Correlation of in vivo collagen degradation rate with in vitro measurements.

The rate of in vitro enzymatic degradation of various materials based on collagen has been determined by a novel mechanochemical method, and has been compared with the extent of degradation resulting from subcutaneous implantation in guinea pigs. In vitro data correlate well with in vivo data. It is suggested that the simple in vitro method described is an effective means of screening a large number of materials based on collagen for their ability to resist degradation during implantation in animals.     

Heterogeneity in the ability of cytotoxic murine NK cell clones to enhance Ig secretion in vitro

We recently described a panel of cytotoxic murine NK cell clones that also enhanced Ig secretion by B cells activated in an in vitro model of T cell-independent type 2 (TI-2) responses. We employed dextran-conjugated anti-IgD (alphadelta-dex) as a model antigen. Here we study the mechanism of Ig induction by these clones. Addition of the various NK clones to sort-purified B cells stimulated with alphadelta-dex and IL-2 resulted in a markedly heterogeneous increase in Ig secretion, which varied from 3-fold, as mediated by clone PKO 56, to 15-fold, as induced by clone PKO 101. The other NK cells showed intermediate levels of Ig induction. Furthermore, while addition of as few as 0.04% of PKO 101 cells stimulated significant increases and 1% induced near maximum Ig production, a 3% addition of PKO 56 cells was required for significant enhancement of Ig secretion. Supernatant material collected from the NK clones mediated Ig production at levels that mirrored the induction by the corresponding cells. Cytokine analysis showed that while all members of the NK panel produced IFN-gamma only two secreted granulocyte macrophage colony stimulating factor and that the levels of Ig induction mediated by the NK clones correlated only with their levels of IFN-gamma secretion. Culture of B and NK cells in the presence of anti-IFN-gamma demonstrated that IFN-gamma was the critical cytokine in NK-induced Ig production. These findings establish heterogeneity in the ability of NK cells to increase Ig secretion in vitro and show that NK-produced IFN-gamma is an important factor in determining this heterogeneity.     

The in vitro and in vivo pharmacology of antisense oligonucleotides targeted to murine Stat6

OBJECTIVE AND DESIGN: This study was designed to establish whether phosphorothioate (PS) antisense oligonucleotides (AS-ODN) targeted to Stat6 were active in vivo in a mouse model of active sensitisation. MATERIALS: Female Balb/c mice (6-8) per group were used for in vivo study. TREATMENT: Mice were treated with active PS AS-ODNs determined in initial in vitro studies. Compounds were dosed daily (3-30mg/kg i.v.) over the course of sensitisation to ovalbumin. METHODS: Stat6 mRNA and protein levels were determined in the spleen after treatment (quantitative northern and western analysis respectively), in addition to serum IgE (ELISA). ANOVA was used to determine any significant differences between groups. RESULTS: Both of the AS-ODNs tested in vivo, down regulated Stat6 mRNA and protein levels in the spleen by 40-50% although there was no effect on serum IgE. These treatments also induced splenomegaly in vivo and caused splenocyte proliferation in vitro. CONCLUSIONS: The AS-ODNs used can down regulate Stat6 mRNA and protein although not sufficiently to influence IgE-levels. These effects are likely to be complicated in vivo by the immune-stimulation evident as splenomegaly.     

Trichomonas vaginalis: strain differences in adhesion to plastic and virulence in vitro and in vivo

Trichomonas vaginalis isolated from clinical cases were grown axenically in TYI-S-33 medium. Various strains of this flagellate showed different adhesion characteristics in medium containing bovine serum or Cohn fractions thereof. These were not inversely related to the time in axenic culture. The adsorption of serum components to the parasites was assessed in relationship to adhesion but in many cases was probably not adhesion-related. All strains tested for virulence in vitro were almost equally cytotoxic for HeLa tissue cultures. However, in infectivity trials, one of these strains exhibited the highest adhesion capability and proved to be the most virulent for mice, and another (cloned) strain with the poorest adhesion capability failed to cause infection. Other strains exhibited lessened virulence following their extended axenic cultivation. It therefore appears that the in vivo pathogenic potential of the parasites growing in axenic culture is inherently strain-dependent. The findings suggest that although adhesion in whole serum-containing medium is sufficient to differentiate between variousTrichomonas isolates, it is insufficiently sensitive to correlate adhesion with virulence. It apparently is important to identify the adhesion-mediating factor(s) in serum or in its Cohn fractions IV-1 and IV-4 and to use it (them) to elucidate the possible correlation between the parasite's capacity to adhere and its pathogenicity.     

