Transoral laser microsurgery for polydimethylsiloxane (VOX Implant®) explantation.

INTRODUCTION: Polydimethylsiloxane (VOX Implant®) injection into the larynx is a surgical technique used to restore volume to paralysed or atrophic vocal folds. We present a case series that demonstrates the feasibility of explantation of this non-resorbable material in the event of complications secondary to this injection. DESCRIPTION: We report four cases of VOX Implant® explantation. In every case, explantation was performed following complications such as dysphonia and/or dyspnoea secondary to injection of this material. VOX Implant® explantation was performed via cordotomy using transoral lasermicrosurgical techniques. DISCUSSION: VOX Implant® explantation improved voice and breathing parameters in all patients. Explantation can be performed by transoral microsurgery in the presence of complications related to polydimethylsiloxane injection.

[Saccharomyces boulardii modulates dendritic cell properties and intestinal microbiota disruption after antibiotic treatment]. / Saccharomyces boulardii module les propriétés descellules dendritiques et le déséquilibre du microbioteintestinal après un traitement antibiotique.

Saccharomyces boulardii is a non-pathogenic yeast with biotherapeutic properties that has been used successfully to prevent and to treat various infectious and antibiotic-associated diarrheas. The intestinal microbiota is responsible for colonization resistance and immune response to pathogens but can be disrupted by antibiotics and lose its barrier effect. Dendritic cells (DCs) are professional antigen-presenting cells of the immune system with the ability to initiate a primary immune response or immune tolerance. In a human microbiota-associated mouse model, we evaluated the influence of S. boulardii on the composition of the microbiota and on the properties of dendritic cells in normal homeostatic conditions and after antibiotic-induced stress. The DCs were derived from splenic precursors. Membrane antigen expression and phagocytosis of FITC-latex beads by DCs were evaluated by flow cytometry. The molecular analysis of the microbiota was performed with fluorescence in situ hybridization (FISH) combined with flow cytometry or confocal microscopy using group specific 16S rRNA targeted probes. This evaluation was conducted during and after a 7-day oral treatment with amoxicillin-clavulanic acid alone and in combination with the administration of the yeast. The antibiotic treatment increased the phagocytic activity of DCs. Their antigen presenting function (MHC class II antigen and CD 86 costimulatory molecule membrane expression) was up-regulated. This reflects a functional activation of DCs. In the presence of S. boulardii, the modification of membrane antigen expression was down regulated. To correlate these modifications to the microbiota disruption, we analyzed in parallel the composition of the intestinal microbiota. As previously shown, the amoxicillin-clavulanic acid treatment, both alone and with S. boulardii, did not quantitatively alter the total microbiota. In contrast, after one day of the antibiotic treatment the Clostridium coccoides group decreased dramatically in the two groups of mice treated with the antibiotic. The level then increased regularly, and at days 17, 22 and 24 it increased faster (P < 0.05) in the AB+ Sb group than in the AB group, reaching the initial level at day 29. The Bacteroides group in the two groups of mice increased during the antibiotic treatment and decreased after the antibiotic was stopped, reaching the initial level. The rate of decrease was faster for the AB+ Sb group than for the AB group, with a significant difference (P < 0.05) at days 17 and 22. During antibiotic treatment, the Enterobacteriaceae group became detectable and its level increased in both groups of mice. After discontinuation of the antibiotic, its level decreased to become undetectable at day 29, without significant difference between the two groups. These results showed that S. boulardii treatment tends to restore the balance of the dominant anaerobic microbiota more rapidly in human microbiota associated-mice treated with amoxicillin-clavulanic acid; the results also suggest that the yeast has a role in modulating the specific immune response to microbial associated-molecular patterns. This may explain, at least in part, the beneficial effects of S. boulardii in preventing antibiotic-associated diarrhea. This also suggests that the yeast plays a role in maintaining intestinal homeostasis.

Cobalt toxicity: chemical and radiological combined effects on HaCaT keratinocyte cell line.

Cobalt (Co) is an essential trace element well known as a constituent of vitamin B(12), but different compounds of Co are also described as highly toxic and/or radiotoxic for individuals or the environment. In nuclear power plants, (58)Co and (60)Co are radioactive isotopes of cobalt present as activation products of stable Co and Ni used in alloys. Skin exposure is a current occupational risk in the hard metal and nuclear industries. As biochemical and molecular cobalt-induced toxicological mechanisms are not fully identified, we investigated cobalt toxicity in a model human keratinocyte cell line, HaCaT. In this study, we propose a model to determine the in vitro chemical impact on cell viability of a soluble form of cobalt (CoCl(2)) with or without gamma-ray doses to mimic contamination by (60)Co, to elucidate the mechanisms of cobalt intracellular chemical and radiological toxicity. Intracellular cobalt concentration was determined after HaCaT cell contamination and chemical toxicity was evaluated in terms of cellular viability and clonogenic survival. We investigated damage to DNA in HaCaT cells by combined treatment with chemical cobalt and a moderate gamma-ray dose. Additive effects of cobalt and irradiation were demonstrated. The underlying mechanism of cobalt toxicity is not clearly established, but our results seem to indicate that the toxicity of Co(II) and of irradiation arises from production of reactive oxygen species.

