MICI-MINOTS: Linguistic and metric validation of a pediatric questionnaire on knowledge about inflammatory bowel disease.

BACKGROUND: Therapeutic education is an essential part of the treatment of chronic diseases, such as inflammatory bowel disease (IBD). The IBD-KID, developed in Canada in English, assesses children's and adolescents' acquired knowledge about their condition and has been validated in Canadian and Australian populations. However, there is no pediatric questionnaire in French to assess patients' knowledge about IBD. OBJECTIVE: To report the linguistic validation process and metric validity of the MICI-MINOTS, the French version of the IBD-KID. METHOD: The translation process consisted of three consecutive steps: forward-backward translation, acceptability testing, and cognitive interviews. The IBD-KID consists of 23 questions, but a 24th question about immunomodulatory therapy was added in the MICI-MINOTS. Psychometric testing was conducted with five groups: children with IBD, their parents, children in a control group, their parents, and health workers recruited from the Timone Pediatric Hospital and the Saint-Sébastien Maternal and Child Protection Center, Marseille, France. A total of 15 individuals completed the tool twice, with a 15-day interval. Internal consistency, reliability, external validity, reproducibility, and sensitivity to change were tested. RESULTS: A total of 38 children with IBD (sex: 20 boys, 18 girls; age: 13.90 [±2.88] years; 25 with Crohn's disease), 20 children in the control group, 58 parents (every child was included with one parent), and 62 health workers were included in the analysis. Intraclass correlation was 0.94 (95% confidence interval 0.83-0.98) for test-retest assessment. Readability using the Scolarius score corresponded to elementary school level. Among the children with IBD, 89.5% answered all 24 questions. For 23 questions, the mean score of children with IBD was higher than among children in the control group: 9.58 (±3.01) versus 5.47 (±3.56), respectively (P<0.01). Parents of children with IBD scored higher than parents of children in the control group: 10.63 (±3.16) versus 8.4 (±3.07), respectively (P=0.012). In the health workers' group, pediatric residents (17.82±3.46) scored higher than nurses 11.75 (±3.4) and ward clerks (8.67±2.40; P<0.01). Patients' knowledge score was significantly related to their parents' knowledge score (r=0.402, P=0.012) for 23 questions. CONCLUSION: The French version of the IBD-KID showed satisfactory psychometric properties to assess knowledge about the disease in French-speaking children.

Phylogeography Approach of Diloboderus abderus (Coleoptera: Melolonthidae) in the Southern Cone of America.

Diloboderus abderus (Sturm, 1826) (Coleoptera: Melolonthidae) is a serious soil pest of corn, wheat, oat, and natural and cultivated pastures in Argentina, Paraguay, Uruguay, and southern Brazil. Despite its economic importance, the genetic diversity and population structure of D. abderus remain unknown. We sequenced a fragment of the mitochondrial gene cytochrome oxidase I region (COI), of six populations of D. abderus from the Southern Cone of America. The mtDNA marker revealed a high haplotype diversity, high pairwise FST values, and significant genetic variations among populations. No correlation was found between genetic and geographical distances, yet the most common haplotype (Dab01) was present in four out of the six populations. Analysis of molecular variance showed that most of the variation was within populations of D. abderus. Tajima's D and Fu's FS tests indicated no evidence that D. abderus populations are under recent expansion. Our results indicate that genetic-based traits will likely remain localized or spread slowly, and management strategies need to be undertaken on a small scale.

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

Impaired wound healing in diabetes: the rationale for clinical use of hyaluronic acid plus silver sulfadiazine.

Diabetes-related chronic cutaneous lesions are a serious and common problem, as well as a major cause for hospital admissions, although no general consensus has been reached on the best available treatment for this frequent pathological condition. The primary objective of this review is to analyze the most recent evidence supporting the clinical use of a formulation containing hyaluronic acid (HA) and silver sulfadiazine (SSD) in the diabetic patient. This formulation has been widely used in cutaneous lesions of various etiology, both acute and chronic. The mechanisms underlying tissue repair are altered in the diabetic patient with respect to a healthy individual, namely for a diminished response of the keratinocytes and a reduced capacity of the endothelial cells to form new vessels (neoangiogenesis). Since HA favours the tissue repair process through various mechanisms, among these an increased angiogenic response and an activation of the keratinocytes, its application in diabetic lesions is a rational choice. SSD has been widely used in acute cutaneous lesions, particularly in burns, where it is considered the "gold standard" by which other treatments are measured. The efficacy of SSD in terms of antibacterial activity spectrum on various types of microorganisms, with a favourable safety profile, supports the potential use of SSD in diabetic lesions, where the presence of infection caused by bacteria resistant to most available antibiotics, but not to SSD, is rather frequent. In conclusion, the combined use of HA and SSD in the diabetic patient proves a rational choice and is potentially capable of improving the general clinical situation, on the basis of the synergic effect to control infection and accelerate the tissue repair process.