In vivo and in vitro characterization of murine T-cell clones reactive to Mycobacterium tuberculosis.

Seventeen helper T-cell clones were derived by stimulating lymph node cells from sensitized C57BL/6 mice with Mycobacterium tuberculosis H37Ra, M. tuberculosis H37Rv, or purified protein derivative. Most clones cross-reacted with Mycobacterium bovis BCG, H37Ra, H37Rv, and purified protein derivative. However, four clones were able to differentiate H37Rv from H37Ra, or BCG from H37Ra and H37Rv. In addition, four other T-cell clones recognized recombinant antigens of 19 and 65 kilodaltons isolated from a genomic expression library of M. tuberculosis by using monoclonal antibodies. All clones were Ia restricted and had the Thy-1.2+ Lyt-1+ L3T4+ Lyt-2- phenotype. On stimulation with antigen, all of the clones tested secreted interleukin-2 and gamma interferon but not B-cell stimulatory factor 1. All of the clones tested induced an antigen-specific delayed-type hypersensitivity response upon local cell transfer, although the magnitude of this response differed markedly among clones.     

In vitro and in vivo evidence that autoimmune reactivity to collagen develops spontaneously in Schistosoma mansoni-infected mice

Egg-induced granuloma formation in murine schistosomiasis mansoni results from vigorous anti-parasite reaction by activated T cells, macrophages, eosinophils, and fibroblasts. The present study suggests that strain-specific, autoimmune T-cell reactivity directed against host matrix proteins might also contribute to granulomatous hypersensitivity. T cells from infected C57B1/6, but not from CBA or BALB/c mice, proliferative in vitro in response to denatured collagen. T cells from uninfected mice, previously immunized with soluble egg antigen (SEA), did not respond in vitro to collagen. Spleen cells from acutely infected mice, but not chronically infected or uninfected animals, formed granulomas around collagen-coupled polyacrylamide beads in vitro. This response was blocked by anti-collagen antibodies that had no inhibitory effect on in vitro granuloma formation around SEA-coupled beads. In related in vivo studies, granuloma formation was quantitated after iv injection of SEA-, collagen-, or uncoated beads into normal or infected recipients. The mean diameter of lung granulomas induced by collagen-coupled beads in infected mice was significantly greater than the diameter of granulomas around either collagen beads in uninfected mice or uncoated beads in infected mice. these observations indicate that anti-collagen responses develop spontaneously in Schistosoma-infected mice and suggest that such reactivity might play a secondary role in granuloma formation and the pathogenesis of hepatic fibrosis.     

载穿心莲内酯胶原缓释支架促进体外炎症环境下软骨细胞表型维持的研究

本文主要研究载穿心莲内酯胶原缓释支架对炎症环境下软骨细胞维持表型的作用。通过物理共混结合真空冷冻干燥的方法制备载穿心莲内酯胶原缓释支架,采用环境扫描电子显微镜(ESEM)、真密度仪和紫外可见分光光度计表征载药支架的形貌、开孔率和药物释放情况。将经分离、扩增培养后的兔关节软骨细胞接种于载穿心莲内酯胶原缓释支架上,在常规培养液条件下培养3 d、白细胞介素1β(IL-1β)模拟的炎症环境下培养7 d,期间采用阿尔玛蓝(Alamar Blue)、荧光素二乙酸(FDA)染色、实时定量聚合酶链式反应(RT-qPCR)等方法研究所制备支架对软骨细胞的增殖和表型的影响。实验结果表明,真空冷冻干燥法制备的胶原支架孔连通性良好,平均孔径为(120.7±17.8)μm,开孔率可达96%,该载药支架可在两周内实现药物的持续释放。Alamar Blue实验结果表明,质量分数为2.22%的载穿心莲内酯胶原缓释支架可显著抑制软骨细胞增殖,而其它载药浓度的胶原缓释支架对炎症环境下软骨细胞的增殖无显著影响。FDA染色结果表明,质量分数为0.44%的载穿心莲内酯胶原缓释支架可降低IL-1β对软骨细胞形貌的改变,且该浓度载药支架可显著抑制基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶-13(MMP-13)的转录,促进基质金属蛋白酶抑制剂-1(TIMP-1)、软骨细胞外基质相关基因II型胶原(COL II)和聚集蛋白聚糖(Aggrecan)的转录,提高COL II/I型胶原(COL I)的比值。基于以上研究结果,表明质量分数为0.44%的载穿心莲内酯胶原缓释支架可以抑制IL-1β模拟的炎症环境下软骨细胞内不利于维持表型的基因(如:MMP-1、MMP-13)的转录,促进利于维持表型的基因(如:COL II、Aggrecan、TIMP-1)的转录,从而增强炎症环境下的软骨细胞维持其表型的能力,有望用于关节炎软骨损伤修复。     