An interdisciplinary approach to investigate the impact of cobalt in a human keratinocyte cell line.

Since in nuclear power plants, risks of skin contact contamination by radiocobalt are significant, we focused on the impact of cobalt on a human cutaneous cell line, i.e. HaCaT keratinocytes. The present paper reports an interdisciplinary approach aimed at clarifying the biochemical mechanisms of metabolism and toxicity of cobalt in HaCaT cells. Firstly, a brief overview of the used instrumental techniques is reported. The following parts present description and discussion of results concerning: (i) toxicological studies concerning cobalt impact towards HaCaT cells (ii) structural and speciation fundamental studies of cobalt-bioligand systems, through X-ray absorption spectroscopy (XAS), ab initio and thermodynamic modelling (iii) preliminary results regarding intracellular cobalt speciation in HaCaT cells using size exclusion chromatography/inductively coupled plasma-atomic emission spectroscopy (SEC/ICP-AES) and direct in situ analysis by ion beam micropobe analytical techniques.

Alterations of antioxidant trace elements (Zn, Se, Cu) and related metallo-enzymes in plasma and tissues following burn injury in rats.

To improve the nutritional support for burn patients, we evaluated the alterations of selenium, zinc and copper (Se, Zn and Cu) and their possible contributions to an unbalanced antioxidant response to burn injury. These trace elements and the related antioxidant enzymes, glutathione peroxidase (GPx) and superoxide dismutase (SOD), were studied both in plasma (or serum) and tissues of 20% total body surface area (TBSA) burned rats for 10 days. While plasma Se and serum Zn levels significantly decreased 6 h after burn injury, serum Cu levels increased after 1 day and remained elevated the following 9 days. Selenium levels increased in kidney but decreased progressively in liver. The hepatic Zn and Cu concentrations followed a biphasic increase following burn injury. During the first day, GPx activity decreased in plasma and remained unchanged in the organs, except for a moderate diminution in the liver. Liver Cu/Zn SOD activity increased from 6 h to 4 days. In summary, following burn injury, copper and zinc were redistributed to the liver and selenium to the kidney with non-detectable changes in the muscle and brain. Changes in antioxidant enzyme activities following burn injury were significant mainly in the plasma. Early combined antioxidant supplementation to maintain and restore antioxidant status in burn patients requires further study.

A peptide derived from bovine beta-casein modulates functional properties of bone marrow-derived macrophages from germfree and human flora-associated mice.

The purpose of this study was to evaluate the immunomodulatory activity of a peptide derived from bovine beta-casein (beta-CN), the beta-CN (193-209) peptide, on mouse macrophages that were obtained either from germfree (GF) or from human flora-associated (HF) mice. Macrophages were derived from bone marrow (BMDM) in the presence of recombinant macrophage colony-stimulating factor and exposed to the peptide or lipopolysaccharide (LPS). Membrane marker expression [F4/80, Mac-1, major histocompatibility complex (MHC) class II antigens] and phagocytic activity were assessed by flow cytometry. Production of tumor necrosis factor-alpha and interleukin (IL)-6 was measured by bioassays and production of IL-1alpha, IL-1beta and IL-12 by ELISA. The expression of cytokine mRNA was determined using semi-quantitative reverse transcription-polymerase chain reaction. The beta-CN (193-209) peptide up-regulated MHC class II antigen expression and phagocytic activity of BMDM from GF and HF mice. Its enhancing effect on phagocytosis was greater than that after LPS stimulation (P < 0.01). The peptide induced notable levels of cytokine mRNA in BMDM from GF and HF mice, but it was a significantly weaker inducer of cytokine secretion than LPS. Nevertheless, although flora implantation had no stimulatory influence on basal MHC class II and basal cytokine levels, cells from HF mice were more susceptible than those from GF mice to the peptide effects on these variables. These results indicate that the beta-CN (193-209) peptide could enhance antimicrobial activity of macrophages without proinflammatory effects.

The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.

Influence of intestinal microflora on murine bone marrow and spleen macrophage precursors.