Goat α(s1)-casein genotype affects milk fat globule physicochemical properties and the composition of the milk fat globule membrane.

Milk fat secretion is a complex process that initiates in the endoplasmic reticulum of the mammary epithelial cell by the budding of lipid droplets. Lipid droplets are finally released as fat globules in milk enveloped by the apical plasma membrane of the mammary epithelial cell. The milk fat globule membrane (MFGM) thus comprises membrane-specific proteins and polar lipids (glycerophospholipids and sphingolipids) surrounding a core of neutral lipids (mainly triacylglycerols and cholesterol esters). We have recently described major proteins of the MFGM in the goat and we have highlighted prominent differences between goats and bovine species, especially regarding lactadherin, a major MFGM protein. Here, we show that, in the goat species, the well-documented genetic polymorphism at the α(s1)-casein (CSN1S1) locus affects both structure and composition of milk fat globules. We first evidenced that both milk fat globule size and ζ-potential are related to the α(s1)-casein genotype. At midlactation, goats displaying strong genotypes for α(s1)-casein (A/A goats) produce larger fat globules than goats with a null genotype at the CSN1S1 locus (O/O goats). A linear relationship (R(2)=0.75) between fat content (g/kg) in the milk and diameter of fat globules (µm) was established. Moreover, we found significant differences with regard to MFGM composition (including both polar lipids and MFGM proteins) from goats with extreme genotype at the CSN1S1 locus. At midlactation, the amount of polar lipids is significantly higher in the MFGM from goats with null genotypes for α(s1)-casein (O/O goats; 5.97±0.11mg/g of fat; mean ± standard deviation) than in the MFGM from goats with strong genotypes for α(s1)-casein (A/A goats; 3.96±0.12mg/g of fat; mean ± standard deviation). Two MFGM-associated proteins, namely lactadherin and stomatin, are also significantly upregulated in the MFGM from goats with null genotype for α(s1)-casein at early lactation. Our findings are discussed with regard to techno-functional properties and nutritional value of goat milk. In addition, the genetic polymorphism in the goat species appears to be a tool to provide clues to the lipid secretion pathways in the mammary epithelial cell.

Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection.

Mastitis, an inflammation of the mammary gland, is the most costly infectious disease of dairy ruminants worldwide. Although it receives considerable attention, the early steps of the host response remain poorly defined. Here, we report a noninvasive method using milk fat globules (MFG) as a source of mammary RNA to follow the dynamics of the global transcriptional response of mammary epithelial cells (MEC) during the course of a bacterial infection. We first assessed that RNA isolated from MFG were representative of MEC RNA; we then evaluated whether MFG RNA could be used to monitor the MEC response to infection. Sufficiently high yields of good-quality RNA (RNA integrity numbers ranging between 6.7 and 8.7) were obtained from goat MFG for subsequent analyses. Contamination of MFG by macrophages and neutrophils, which can be trapped during creaming, was assessed and when using quantitative real-time PCR for cell-type specific markers, was shown to be weak enough (<8%) to affect MFG gene expression profiling. Using microarrays, we showed that RNA extracted from MFG and from mammary alveolar parenchyma shared approximately 90% of the highlighted probes corresponding in particular to genes encoding milk proteins (CSN, BLG, LALBA) and enzymes involved in milk fat synthesis and secretion (FASN, XDH, ADRP, SCD, and DGAT1). In addition, a gene involved in the acute-phase reaction, coding for the serum amyloid A3 (SAA3) protein, was found within the first 50 most highly expressed genes in a noninfectious context in both mammary alveolar parenchyma and MFG, strongly suggesting that SAA3 is expressed in MEC. We took advantage of this noninvasive RNA sampling to follow the early proinflammatory response of MEC during the course of a bacterial infection and showed that the levels of mRNA encoding SAA3 sharply increased at 24h postinfection. Taken together, our results demonstrate that MFG represent a unique source of MEC RNA to noninvasively sample sufficient amounts of high-quality RNA to assess the dynamics of MEC gene expression in vivo, especially during the first steps of infection, thereby paving the way for the discovery of early biomarkers for the control of intramammary infections. Furthermore, this noninvasive technique could be used to provide mammary transcriptomic data on a large scale, thus filling the gap between genomic and phenotypic data.

