Diffusion of ionic and non-ionic contrast agents in articular cartilage with increased cross-linking--contribution of steric and electrostatic effects.

OBJECTIVE: To investigate the effect of threose-induced collagen cross-linking on diffusion of ionic and non-ionic contrast agents in articular cartilage. DESIGN: Osteochondral plugs (Ø=6mm) were prepared from bovine patellae and divided into two groups according to the contrast agent to be used in contrast enhanced computed tomography (CECT) imaging: (I) anionic ioxaglate and (II) non-ionic iodixanol. The groups I and II contained 7 and 6 sample pairs, respectively. One of the paired samples served as a reference while the other was treated with threose to induce collagen cross-linking. The equilibrium partitioning of the contrast agents was imaged after 24h of immersion. Fixed charge density (FCD), water content, contents of proteoglycans, total collagen, hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent) cross-links were determined as a reference. RESULTS: The equilibrium partitioning of ioxaglate (group I) was significantly (p=0.018) lower (-23.4%) in threose-treated than control samples while the equilibrium partitioning of iodixanol (group II) was unaffected by the threose-treatment. FCD in the middle and deep zones of the cartilage (p<0.05) and contents of Pent and LP (p=0.001) increased significantly due to the treatment. However, the proteoglycan concentration was not systematically altered after the treatment. Water content was significantly (-3.5%, p=0.007) lower after the treatment. CONCLUSIONS: Since non-ionic iodixanol showed no changes in partition after cross-linking, in contrast to anionic ioxaglate, we conclude that the cross-linking induced changes in charge distribution have greater effect on diffusion compared to the cross-linking induced changes in steric hindrance.

Computed tomography detects changes in contrast agent diffusion after collagen cross-linking typical to natural aging of articular cartilage.

OBJECTIVE: The effect of threose-induced collagen cross-linking on the mechanical and diffusive properties of cartilage was investigated in vitro. In particular, we investigated the potential of Contrast Enhanced Computed Tomography (CECT) to detect changes in articular cartilage after increased collagen cross-linking, which is an age-related phenomenon. METHODS: Osteochondral plugs (Ø=6.0 mm, n=28) were prepared from intact bovine patellae (n=7). Two of the four adjacent samples, prepared from each patella, were treated with threose to increase the collagen cross-linking, while the other two specimen served as paired controls. One sample pair was mechanically tested and then mechanically injured using a material testing device. Contrast agent [ioxaglate (Hexabrix™)] diffusion was imaged in the other specimen pair for 25 h using CECT. Water fraction, collagen and proteoglycan content, collagen network architecture and the amount of cross-links [hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent)] of the samples were also determined. RESULTS: Cartilage collagen cross-linking, both Pent and LP, were significantly (P<0.001) increased due to threose treatment. CECT could detect the increased cross-links as the contrast agent penetration and the diffusion flux were significantly (P<0.05) lower in the threose treated than in untreated samples. The equilibrium modulus (+164%, P<0.05) and strain dependent dynamic modulus (+47%, P<0.05) were both significantly greater in the threose treated samples than in reference samples, but there was no association between the initial dynamic modulus and the threose treatment. The water fraction, proteoglycan and collagen contents, as well as collagen architecture, were not significantly altered by the threose treatment. CONCLUSIONS: To conclude, the CECT technique was found to be sensitive at detecting changes in cartilage tissue due to increased collagen cross-linking. This is important since increased cross-linking has been proposed to be related to the increased injury susceptibility of tissue.

Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome.

BACKGROUND: A multispecies probiotic has shown beneficial effects in irritable bowel syndrome. In addition, certain other probiotics have demonstrated advantageous effects, but the mechanisms behind this are poorly understood. AIM: To investigate the mode of action of a multispecies probiotic consisting of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 by monitoring its effects on intestinal microbiota and markers of microbial activity. METHODS: A total of 55 irritable bowel syndrome patients participated in this placebo-controlled double-blind trial. Subjects received either multispecies probiotic or placebo supplementation daily during a 6-month period. The composition of intestinal microbiota was analysed with real-time polymerase chain reaction, short-chain fatty acids with gas chromatography and enzymes with spectrophotometer. RESULTS: Each supplemented probiotic strain was detected in faecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group (P = 0.028). No changes in short-chain fatty acids occurred. A decrease in ss-glucuronidase activity was detected in 67% of the subjects in the probiotic group vs. 38% in the placebo group (P = 0.06). CONCLUSIONS: Factors other than the microbial groups and metabolites studied herein seem responsible for the alleviation of irritable bowel syndrome symptoms by the multispecies probiotic.

Neuronal cells show regulatory differences in the hsp70 gene response.