Assembly of collagen-binding peptide with collagen as a bioactive scaffold for osteogenesis in vitro and in vivo

Bioactive scaffolds inducing cell adhesion, differentiation have been premise for optimal formation of target tissue. Collagen has been employed as a tissue regenerative scaffold especially for bone regeneration and has been chemically surface-modified to present bioactivity. Herein, we show that peptide, denoted as collagen-binding motif (CBM, GLRSKSKKFRRPDIQYPDATDEDITSHM) identified from osteopontin (OPN) protein, was able to specifically bind collagen without chemical conjugation, while presenting apatite forming capability in vitro and in vivo. Collagen surface alone was not able to induce noticeable apatite nucleation however, mineralization was evident when assembled with CBM peptide, implying that the collagen-CBM assembly played a pivotal role in biomineralization. In vivo result further demonstrated that the CBM peptide in complex with material was able to induce bone formation by helping mineralization in the bone defect. Taken together, the CBM peptide herein and its assembly with collagen can be applied as an inducer of biomineralization as well as a bioactive scaffold for bone regeneration.     

Rapid development in vitro and in vivo of resistance to ceftazidime in biofilm-growing Pseudomonas aeruginosa due to chromosomal beta-lactamase

The aim of this study was to examine the development of resistance of biofilm-growing P. aeruginosa during treatment with ceftazidime. Biofilms were established in vitro using a modified Robbins device (MRD) and in vivo in the rat model of chronic lung infection. Three P. aeruginosa strains isolated from the lungs of cystic fibrosis (CF) patients (MICceftazidime-basal/induced beta-lactamase activity: PAO 579= 0.8 mg/l-19/550 milliunits, 19676A=50 mg/l-38/957 milliunits, 17107B=100 mg/l-504/947 milliunits) were studied. After 1 or 2 weeks of continuous or intermittent (4 h/day) administration of ceftazidime to biofilms established in MDR, a statistically significant development of resistance to ceftazidime in PAO 579 or 19676A bacterial populations occurred. When ceftazidime was administered 4 h/day (200 mg/l) for 2 weeks, the frequency of resistant 19676A having MIC>25 mg/l was 4.4 10(-1) compared to 6.0-10(-5) in the control biofilm. The same trend was observed after continuous administration of ceftazidime. MICceftazidime of the more resistant variants was increased 500-fold for PAO 579 and 8-fold for 19676A, and the specific basal beta-lactamase activities from 19 to 1,400 units for PAO 579 and from 38 to 10,000 units for 19676A. Chronic P. aeruginosa lung infection was established with alginate-embedded PAO 579, 19676A or 17107B in 146 Lewis rats which were treated with ceftazidime 4 g/kg intraperitoneally twice a day for 1 week. A statistically significant development of resistance was observed for all three strains. The MICceftazidime of the more resistant variants was increased 15-fold for PAO 579, 8-fold for 19676A, and 4-fold for 17107B, and the specific basal 3-lactamase activity from 19 to 100 units for PAO 579, from 38 to 1,300 units for 19676A, and from 500 to 1,300 units for 17107B. It was shown that, during treatment with ceftazidime, biofilm-growing P. aeruginosa had the capacity to develop resistance due to the production of chromosomal beta-lactamase.     

Inhibition of in vitro gamete adhesion and in vivo fertility in mice by immunization with a synthetic peptide

PROBLEM: To evaluate the contraceptive ability of a synthetic peptide in in vitro and in vivo fertility in the mouse. METHOD OF STUDY: A synthetic peptide segment: GELRERAPGQGTNG (SP) was used to immunize female B6CF1 (C57BL/6 x BALB/c) mice. A peptide with an amino acid sequence QQPLSIQQHERG (p2control) was used as control. Anti-SP and anti-p2control antisera were used to evaluate sperm function inhibition in vitro. Fertility of immunized mice was determined by microscopic evaluation of the number and state of preimplantation embryos (8-16 cell stage). RESULTS: In the mouse, anti-SP antisera recognized surface antigens in the acrosome region of mature and capacitated sperm. Anti-SP antisera inhibited in vitro sperm binding to zona pellucida. In vivo, immune response against SP in Freund's adjuvant resulted in a significant increase (P < 0.01) in the number of dead embryos and eggs (a mean of 66%, in contrast with < 25% in control mice). Fertility inhibition in vivo and in vitro was not observed when the p2control peptide was used in the immunizations. CONCLUSIONS: These results would suggest that the SP sequence is involved in gamete adhesion, and an antifertility vaccine against the SP peptide segment could be feasible.     

