimbriae conferred adherence to rat colonic epithelial cells. With two mutant strains from a pyelonephritogenic isolate of serotype O75:K5:H-, the P-fimbriated strain 824 attained much higher numbers than its type 1-fimbriated counterpart when colonized in vivo for 2 weeks (10(10) versus 10(6) bacteria per g, respectively; P < 0.0001). The expression of P fimbriae by 824 was also retained during colonization. With transformant isogenic strains obtained from a normal fecal isolate incapable of phase variation, no benefit of P fimbriae was seen and most bacteria lost their plasmids during in vivo colonization. When the pyelonephritogenic mutant and fecal transformant strains were combined, the former colonized at high levels while the latter were suppressed. In contrast, no suppression was seen when the transformant E. coli strains colonized in combination with Lactobacillus acidophilus or Peptostreptococcus sp. The results indicate that P fimbriae, but also other bacterial traits linked to uropathogeneicity, could play an important role for persistence in the gut normal microbiota. Neither P nor type 1 fimbriae seemed to contribute to the ability to translocate to the mesenteric lymph nodes.

The effect of caloric supplementation on selected milk protective factors in undernourished Guatemalan mothers.

The level and avidity indices of specific antibodies against tetanus toxoid, Escherichia coli O6 and a pool of 10 common E. coli O antigens, as well as the concentration and daily output of lactoferrin and total secretory IgA (SIgA), were evaluated in the milk of moderately undernourished mothers who were in a random blind design divided into two groups and given different caloric supplementations. Group A received a high caloric supplement (500 kcal/d), and group B received a low caloric supplement (140 kcal/d). Determinations were done using ELISA in various modifications, except for lactoferrin, which was quantified by single radial immunodiffusion. The avidity indices were investigated as an evaluation of the antibody quality. In all the parameters evaluated, the only difference found between the two groups at the end of the supplementation period was in the content of total SIgA, which was lower in group B, both in concentration and daily output. However, the SIgA remained within the normal range. Increases as well as decreases in the levels of specific IgA antibodies occurred within both groups. Avidity was decreased in group B only against one of the antigens tested. We conclude that moderate undernutrition does not impair the levels of milk antibodies, and supplementation does not enhance them but prevents the decrease in the content of total milk SIgA. There is a suggestion that the avidity of certain antibody specificities could be hampered.

Fructose 2,6-bisphosphate levels and modulation of glycolysis by histamine, cholecystokinin, and forskolin in isolated rabbit gastric glands.

In isolated rabbit gastric glands incubated in the presence of 1 mmol/L glucose, the content of fructose 2,6-bisphosphate (F-2,6-P2) was 5.7 +/- 0.5 pmol/mg dry weight. This value was progressively incremented by increasing glucose concentration in the incubation medium, and was almost doubled at 10 mmol/L glucose. Under these conditions, a close correlation could be established between the levels of F-2,6-P2 and the rate of L-lactate formation (r = .98; P less than .05). Both histamine (0.1 mmol/L) and cholecystokinin octapeptide (CCK-OCT; 0.1 mumol/L) increased L-lactate production, without significant changes in either F-2,6-P2 concentration or the amount of 6-phosphofructo-2-kinase in active form. In contrast, forskolin, which markedly increased the glandular content of cyclic adenosine monophosphate (cAMP), partially blocked glucose consumption and caused a significant reduction in both F-2,6-P2 levels and the proportion of 6-phosphofructo-2-kinase in active form. Furthermore, forskolin partially blocked the rate of glucose uptake by isolated gastric glands. Our results suggest a regulatory role of F-2,6-P2 in the control of the glycolytic flux in response to glucose, but not in its response to histamine or CCK-OCT.