The synthesis of heat shock proteins (Hsps), encoded by heat shock genes, is increased in response to various stress stimuli. Hsps function as molecular chaperones, they dissociate cytotoxic stress-induced protein aggregates within cells and ensure improved survival. Induction of heat shock genes is mainly regulated at the transcriptional level. The stress responsive transcription factor, heat shock factor 1 (HSF1), is involved in the transcriptional induction of the heat shock genes. Our objective was to examine how hsp70 genes are regulated in different transformed and primary neurons upon exposure to elevated temperature. Our findings reveal that the Hsp70 response is regulated at the translational level in Neuro-2a neuroblastoma cells, while the IMR-32 neuroblastoma cells respond to stress by the classical HSF1-driven transcriptional regulatory mechanism. Primary rat hippocampal neurons show a lack of HSF1 and induction of the hsp70 gene. These observations suggest that neuronal cells display different hsp70 gene expression patterns which range from undetected response to transcriptional and posttranscriptional regulation during heat stress.

Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina.

A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri artificial vagina than by the Equidame phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame phantom had lower mean volumes (22.1 +/- 2.3 mL [+/- SEM] versus 101.6 +/- 9.3 mL) and significantly higher mean sperm concentrations (218.0 +/- 25.8 x 10(6) vs 86.2 +/- 8.1 x 10(6) cells/mL; P<0.05) than the total ejaculates collected by the Missouri device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri artificial vagina.

Recombinant allergen fragments as candidate preparations for allergen immunotherapy.

BACKGROUND: Lately, renewed interest has arisen in the new forms of allergen immunotherapy because they may offer alternatives for drug treatment. OBJECTIVE: The purpose of this study was to develop a well-characterized preparation of the main respiratory cow dander allergen, Bos d 2, with attenuated allergenic activity. METHODS: The immunologic characteristics of Bos d 2 preparations were studied by indirect IgE ELISA, ELISA inhibition, Western blotting, histamine release, skin prick tests, and the proliferation tests of allergen-specific T-cell clones. RESULTS: The complete recombinant Bos d 2 was observed to bind effectively, IgE of cow-allergic patients in indirect ELISA. In other experiments, the IgE-binding capacity of recombinant Bos d 2 proved to be lower compared with native Bos d 2. When the two overlapping recombinant fragments of Bos d 2 (corresponding amino acids 1-131 and 81-172, respectively) covering the whole molecule were compared with the complete recombinant Bos d 2 with several methods, only a low level of residual reactivity was observed. For example, recombinant fragments could not bind antibody at all in ELISA inhibition tests retaining, however, some reactivity in skin prick tests. In contrast, the fragments were able to stimulate vigorously Bos d 2-specific T-cell clones. CONCLUSION: The approach we have taken may offer a simple and reproducible way to produce hypoallergenic preparations for immunotherapy, circumventing simultaneously some of the problems of other experimental methods such as individual T-cell epitope recognition in peptide-based immunotherapy.

Immuno-electron-microscopic localization of types III pN-collagen and IV collagen, laminin and tenascin in developing and adult human spleen.

The distribution of the extracellular matrix proteins types III pN-collagen and IV collagen, laminin and tenascin was investigated in fetal, infant, and adult human spleens by using immuno-electron microscopy. The presence of type III pN-collagen was assessed by using an antibody against the aminoterminal propeptide of type III procollagen. All the proteins other than type III pN-collagen were found in reticular fibers throughout development. In the white pulp of the fetus aged 16 gestational weeks, only an occasional type III pN-collagen-containing fibril was present, although type III pN-collagen was abundant in the reticular fibers of the red pulp. Conversely, in adults, most of the reticular fibers of the white pulp, but not of the red pulp, were immunoreactive for type III pN-collagen. Ring fibers, the basement membranes of venous sinuses, were well developed in both infant and adult spleens. The first signs of their formation could be seen as a discontinuous basement membrane, which was immunoreactive for type IV collagen, laminin, and tenascin in the fetus aged 20 gestational weeks. Intracytoplasmic immunoreactivity for all the proteins studied was visible in the mesenchymal cells of the fetus aged 16 gestational weeks and in the reticular cells of the older fetuses, which also showed labeling for type IV collagen and laminin in the endothelial cells. The results suggest that proteins of the extracellular matrix are produced by these stationary cells.

Embolization for the treatment of posterior epistaxis. An analysis of 31 cases.

Thirty-one patients with posterior epistaxis refractory to nasal packing alone or in combination with surgical ligation (n = 8) underwent diagnostic angiography and therapeutic embolization of the internal maxillary artery. Embolization resulted in the cure of epistaxis in 22 cases (71.0%). Of the nine failures (29.0%), seven underwent successful surgical clipping of the ethmoid arteries, and two were treated conservatively and died of their primary hematologic disease within 33 days. Late rebleeding occurred in two patients: one underwent re-embolization and the other was treated surgically. No severe or permanent complications occurred. The results indicate that embolization is a feasible alternative to surgical intervention for patients with posterior epistaxis, and we recommend it as the treatment of choice in cases with high surgical risk or failure of prior arterial ligation.