A survey of pathological changes associated with long-term high tar tobacco smoke exposure in a murine model

Balb/c mice were exposed to the fresh smoke of a daily equivalent of thirty high tar filtered cigarettes for periods of up to 95 weeks. Detailed surveys of the gross and histopathological data are presented which indicates that there is the induction or production of significant numbers of malignant tumours of several types in the animals exposed to tobacco smoke. There are also significant histopathological changes which consist mainly of interstitial pneumonia and focal low grade emphysema. These features contribute significantly to the reduction in life span of the test animals in which the main causes of death are malignancies and inflammatory diseases of the lungs.     

Capacity of Mycobacterium avium isolates to grow well or poorly in murine macrophages resides in their ability to induce secretion of tumor necrosis factor.

The results of this study show that clinical isolates of Mycobacterium avium fall into two categories in terms of their capacity to grow within murine bone marrow-derived macrophage cultures: those that grow progressively and those that are incapable of growing within such cells. Members of the first category were invariably of the smooth-transparent colonial type, while most of the second were of the smooth-doomed type. In addition, this paper shows that although all isolates induced tumor necrosis factor (TNF) secretion by host cells to some extent, this production was always delayed in isolates that subsequently grew well in the host cells. This observation, coupled with the demonstration that the growth of the latter isolates was inhibited by the exogenous addition of TNF, leads us to hypothesize that the ability of a given isolate to somehow avoid host macrophage TNF production early during the course of the infection is a key factor in the pathogenesis of the disease.     

Exogenously administered gangliosides fail to increase in vivo metastatic frequency or in vitro growth of murine neoplastic cells

The influence of gangliosides on tumor growth and frequency of metastasis in vivo, as well as growth of neoplastic cells in vitro, was tested utilizing the mouse fibrosarcoma cell line MN4. In mice receiving intramuscular tumor transplants, injections of a ganglioside mixture twice daily did not influence the tumor volume or the number of spontaneous metastases per animal. Furthermore, in mice receiving the cells by tail vein injection, injections of a ganglioside mixture once or twice daily did not affect the number of metastatic foci in the lungs. Preincubation of neoplastic cells with the ganglioside mixture decreased the number of metastatic foci in the lungs of mice receiving the cells by tail i.v. injection. The addition of ganglioside mixture to the culture medium for up to a 48-h incubation period had no effect, independently of the cell density utilized, on either the rate of DNA synthesis or the relative numbers of neoplastic cells as compared to controls; at higher ganglioside concentrations, growth was actually reduced. These results are interpreted to indicate that gangliosides, under the present conditions, do not influence tumor growth and metastatic neoplastic capacity in vivo, and growth in vitro.     

Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo

Integrin alpha4beta 7 is the principal gut-homing receptor, and it is assumed that expression of this specific integrin directs lymphocytes to the gut in vivo. Adoptive cellular immunotherapy against inflammatory bowel disease (IBD) may depend on the expression of integrin alpha4beta 7 to accomplish local delivery of intravenously injected regulatory T cells in inflamed gut mucosa. The present study aimed to investigate whether in vitro expanded human T cells from the colonic mucosa maintain integrin expression, show in vitro adhesion and retain in vivo gut-homing properties during cultivation. Whole colonic biopsies from healthy subjects were cultured in the presence of interleukin-2 (IL-2) and IL-4. The integrin expression of the cultured T cells was determined by flow cytometry and in vitro adhesion was assessed in a mucosal addressin cell adhesion molecule 1 (MAdCAM-1) adhesion assay. We studied the homing pattern after autologous infusion of 3 x 10(8 111)Indium ((111)In)-labelled T cells in five healthy subjects using scintigraphic imaging. The cultured CD4(+)CD45RO(+) gut-derived T cells express higher levels of integrin alpha4beta 7 than peripheral blood lymphocytes (PBLs) and show strong adhesion to MAdCAM-1 in vitro, even after (111)In-labelling. Scintigraphic imaging, however, showed no gut-homing in vivo. After prolonged transit through the lungs, the T cells migrated preferentially to the spleen, liver and bone marrow. In conclusion, it is feasible to infuse autologous T cells cultured from the gut mucosa, which may be of interest in adoptive immunotherapy. Despite high expression of the gut-homing integrin alpha4beta 7 and adhesion to MAdCAM-1 in vitro, evaluation by (111)In-scintigraphy demonstrated no gut-homing in healthy individuals.     