Exogenous hyaluronic acid and wound healing: an updated vision.

Hyaluronic acid (HA), an endogenous substance whose concentration increases during the process of wound repair, can be manufactured in order to use it as an exogenous intervention able to reduce the time to wound repair and improve the quality of the scar. The role of HA as a key component of the extracellular matrix structure has been recognized for many decades, while its actions on cells involved in the process of tissue repair has been partly clarified only in the last few years. Fibroblasts, endothelial cells and macrophages are key players in the tissue repair process and a concerted activation of specific functions of these cells may substantially improve the process of wound closure. Hyaluronan, as well as its degradation products that are generated in the wounds, are capable to activate specific responses in all the cells involved in the process; in particular, fibroblast proliferation and new vessel formation have been extensively studied. The molecular patterns leading to cell activation have been substantially clarified and it is now widely accepted that cellular actions of hyaluronic acid are mediated by specific surface receptors, including CD44, RHAMM and toll like receptors. Elucidation of the mechanisms of cellular activation will allow an optimal use of exogenous hyaluronan and its derivatives in the wound care setting.

Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4.

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

Terminal differentiation of goat mammary tissue during pregnancy requires the expression of genes involved in immune functions.

Terminal differentiation of mammary tissue into a functional epithelium that synthesizes and secretes milk occurs during pregnancy. The molecular mechanisms underlying this complex process are poorly understood, especially in ruminants. To obtain an overview of the ruminant mammary gland's final differentiation process, we conducted time-course gene expression analysis of five physiological stages: four during pregnancy (P46, P70, P90, and P110) and one after 40 days of lactation (L40). An appropriate loop experimental design was used to follow gene expression profiles. Using three nulliparous (pregnancy) or primiparous (lactation) goats per stage, we performed a comparison starting from nine dye-swaps and using a 22K bovine oligoarray. Statistical analysis revealed that the expression of 1,696 genes varied significantly at least once in the study. These genes fell into 19 clusters based on their expression profiles. Identification of biological functions with Ingenuity Pathway Analysis software revealed several similarities, in keeping with physiological stages described in mice. As in mice, expression of milk protein genes began at midpregnancy, and genes regulating lipid biosynthesis were induced at the onset of lactation. During the first half of pregnancy, the molecular signature of goat mammary tissue was characterized by the expression of genes associated with tissue remodeling and differentiation, while the second half was mainly characterized by the presence of messengers encoding genes involved in cell proliferation. A large number of immune-related genes were also induced, supporting recent speculation that mammary tissue has an original immune function, and the recruitment of migrating hematopoietic cells possibly involved in the branching morphogenesis of the mammary gland. These data hint that the induction of differentiation occurs early in pregnancy, very likely before P46. This period is therefore crucial for obtaining a healthy and productive mammary gland.

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials.

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.

Identification of differentially expressed genes in spontaneously regressing melanoma using the MeLiM swine model.

Partial and some few cases of complete spontaneous regression have been observed in cutaneous melanoma patients but little is known about the molecular mechanisms involved. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a suitable animal model to study the phenomenon of spontaneous regression because MeLiM pigs exhibit naturally occurring melanomas which regress completely 6 months after birth. In this study, we used suppression subtractive hybridization (SSH) to identify molecular determinants of melanoma regression within swine melanoma tissues and melanoma cell cultures. Several markers involved in cell-adhesion, -communication, -motility, signal transduction, negative regulation of cell proliferation, transport and immune response were identified that correlated with melanoma regression whereas the main genes involved in melanin synthesis showed a strong downregulation. For the most differentially expressed genes, we validated the results obtained by SSH with qRT-PCR and with immunohistochemistry for some of them (CD9, MITF, RARRES1). Most notable, for the first time in melanoma, we identified the retinoic acid responder 1 gene (RARRES1) as a main actor of the regression process in melanoma. This first gene expression study in swine melanoma regression, may contribute to the finding of new therapeutic targets for human melanoma treatment.

Effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage.