EMLA in local anaesthesia of the tympanic membrane.

An ideal agent for local anaesthesia of the tympanic membrane has been missing so far. Recently, however, a eutectic mixture of lignocaine and prilocaine (EMLA, Astra, Södertälje, Sweden) has proved promising. We compared the anaesthetizing efficacy of EMLA, Bonain's solution (cocain, menthol, phenol ana partes) and Xylocain-spray (Astra, Södertälje, Sweden) in 42 voluntary subjects. EMLA was applied on one tympanic membrane and either one of the other two agents in the other ear of each subject. Small cotton pledgets were used for application. Sensitivity of the ear drum was tested under otomicroscope with a cotton tipped wire before and after each local anaesthesia. Full anaesthesia could be reached with EMLA very significantly (p less than 0.001) more often than with Xylocain and almost significantly (p = 0.057) more often than with Bonain's solution. Most of the test subjects preferred EMLA to Bonain's solution of Xylocain. Undesired side effects, including two tympanic membrane perforations, appeared in most of the ears anaesthetized with Bonain's solution. In the clinical part of the study, EMLA topical anaesthesia was used in 127 minor policlinical tympanic membrane procedures like myringotomy and tympanostomy tube assembling. Eighty-three of the procedures were assessed as painless, 36 unpleasant and 8 painful. A 30-min action time of EMLA was considered sufficient in most cases. No EMLA related side effects appeared. In conclusion, Bonain's solution is recommended to be replaced by EMLA or a corresponding agent for local anaesthesia of the tympanic membrane.

Secretory antibodies specific to Streptococcus pneumoniae, Haemophilus influenzae and Branhamella catarrhalis in middle ear effusion during acute otitis media.

The occurrence of specific secretory antibodies against the type-specific capsular polysaccharide of Streptococcus pneumoniae (Pn) and against the whole cell antigen of Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) were measured by the ELISA method in 211 middle ear effusion (MEE) samples of 85 children with acute otitis media (AOM) during the course of the disease. Antibodies against at least one of those bacteria were detected at the initial visit in 33.6% of the ears and later in 20%. All in all, such antibodies could be found in 50% of the ears during the follow-up. Pneumococcal secretory antibodies were found in 5 out of 116 ears only, anti-Hi antibodies in 52 and anti-Br antibodies in 42 ears. The specific secretory antibodies were detected against all these bacteria regardless of the bacterial etiology of the AOM attack in question. The AOM attack was prolonged more often if such antibodies were not found in the MEE sample taken at the initial visit. The appearance of such antibodies during the disease seemed to imply termination of the AOM episode in question. The conclusions of this study are that during an AOM attack a local production of antibodies in middle ear against the three most common bacteria. Pn, Hi and/or Br, causing AOM may be induced. The appearance of such antibodies in MEE seems to be beneficial for the resolution of AOM.

Occurrences of antibodies against Streptococcus pneumoniae, Haemophilus influenzae and Branhamella catarrhalis in middle ear effusion and serum during the course of acute otitis media.

The occurrence of IgG, IgM and IgA class antibodies against a type-specific capsular polysaccharide of Streptococcus pneumoniae (Pn) and against a whole cell antigen of Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) was studied using the ELISA method on middle ear effusion (MEE) samples of 85 patients and paired serum samples of 40 patients during the course of acute otitis media (AOM). Although specific antibodies to all of these three bacteria appeared in MEE during the course of an AOM episode, antibodies against the infecting bacteria of that particular AOM episode were more often prominent. The antibodies were also detectable in the MEE without simultaneous presence in the serum. The middle ear infection was prolonged more often if specific antibodies to the infecting bacterium could not be detected in the MEE obtained at the beginning of the AOM attack. The present study indicates that AOM caused by Pn, Hi or Br may induce both a systemic and a local production of specific antibodies against the causative organisms during the course of otitis media. The occurrence of such antibodies in MEE seems to play a major role in the resolution of AOM.

Cellular events in relation to bacteria-specific antibodies in middle ear effusion during acute otitis media.

The proportions of neutrophils and lymphocytes, the total number of viable lymphocytes and their spontaneous proliferating activity as well as the proportion of B-cells were determined in 238 middle ear effusion (MEE) samples from 124 ears and in 40 serum samples of 85 children in relation to the presence of bacteria-specific antibodies in MEE and the clinical outcome of acute otitis media (AOM) during the course of AOM. The percentage of lymphocytes was higher, and that of the neutrophils lower, in the ears with bacteria-specific antibodies than in the ears with no antibodies. The higher proportion of MEE lymphocytes and the presence of antibodies correlated to the faster resolution of AOM. Moreover, the total number of viable lymphocytes and the proliferating activity of these cells were related to the presence of specific antibodies in MEE. The findings of this study underline the importance of local mucosal immunity taking place in the middle ear in connection with bacteria-specific antibodies in resting AOM.