A 19-year-old man with leucocyte adhesion deficiency. In vitro and in vivo studies of leucocyte function

We describe a male patient with leucocyte adhesion molecule deficiency (LAD) of moderate phenotype. Although diagnosis was made only 2 years before his death, the patient survived until 19 years of age. This enabled us to perform a number of novel investigation, both in vivo and in vitro, relating to his leucocyte biology. Monocytes cultured in vitro matured into morphologically normal, phagocytically capable macrophages, which were able to recognize aged 'apoptotic' neutrophils. By injection of radiolabelled autologous neutrophils we demonstrated a prolonged neutrophil half-life, but normal margination, de-margination on exercise, and splenic pooling. Neutrophil adherence in vitro to vascular endothelium was normal. Histological examination of the patient's lungs at post-mortem showed intravascular aggregation of polymorphonuclear leucocytes but a paucity of cells in the interstitium and alveolar spaces. These findings indicate that the peripheral blood leucocytosis commonly observed in these patients may be due to prolonged intravascular neutrophil survival, and suggest that CD11/18 molecules have an important role in facilitating neutrophil emigration from blood vessels at sites of inflammation.     

The in vitro mitogenic response to intact bacteria by murine B cells does not predict in vivo susceptibility to Salmonella typhimurium

We are interested in developing in vitro culture systems that will permit immune responses to intact Salmonella typhimurium, since these systems would have certain advantages over in vivo infection models for the characterization of the host's responding cell types. In this report, the in vitro proliferative response of nonimmune murine spleen cells to four different killed preparations of S. typhimurium, strain TML (TML), are examined. These studies show that UV-killed TML, heat-killed TML, glutaraldehyde-killed TML, and acetone-killed and dried TML, all elicit a nonspecific mitogenic spleen cell response in vitro, as does a live, avirulent, temperature-sensitive mutant of TML, TS27. This response reaches a maximum on day 2 after initiation of culture, which is similar to the time course of a conventional lipopolysaccharide (LPS) response. Unlike the LPS response, little 3H-thymidine incorporation is observed in low-density cultures (2 X 10(5) cells/well), which suggests a critical role for accessory cells. The responding cell types include, but are not necessarily limited to, the B-cell population. The response cannot be readily inhibited by polymyxin B, which binds specifically to the lipid A portion of LPS. Thus, the bacterial components required for mitogenicity are not yet definitively identified. A survey of the mitogenic responses of lymphocytes from various inbred mouse strains, including the C3H/HeJ LPS hyporesponsive strain, indicates that all B cells tested are capable of proliferating vigorously in response to intact TML, regardless of the in vivo susceptibility to virulent infection. These results also emphasize the importance of assessing the nonspecific components of the immune response when studying the specific immune response to intact S. typhimurium.     

Deficient in vitro anti-mycobacterial immunity despite successful long-term highly active antiretroviral therapy in HIV-infected patients with past history of tuberculosis infection or disease

We evaluated the anti-Mycobacterium tuberculosis (Mtb) immune responses of HIV patients after long-term successful HAART, presenting >500 TCD4+ cells/microl, undetectable viral load, and past history of tuberculosis infection (HIV+PPD+, n=14) or disease (HIV+CTB, n=17). Their lymphoproliferative and IFN-gamma responses were compared with those from HIV-uninfected controls either PPD+ (HIV-PPD+, n=17) or with past history of pulmonary tuberculosis (n=15). Most HIV-infected patients presented normal PHA responses while responses to the Mtb recombinant polypeptides ESAT-6 and Ag85B were markedly reduced. Responses to a whole Mtb lysate (S-Mtb) in HIV+PPD+ patients were lower than in HIV-PPD+ controls, while in HIV+CTB patients these responses were similar to that of past-tuberculosis controls. Comparison between the two HIV groups also suggested better S-Mtb responses in those cured from tuberculosis. Thus, while immune responses to single Mtb proteins are depressed even after successful HAART, reactivity to S-Mtb is high, specially in those cured from tuberculosis, possibly as a result of the survival of higher numbers of mycobacteria-specific T cell clones during the immunosuppression phase, which may afford sufficient protection against new Mtb challenges.