Umbilical Cord Prolapse in the Prehospital Setting: A Case Report.

Umbilical cord prolapse is an acute obstetric emergency associated with high fetal morbidity and mortality. To avoid poor outcomes, rapid diagnosis with immediate intervention is required, especially in the prehospital setting where resources are limited. In this case report, we describe a 38-year-old woman with umbilical cord prolapse, with a review of appropriate prehospital maneuvers and treatment.

Multiple wavelength reflectance microscopy to study the multiphysical behavior of microelectromechanical systems.

In order to characterize surface chemomechanical phenomena driving microelectromechanical systems behavior, we propose herein a method to simultaneously obtain a full kinematic field describing the surface displacement and a map of its chemical modification from optical measurements. Using a microscope, reflected intensity fields are recorded for two different illumination wavelengths. Decoupling the wavelength-independent and -dependent contributions to the measured relative intensity changes then yields the sought fields. This method is applied to the investigation of the electroelastic coupling, providing images of both the local surface electrical charge density and the device deformation field.

Cushing's disease and cutaneous alternariosis.

Alternaria species are common plant pathogens, but a rare cause of human infection. We present a patient with cutaneous alternariosis that revealed a relapse of an old case of Cushing's disease. Immunosuppression following the excessive glucocorticoid production seemed to contribute to the development of dermatosis. We also present a review of the literature on the association of Cushing's disease and cutaneous alternariosis. Our case is unique because the ketoconazole therapy that we used was successful in the treatment of both diseases.

Accumulation of mature mRNA in the nuclear fraction of mammalian cells.

Little is known about the nuclear mRNA content of mammalian cells. In this study, we analyzed by Northern blotting with a panel of probes the nuclear and cytoplasmic fractions derived from several rodent cell lines. For most of the genes under study, mature mRNAs could easily be detected in the nuclear fraction and accumulated to higher levels than the corresponding precursors. In addition, significant differences in the nucleo-cytoplasmic partition of mature mRNAs were observed between genes as well as between cell types (NIH 3T3, CTLL-2, D3-ES, PC-12), indicating that this nuclear accumulation of mRNA is regulated. Thus, while it is usually considered that splicing is the limiting step of pre-mRNA processing, these results point towards transport or nuclear retention of mRNA as a key determinant of nuclear mRNA metabolism.

Transcription of unr (upstream of N-ras) down-modulates N-ras expression in vivo.

The ras proteins (Harvey, Kirsten and N-ras) are key regulators of signal transduction and a perturbation of their GDP/GTP cycle is frequently observed in tumors. In mammals, N-ras constitutes with unr (upstream of N-ras) a tightly linked tandem of ubiquitously expressed genes. Although unr and N-ras appear to be involved in distinct functions, this unusual genetic organization could be important for the regulation of N-ras expression. Specifically, transcription of unr could negatively regulate that of N-ras by transcriptional interference. To investigate this possibility, we have deleted the unr promoter by homologous recombination in murine embryonic stem cells. Analysis of tissues of heterozygous mice revealed an increase in N-ras mRNA accumulation ranging between 20 and 65%, in agreement with the suppression of a transcriptional interference.

Abnormal organisation of the splenic marginal zone and the correlated leukocytosis in lymphotoxin-alpha and tumor necrosis factor alpha double deficient mice.

In this study we further characterized the phenotype at the homeostasis of mice genetically deficient in Tumor Necrosis Factor-alpha and Lymphotoxin-alpha (LT-alpha TNF-alpha -/-). As initially observed in LT-alpha -/- mice, these mice are devoid of lymph nodes and Peyer's patches, while in their spleen the white and red pulp domains are no more detectable. In the blood the leukocytosis dominated by lymphocytosis is not solely due to the absence of lymph nodes. Indeed, this abnormality was shown to be correctable by the transfer of wild type bone marrow in the absence of lymph node. We now report that the metallophilic macrophages of the marginal zone are no more detectable with an antibody reactive to sialoadhesin, a macrophage restricted transmembrane molecule known to bind myeloid and lymphoid cells. The absence of sialoadhesin within the marginal zone, a critical domain for lymphocyte trafficking towards the white pulp suggests a possible cellular basis for the observed blood leukocytosis. In addition, in the peritoneal cavity of LT-alpha TNF-alpha-/- mice, the size of the resident leukocyte population is increased. By their amplitudes these leukocytosis are similar within the blood and the peritoneal compartments.

Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences.

In all the organisms, homologous recombination (HR) is involved in fundamental processes such as genome diversification and DNA repair. Several strategies can be devised to measure homologous recombination in mammalian cells. We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination. The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells. The possibility to use two different orientations of tandem repeats (direct or inverted repeats) in parallel constitutes also an advantage. While inverted repeats measure only events arising by strand exchange (gene conversion and crossing over), direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage. In yeast, these processes depend on different pathways, most of them also existing in mammalian cells. These data permit to devise substrates adapted to specific questions about HR in mammalian cells. The effect of substrate structures (heterologies, insertions/deletions, GT repeats, transcription) and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease (especially the rare-cutting endonuclease ISce-I) on HR are discussed. Finally, transgenic mouse models using tandem repeats are briefly presented.

Gene expression profiling in blood cells of endurance horses completing competition or disqualified due to metabolic disorder.

REASONS FOR PERFORMING STUDY: Genomics using cDNA microarrays could provide useful information about physiological adaptations and metabolic disorders in endurance horses. OBJECTIVES: In order to show that genes are modulated in leucocytes in relationship with performance and clinical status of the horses, gene expression in leucocytes, haematological and biochemical parameters were compared between successful and disqualified endurance horses. METHODS: Blood samples were collected at rest (TO) and just after a 140-160 km endurance race (T1) in 2 groups of horses: 10 continuing successful (S) and 10 disqualified horses stopped at a vet-gate for metabolic disorders (D). Total RNA was extracted from the blood cells (leucocytes), checked for purity, amplified and hybridised using mouse cDNA microarrays including 15,264 unique genes. Differential gene expressions were studied by hybridisation of each sample T1 vs. a control sample collected at TO (pool of 20 sound horses). RESULTS: Some significant differences were observed in the haematology and biochemistry of the 2 groups (S vs. D). In Group D, rhadomyolysis was confirmed with CK 13,124 u/l and AST 1242 u/l. The list of 726 (including 603 annotated genes) significant genes was filtered according to a high P-value cut-off (P<0.00001). Among them, 130 were upregulated (expression ratio>1.5) and 288 were down-regulated (<1/1.5). Analysis of variance revealed 62 genes differentially expressed (P<0.05) in Groups D and S. The expression levels of 28 and 50 genes were significantly correlated (r>0.75) with CK and AST level in Group D, respectively. The gene ontology classification showed that more genes were up-regulated in S than in the D. More genes were down-regulated in the disqualified horses. CONCLUSIONS: Long exercise induced many significant gene modulations in leucocytes. Some genes were expressed in relationship with the clinical phenotype observed in Group D: rhabdomyolysis and haemolysis. POTENTIAL RELEVANCE: Some of these genes could be candidates to explain poor performance or pathologies. Further association studies with a greater number of genes should be conducted.

Gene expression profiling in equine muscle tissues using mouse cDNA microarrays.