The use of autologous chondrocytes seeded onto a biological scaffold represents a current valid tool for cartilage repair. However, the effect of the contact of blood to the engineered construct is unknown. The aim of this work was to investigate in vitro the effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage. Articular chondrocytes were enzymatically isolated from swine joints, expanded in monolayer culture and seeded onto collagen membranes for 2 weeks. Then, the seeded membranes were placed for 3 days in contact with peripheral blood, which was obtained from animals of the same species and diluted with a standard medium. As controls, some samples were left in the standard medium. After the 3 days' contact, some samples were retrieved for analysis; others were returned to standard culture conditions for 21 additional days, in order to investigate the "long-term effect" of the blood contact. Upon retrieval, all seeded samples showed increasing sizes and weights over time. However, the samples exposed to blood presented lower values with respect to the controls. Biochemical evaluation demonstrated a reduction in the mitochondrial activity due to blood contact at the early culture time (3 days post blood contact), followed by a partial recovery at the longer culture time (21 days post blood contact). Histological evaluation demonstrated evident cartilage-like matrix production for both groups. Biomechanical data showed a reduction of the values, followed by stabilization, regardless of the presence of blood. Based on the data obtained in this study, we can conclude that blood contact affects the chondrocyte activity and determines a delay in the dimensional growth of the engineered cartilage; however, at the experimental times utilized in this study, this delay did not affect the histological pattern and the biomechanical properties of the construct.

Safety and immunogenicity of the RTS,S/AS02A candidate malaria vaccine in children aged 1-4 in Mozambique.

BACKGROUND: The development of a malaria vaccine remains a public health priority for sub-Saharan Africa. RTS,S/AS02A candidate malaria vaccine has been shown to be safe and immunogenic in previous studies in adults and staggered dose-escalation studies in children in The Gambia. However, genetic features and the intensity of malaria transmission may modify the safety and immune response of a vaccine. OBJECTIVE: We carried out a phase I, double-blind randomized controlled trial in 60 children aged 1-4 in Mozambique to evaluate the safety, reactogenicity and immunogenicity of the paediatric vaccine dose (fixed 25 microg RTS,S in 0.25 ml) of RTS,S/AS02A, prior to undertaking a planned larger phase IIb proof-of-concept of efficacy study in the same population. METHOD: Children were randomized to receive either RTS,S/AS02A or Engerix-B vaccine. Monitoring of safety and reactogenicity included detailed clinical and laboratory analyses and assessment of adverse events (AEs). RESULTS: The RTS,S/AS02A was found to be safe and well tolerated. Serious adverse events were balanced between both groups and none was related to vaccination. The frequency of adverse events reported with RTS, S/AS02A was comparable to previous studies in children. Grade 3 AEs were infrequent (one case of pain, one of fever in each group and some swelling greater than 20 mm in diameter), transient and resolved without sequelae. RTS,S/AS02A was highly immunogenic for anti-circumsporozoite protein antibody response and induced a strong anti-hepatitis-B surface antigen response.

Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice.

BACKGROUND: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. METHODS: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon gamma (IFN-gamma) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. RESULTS: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-gamma) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-gamma (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. CONCLUSIONS: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.

Expression of NGF, Trka and p75 in human cartilage.

Nerve growth factor (NGF) exerts its action through two types of receptor: high-affinity tyrosine kinase A receptor (trkA) and low-affinity p75 receptor. NGF has a neurotrophic role in central and peripheral nervous system development, but there is also clear evidence of its involvement in the developing skeleton. The aim of the present immunohistochemical study was to investigate the expression and distribution of NGF, trkA, and p75 in normal cartilaginous tissues from adult subjects: articular and meniscal cartilage of the knee, cartilage from the epiglottis, and intervertebral disc tissue. Detection of NGF mRNA was also performed by in situ hybridization. Immunoreaction for NGF and the two receptors in articular chondrocytes, chondrocyte-like cells of meniscus and annulus fibrosus, and chondrocytes of the epiglottis demonstrated that they are all expressed in hyaline, fibrous and elastic cartilaginous tissues, suggesting that they could be involved in cartilage physio-pathology.

Anti-Fas-induced apoptosis in chondrocytes reduced by hyaluronan: evidence for CD44 and CD54 (intercellular adhesion molecule 1) invovement.