To investigate the adjuvant effect of intestinal flora on macrophage-colony-stimulating factor-responsive macrophage progenitors from spleen and bone marrow, we compared progenitor numbers and phenotypic characteristics of in vitro matured macrophages in germ-free and flora-associated mice (conventional, Escherichia coli-monoassociated and conventionalized mice). The data obtained show that the flora affected differentially bone marrow and spleen progenitors. It increased the numbers of progenitors in the spleen but not in the bone marrow. It did not modify the expression of F4/80, Mac-1 and major histocompatibility complex (MHC) class II on bone-marrow-derived macrophages (BMDM), while it clearly up-regulated MHC class II expression on spleen-derived macrophages (SDM). This effect was more pronounced in flora-associated ex germ-free mice than in conventional mice and it was greatly enhanced in the absence of M-CSF. In vitro stimulation by lipopolysaccharide had no effect on marker expression of BMDM, while it decreased F4/80 and enhanced MHC class II molecules on SDM from germ-free and flora-associated mice. However, the expression of MHC class II remained lower in germ-free mice. Enhancement of MHC class II molecule expression on SDM may contribute to the protective role of flora, because successful immune responses are dependent on the expression of these molecules.

The influence of E. coli implantation in axenic mice on cytokine production by peritoneal and bone marrow-derived macrophages.

To assess the involvement of bacterial microflora in the development of host defenses, we compared in vitro LPS-induced cytokine production by macrophages in germ-free and E. coli monoxenic mice. E. coli implantation significantly increased IL-1 and IL-6 and, to a lesser extent, TNF activities of peritoneal and bone marrow-derived macrophages. These results suggest that exposure to microflora primes macrophages for an enhanced cytokine production, which may contribute to the activation of the antiinfectious defense. The priming was not restricted to peritoneal macrophages but was associated with a more general effect of the flora since the enhanced response of bone marrow-derived macrophages indicates an effect on macrophage precursors. Furthermore, a higher ability of peritoneal macrophages to produce IL-1 in axenic and monoxenic mice was observed as compared to bone marrow-derived macrophages. In contrast, bone marrow-derived macrophages demonstrated a higher ability to produce IL-6 and TNF but only 3 weeks after bacterial administration.

Immunoreactivity of endogenous digitalis-like substances in cord blood sera studied with antidigitoxin monoclonal antibodies.

The presence of digitoxin-like immunoreactive substances, whose nature is yet unknown, has been demonstrated in the umbilical cord blood. We selected six antidigitoxin monoclonal antibodies (MAb) having different specificity profiles concerning digitoxin analogs and steroid hormones. These antibodies were tested in a digitoxin radioimmunoassay (RIA). With the help of this technique, we measured the concentrations of apparent digitoxin in the cord blood drawn either at birth or in utero from mothers not undergoing any digitalis treatment. In the cord blood of newborns, the concentrations of apparent digitoxin, measured by the two MAbs that have the highest cross-reactions with dehydroepiandrosterone (DHEA) (123A23 and 145A41), were two or three times higher than with the other antibodies. In the fetal cord blood, where the concentration of DHEA is five to seven times lower than that observed at birth, these antibodies revealed a threefold lower concentration of apparent digitoxin than that observed in blood drawn at birth. Furthermore, MAbs that had similar specificities towards digitoxin analogs and steroids showed different measurements of digitoxin-like concentrations. These observations suggest that digitoxin-like immunoreactive compounds detected by the RIA may constitute a group of different molecules, one of which would be the DHEA.

Specific binding characteristics of high affinity monoclonal antidigitoxin antibodies.

The specificity of various monoclonal antidigitoxin antibodies was characterized using 6 cardiac glycoside analogs. Spleen cells from BALB/c mice, immunized with BSA- or KLH-digitoxin conjugates, were fused with NS1 myeloma cells, and antibody-producing hybrids were identified by radioimmunoassay. Twenty-one monoclonal antidigitoxin-specific antibodies were obtained, 10 of which were cloned and characterized for affinity and specificity. All the antibodies had a high affinity constant, ranging from 8.10(8) to 2.5.10(10) 1/M. On the basis of their binding specificities, the antibodies could be classified into 3 groups: the first contained 7 antibodies exhibiting high cross reactivity (42-100%) with digitoxigenin, whereas the second and third groups did not recognize this analog (cross-reactivity of 1%). In the former group, the absence of the sugar moiety only slightly affected the binding reaction, although for the two other groups, this structure did appear to be involved in antibody recognition. Changes in the functional groups of the hapten molecule led to considerable changes in the antibody-antigen reaction. For all the antibodies except one, saturation of the lactone ring considerably affected binding. These results demonstrated that monoclonal antibodies of different specificities with respect to both the steroid backbone and the sugar moiety of digitoxin can be induced using a digitoxin-protein conjugate.