REASONS FOR PERFORMING STUDY: Progress could be achieved by using microarrays to understand metabolic adaptations and disorders in equine muscle in response to exercise. OBJECTIVES: To test the feasibility of using mouse cDNA microarrays to analyse gene expression profile in normal equine muscles. METHODS: Muscular biopsies of dorsal gluteus medius and longissimus lumborum were done in 4 healthy Standardbreds. Total RNA was extracted from the muscle samples. The concentration and quality of RNA were measured before and after amplification. Gene expression profiles were measured using mouse cDNA microarrays including 15,264 unique genes representing about 11,000 documented genes. Three hybridisation tests were performed to check interspecificity, reproducibility and to compare gene expression in these muscles. For each test, a dye-swap hybridisation with Cy3 and Cy5 fluoromarkers were done and the gene list filtered according the signal level. RESULTS: According to the specificity test, the mouse cDNA microarrays were correctly hybridised by equine muscle cDNA. All positive control genes (GAPDH, HPRT and beta-Actin) and no negative control gene (yeast, plant) hybridised. The reproducibility test demonstrated a good linearity between the duplicate hybridisations: 99.99% of the significant expressed genes have an expression ratio between 1.4 and 1/1.4 = 0.71. These limits can be considered as the thresholds to qualify as up-regulated (ratio >1.4) or downregulated (ratio <0.71). In the muscle comparison test between gluteus medius vs. longissimus lumborum, 63 genes were found up-regulated and 8 genes down-regulated. The range of gene expression ratios in the gluteus medius was 0.61-8.31 x the longissimus lumborum. This list of modulated genes was classified by functions using a gene ontology data basis. CONCLUSION: Mouse microarrays could be used to hybridise equine RNA extracted from muscle tissues. For many genes there are large sequence identities that allowed interspecific cDNA hybridisation. The sensitivity of the method allowed quantification of up- and down-regulated genes after applying appropriate filters. POTENTIAL RELEVANCE: Expression profiling could be used to explore the muscle metabolism changes related to exercise, training, pathology and illegal medication in horses.

GRP-preferring bombesin receptors increase generation of inositol phosphates and tension in rat myometrium.

In the estrogen-treated rat myometrium, bombesin (Bn) and related agonists triggered contraction and the increased generation of inositol phosphates. The relative order of potencies was identical for both responses: Bn = gastrin releasing peptide (GRP) = litorin = neuromedin C >> neuromedin B. Two specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13) methyl ester and [Leu14,psi 13-14]Bn were inhibitory for both Bn-mediated tension and generation of inositol phosphates. [125I-Tyr4]Bn bound to myometrial membranes with high affinity (Kd = 104 pM) to a single class of sites in a saturable and reversible manner. The relative potencies for inhibiting binding were GRP = litorin = [Tyr4]Bn (Ki = 0.4 to 0.6 nM) >> neuromedin B (Ki = 10.3 nM). The high affinity displayed by [D-Phe6]Bn-(6-13) methyl ester (Ki = 2.8 nM) and [Leu14,psi 13-14]Bn (Ki = 35 nM) for competing for [Tyr4]Bn binding supported the involvement of a GRP-preferring Bn receptor. Guanine nucleotides decreased the binding of [125I-Tyr4]Bn and accelerated the rate of ligand dissociation, reflecting the coupling of receptors to guanine nucleotide regulatory proteins (G proteins). The results demonstrate that rat myometrium expresses functional GRP-preferring Bn receptors whose activation stimulates the phospholipase C pathway, pertussis toxin-insensitive event that contributes to Bn-mediated uterine contractions.

Control of nitric oxide production by endogenous TNF-alpha in mouse retinal pigmented epithelial and Muller glial cells.

Since the induction of nitric oxide synthase (NOS) by lipopolysaccharide (LPS) has been suggested to be partially dependent of the synthesis of tumor necrosis factor alpha (TNF alpha), we have investigated in vitro the production of NO in retinal cells from mice deficient in Lymphotoxin alpha (LT alpha)/TNF alpha. Treatment of retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells from both wild-type and knockout mice with LPS and interferon gamma (IFN gamma) induced NO synthesis as determined by nitrite release into the media and was correlated to an increase in NOS-2 mRNA levels, evaluated by RT-PCR. However, the level of nitrite and the accumulation of mRNA was always less in cells from LT alpha/TNF alpha knockout mice than in wild type mice. Simultaneous addition of TNF alpha restored the level of NO synthesis by RMG and RPE cells from LT alpha/TNF alpha knockout mice stimulated with LPS and IFN gamma to wild type levels. Transforming growth factor beta (TGF beta) blocked LPS/IFN gamma-induced NO production is RMG and RPE cells from wild-type and LT alpha/TNF alpha knockout mice. Our results demonstrate that induction of NO synthesis in RMG and RPE cells by LPS and IFN gamma is dependent in part on endogenous TNF alpha while inhibition of NO production by TGF beta does not require a modulation of TNF alpha synthesis.