Hearing asymmetry among occupationally noise-exposed men and women under 60 years of age.

Interaural asymmetry of hearing thresholds at 4 kHz was analysed in four populations exposed to occupational noise. The left ear was found to be on average significantly worse than the right ear, among both the male and female subjects. In the male population the left ear was twice as often the worse ear as the right one. In the female population the corresponding ratio was 1.5. The average inferiority of the left ear increased as a function of the hearing threshold level. Among subjects with abundant shooting (reindeer herders) the average inferiority of the left ear was close to the average of all male subjects. Interaural difference increased as a function of the hearing threshold level, both among subjects with the left ear and subjects with the right ear being the worse one. In the male population the interaural difference was significantly greater in the former than in the latter group of subjects.

Antibodies against Streptococcus pneumoniae, Haemophilus influenzae and Branhamella catarrhalis in middle ear effusion during early phase of acute otitis media.

Serum type (IgG, IgM and IgA-class) and secretory type antibodies specific to Streptococcus pneumoniae (Pn), Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) were measured by enzyme-linked immunosorbent assay (ELISA) in 46 serum and 114 middle ear effusion (MEE) samples from 85 children with acute otitis media (AOM). The samples were obtained within 12 h from the onset of the ear symptoms. Serum (but not secretory) type antibodies to the infecting Pn serotype were found in 24% of the MEE samples of the patients with Pn AOM and, correspondingly, serum and/or secretory type antibodies to Hi and Br were seen in 54% and 63% of the MEE samples of the patients with Hi or Br AOM, respectively. Moreover, antibodies against bacteria other than the causative one could also be found in the MEE. The occurrence of the serum type antibodies against these bacteria in the MEE was closely correlated with their serum levels. The findings of this study indicate that during the very early phase of AOM, the MEE contains both serum type antibodies originating from the serum, and secretory antibodies of middle ear origin. Among them there are antibodies specific to the three most common bacteria causing AOM (Pn, Hi, and Br) regardless of the bacterial etiology of the AOM attack in question.

Tumorigenicity and H-2 expression of papillomavirus-transformed mouse cell lines.

Tumorigenicity in immunocompetent syngeneic mice and H-2 class I antigen expression of BPV1-transformed mouse cell lines had no correlation. H-2 expression was examined using monoclonal anti-(H-2Kb) and anti-(H-2Db) antibodies in immunofluorescence staining for flow cytometry analysis and by determining the sensitivity of the cells to cytolysis by allostimulated spleen cells. Nontumorigenic cell lines were as resistant as tumorigenic cell lines to natural killer activity. The results indicate that in our model defence by natural killer cells is not a decisive factor. The results also show that instead of or in addition to H-2 class I antigens other factors (e.g. the presence or absence of virus-specific antigens) are important in determining the tumorigenicity of BPV1-transformed cell lines.

Generation of cell-mediated cytotoxicity against bovine-papilloma-virus-transformed primary mouse cell lines.

The immunogenicity and immunosensitivity of primary mouse cell lines transformed by bovine papilloma virus 1 (BPV1) DNA were studied in a syngeneic mouse model by determining cell-mediated cytotoxicity in the spleens of mice immunized with the transformed cells. One of the cell lines induced the generation of cell-line-specific Thy1.2-positive cytotoxic effector cells. However, most of the cell lines tested induced the generation of Thy1.2-positive effector cells, which in addition to BPV1-transformed cells were able to lyse a syngeneic cell line transformed by methylcholanthrene. The lysis of BPV1- and methylcholanthrene-transformed cell lines was mediated by recognition of the same antigenic determinants expressed on these cells, and all the BPV1-transformed cell lines were sensitive to lysis by these nonspecific effector cells of the lymphokine-activated killer (LAK) type.

Phenotypic transformation of primary mouse fibroblasts by BPV 1 DNA.

Cultures of primary fibroblasts of C57BL/6J mice were used as targets for transformation by bovine papillomavirus type 1 (BPV 1) DNA. Although no foci were observed, several lines of transformed cells were established by subculturing. These immortalized cell lines had in vitro growth characteristics in high and low serum media and saturation densities typical of transformed cells. Karyotype analyses revealed extensive aneuploidic changes. In two of the three cell lines analyzed, viral DNA was present in monomeric episomal form, in the third cell line all viral sequences were found in the high molecular weight region of a Southern blot. Despite the transformed phenotype, only one of the cell lines was tumorigenic in nude mice at a low level.

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells.

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.