OBJECTIVE: To investigate the in vitro effect of therapeutic hyaluronan (HA) of 500-730 kd on anti-Fas-induced apoptosis of chondrocytes from osteoarthritis (OA) patients, and to assess its mechanism of action by analyzing the role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecule 1 [ICAM-1]). METHODS: Chondrocytes isolated from human OA knee cartilage were cultured and the effect of HA on both spontaneous and anti-Fas-induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analysis of oligonucleosome), TUNEL assay, and electron microscopy. Blocking experiments with anti-CD44 and anti-CD54 alone or in combination were performed to investigate the HA mechanism of action. RESULTS: Both quantitative tests demonstrated that anti-Fas significantly induced apoptosis of isolated OA chondrocytes. HA at 1,000 microg/ml significantly reduced the anti-Fas-induced apoptosis of chondrocytes but did not affect spontaneous chondrocyte apoptosis. These data were also confirmed by TUNEL staining and by electron microscopy morphologic evaluation. The antiapoptotic effects of HA on anti-FAS-induced chondrocyte apoptosis were significantly decreased by both anti-CD44 (mean +/- SD 57 +/- 12% inhibition) and anti-ICAM-1 (31 +/- 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 +/- 13%. CONCLUSION: These data demonstrate that 500-730-kd HA exerts an antiapoptotic effect on anti-FAS-induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM-1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartilage matrix degradation.

Goats' milk of defective alpha(s1)-casein genotype decreases intestinal and systemic sensitization to beta-lactoglobulin in guinea pigs.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) alpha(s1)-casein content. Parenteral sensitization to GM1 and GM2 proteins as also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (delta Isc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-beta-lg IgG1, with an important cross reactivity between goat and cow beta-lg. However, in guinea pigs fed on GM2, anti-goat beta-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3.5+/-4.5 microA/cm2) compared with that observed in GM1 guinea pigs (8.3+/-7.6 microA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

Autologous chondrocyte implantation: cells phenotype and proliferation analysis.

The phenotype and proliferation of human chondrocytes in culture were analyzed before they were implanted as autologous graft in cartilage lesions. During ten autologous chondrocyte implantations in articular cartilage lesions of the knee in ten patients, small amounts of cells to be implanted were collected and analyzed by morphology, cytochemistry (alcian blue, safranin-O), and immunocytochemistry (antibodies anti-S100 protein, anti-collagen types I and II, anti-chondroitin-S). In four cases the cells were also cultured for 3 weeks. At 1, 10, and 20 days of culture cells were incubated with 1 microCi/ml [3H]thymidine for proliferation analysis. In all cases the cells showed the morphological appearance of mature chondrocytes, stained positively for alcian blue and safranin-O, and revealed a strong immunoreaction for S-100 protein, type II collagen, and chondroitin-S but not for type I collagen. Radioisotope assay of chondrocyte proliferation at 1, 10, and 20 days of culture revealed a progressive increase in [3H]thymidine incorporation. These findings indicate that the cells before autologous implantation maintain their differentiated phenotype of mature chondrocytes and proliferate greatly. This analysis is an essential step preceding wider use of this treatment in humans. However, other biological aspects of the autologous chondrocyte graft remain to be elucidated.

Cloricromene, a semi-synthetic coumarin derivative, inhibits tumor necrosis factor-alpha production at a pre-transcriptional level.

Cloricromene decreases myocardial infarct size after ischemic-reperfusion injury in vivo, and it has been suggested that this is due to inhibition of tumor necrosis factor-alpha (TNF-alpha). The purpose of this work was to characterize the mechanism of cloricromene-induced inhibition of TNF-alpha in rat macrophages. Cloricromene inhibited lipopolysaccharide-induced TNF-alpha release in a dose-dependent manner (IC(50)=5.9 +/- 0.8 microM). This was not due to cytotoxicity, as cloricromene was well tolerated up to 500 microM. Cloricromene inhibited lipopolysaccharide-induced expression of TNF-alpha mRNA, which suggests a pre-transcriptional effect. We then investigated the early signal transduction pathway triggered by lipopolysaccharide. The binding of lipopolysaccharide to its receptor CD14 activates protein kinase C and nuclear factor-kappaB (NF-kappaB). Cloricromene inhibited NF-kappaB activation in a dose-dependent manner, but affected protein kinase C translocation only slightly. We then established that cloricromene inhibited lipopolysaccharide-induced cellular oxidative activity, which is important for NF-kappaB activation. Our results show that cloricromene interferes with the early signal transduction pathway triggered by lipopolysaccharide.