Lipopolysaccharide-induced cytokine cascade and lethality in LT alpha/TNF alpha-deficient mice.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is often considered the main proinflammatory cytokine induced by lipopolysaccharide (LPS) and consequently the critical mediator of the lethality associated with septic shock. MATERIALS AND METHODS: We used mice carrying a deletion of both the lymphotoxin alpha (LT-alpha) and TNF-alpha genes to assess the role of TNF in the cytokine cascade and lethality induced by LPS. RESULTS: Initial production of IL-1 alpha, IL-1 beta, IL-6, and IL-10 is comparable in wild-type and mutant mice. However, at later times, expression of IL-1 alpha, IL-1 beta, and IL-10 is prolonged, whereas that of IL-6 decreases in mutant mice. Expression of IFN-gamma is almost completely abrogated in mutants, which is in agreement with a more significant alteration of the late phase of the cytokine cascade. We measured similar LD50 (600 micrograms) for the intravenous injection of LPS in mice of the three genotypes (+/+, +/-, -/-), demonstrating that the absence of TNF does not confer long-term protection from lethality. However, death occurred much more slowly in mutant mice, who were protected more efficiently from death by CNI 1493, an inhibitor of proinflammatory cytokine production, than were wild-type mice. DISCUSSION: Thus, while TNF-alpha is not required for the induction of these cytokines by LPS, it modulates the kinetics of their expression. The lethality studies simultaneously confirm a role for TNF as a mediator of early lethality and establish that, in the absence of these cytokines, other mediators take over, resulting in the absence of long-term protection from LPS toxicity.

Secondary alveolar echinococcosis in lymphotoxin-alpha and tumour necrosis factor-alpha deficient mice: exacerbation of Echinococcus multilocularis larval growth is associated with cellular changes in the periparasitic granuloma.

The availability of mice carrying a deletion of LT-alpha and tumour necrosis factor (TNF)-alpha genes enabled us to investigate the role of the TNF during alveolar echinococcosis. We compared the growth rate of Echinococcus multilocularis in LT-alphaTNF-alpha +/+ mice to that of mice having either no or only one LT-alphaTNF-alpha functionnal allele. LT-alphaTNF-alpha -/- mice harboured a significantly higher parasite burden than did the other two populations at 5, 10, and 15 weeks of infection, and they did not survive thereafter. Liver metacestodes removed from these mice were alive and the dehydrogenase activities of peritoneal metacestodes were decreased. Liver lesions regressed in most wild-type mice. Indeed, dead parasites were cordoned by granulomas containing numerous macrophages and lymphocytes leading to focal liver fibrosis at an early stage of infection. In contrast, most of LT-alphaTNF-alpha -/- mice harboured metacestodes interspersed with leucocytes, realising purulent abscesses with secondary extensive irregular fibrosis at a late stage of infection. Heterozygous mice had behavioural characteristics intermediate between homozygous mutants and wild-type mice. Levels of E. multilocularis-specific delayed-type hypersensitivity and serum antibodies were slightly decreased in LT-alphaTNF-alpha -/- mice. This study shows that TNF-alpha and/or LT-alpha genes play an essential role in the immune protection mechanisms against E. multilocularis at the site of infection.

Mice heterozygous for a deletion of the tumor necrosis factor-alpha and lymphotoxin-alpha genes: biological importance of a nonlinear response of tumor necrosis factor-alpha to gene dosage.

The tumor necrosis factors (TNF-alpha and lymphotoxin, or LT-alpha) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-alpha/TNF-alpha knockout mice and compared mice having one or two functional LT-alpha/TNF-alpha alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-alpha levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D-galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-alpha levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.

Efficient, repeated adenovirus-mediated gene transfer in mice lacking both tumor necrosis factor alpha and lymphotoxin alpha.

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (LTalpha), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-alpha/LTalpha-/-), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-alpha/LTalpha-/- and TNF-alpha/LTalpha+/- mice versus TNF-alpha/LTalpha+/+ mice links antibody levels to TNF-alpha/LTalpha gene dosage. Due to the absence of neutralizing antibodies, the TNF-alpha/LTalpha knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-alpha in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.

Increased susceptibility of TNF-alpha lymphotoxin-alpha double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans.

TNF-alpha and lymphotoxin-alpha (LT) are members of the TNF family, and these cytokines play crucial roles in the defense against infection with Candida albicans. The aim of the present study was to investigate the role of endogenous TNF and LT during disseminated candidiasis in TNF-/-LT-/- knockout mice. The TNF- and LT-deficient animals had a significantly increased mortality following C. albicans infection compared with control mice, and this was due to a 10- to 1000-fold increased outgrowth of the yeast in their organs. No differences between TNF-/-LT-/- mice and TNF+/+LT+/+ were observed when mice were rendered neutropenic, suggesting that activation of neutrophils mediates the beneficial effects of endogenous TNF and LT. Histopathology of the organs, combined with neutrophil recruitment experiments, showed a dramatic delay in the neutrophil recruitment at the sites of Candida infection in the TNF-/-LT-/- mice. Moreover, the neutrophils of deficient animals were less potent to phagocytize Candida blastospores than control neutrophils. In contrast, the killing of Candida and the oxygen radical production did not differ between neutrophils of TNF-/-LT-/- and TNF+/+LT+/+ mice. Peak circulating IL-6 was significantly higher in TNF-/-LT-/- mice during infection. Peritoneal macrophages of TNF-/-LT-/- mice did not produce TNF, and synthesized significantly lower amounts of IL-1alpha, IL-1beta, IL-6, and macrophage-inflammatory protein-1alpha than macrophages of TNF+/+LT+/+ animals did. In conclusion, endogenous TNF and/or LT contribute to host resistance to disseminated candidiasis, and their absence in TNF-/-LT-/- mice renders the animals susceptible through impaired recruitment of neutrophils and impaired phagocytosis of C. albicans.

Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways.

Despite the differences in the molecular structure between lipopolysaccharides (LPS) isolated from Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium, the potential differences in their biological effects in vivo have not been investigated. In the present study, TNF and LT double knock-out (TNF-/-LT-/-) mice were almost as susceptible as TNF+/+LT+/+ controls to S. typhimurium LPS, but they were significantly more resistant to lethal endotoxemia induced by E. coli or K. pneumoniae LPS. The effect was not due to endotoxin-associated proteins. In the knock-out mice, this difference in lethality was accompanied by decreased interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) production after challenge with E. coli LPS, whereas after S. typhimurium LPS more IL-1 and IFN-gamma were produced. In contrast, more IL-10 was produced after challenge of mice with E. coli LPS than with S. typhimurium LPS. The hypothesis that a combination of pro-inflammatory cytokines is responsible for the mortality after S. typhimurium LPS was suggested by experiments in mice deficient in IL-1beta-converting enzyme (ICE-/- mice). ICE-/-mice, lacking mature IL-1beta and IL-18, but also defective in IFN-gamma and TNF production, were completely protected against both E. coli and S. typhimurium LPS. Experiments in Toll-like receptor (TLR)-4 defective mice suggested that the difference is not due to differential activation of TLR4. In conclusion, TNF and LT play a central role in the lethality due to E. coli LPS, whereas the lethal effects of S. typhimurium LPS are mediated through mechanisms also involving other cytokines such as IFN-gamma, IL-1 and IL-18.