The evaluation of subatmospheric pressure and hyperbaric oxygen in ischemic full-thickness wound healing.

We evaluated the efficacy of subatmospheric pressure and hyperbaric oxygen (HBO) as adjuncts in the treatment of hypoxic full-thickness wounds in a rabbit model. We hypothesized that subatmospheric pressure and HBO independently are effective in improving wound healing in the ischemic wound model and that when they are used in combination there is an increased positive effect on wound healing. Using a standard ischemic wound model four full-thickness wounds were created on each ear of 41 male New Zealand white rabbits (N = 82 ears). On each rabbit one ear was dressed with the vacuum-assisted closure (VAC) device and connected to suction; the other was dressed identically without the suction and suction tubing. Twenty rabbits were treated with HBO daily for 10 days at 2.0 atmospheres absolute for 90 minutes plus descent and ascent times. Necropsy on all rabbits was performed on postoperative day 10. Four ischemic wound treatment groups were evaluated: Group 1 (N = 21) VAC dressing alone; Group 2 (N = 20) VAC dressing plus HBO; Group 3 (N = 21) VAC dressing to suction alone; and Group 4 (N = 20) VAC dressing to suction and HBO. Using light microscopy a veterinary pathologist blinded to treatment groups quantified peak granulation tissue, granulation tissue gap, and epithelialization tissue gap. Data were analyzed by analysis of variance with significance indicated by P < 0.05. Statistical significance was found in a comparison of VAC dressing to suction and VAC dressing alone for peak granulation tissue and granulation tissue gap both with and without use of HBO. VAC device use appears to increase the rate of healing in a rabbit ischemic wound model. HBO therapy did not significantly affect the rate of healing in this model.

The use of controlled subatmospheric pressure to promote wound healing in preparation for split-thickness skin grafting in a fourth degree burn.

Subatmospheric pressure application to acute and chronic wounds has been shown to increase local wound blood flow, increase the rate of formation of granulation tissue, and enhance bacterial clearance. The mechanical forces applied to the wound enhance the rate of granulation tissue formation by the increase intracellular messengers regulating protein production and turnover. This method of wound care is particularly useful for larger wounds that could not be readily closed by local methods, but may also be useful for chronic wounds in debilitated patients who may not be ideal surgical candidates. Further basic scientific research is needed to discern the exact mechanisms of action of subatmospheric pressure, and more clinical experience is needed to establish guidelines for its application. Further clinical studies with larger subject populations in a randomized prospective study would lend support to the utility of this wound management protocol.

Posttraumatic carotid cavernous fistula: frequency analysis of signs, symptoms, and disability outcomes after angiographic embolization.

OBJECTIVE: To increase awareness and understanding of posttraumatic carotid cavernous fistula (PTCCF) with the intent to expedite diagnosis and treatment of this disabling injury, a 14-year retrospective review of patients with angiographically identified PTCCF was conducted at this Level I trauma center. A frequency analysis of signs, symptoms, and disability was performed. The impact on disability of demographics, number of embolization attempts required for closure of the PTCCF, and time from injury to diagnosis was assessed by t test for independent samples. RESULTS: Nine patients were diagnosed with 10 PTCCFs. Mean patient age was 41.5 years. All patients with PTCCF had basilar skull fracture, loss of consciousness, bruit, and chemosis; 90% had exophthalmos; 70% had visual changes; 50% complained of headache; and 80% had some lasting disability. Mean age of patients with partial to total disability was 47 years, while the mean age of patients without lasting disability was 19.5 years (p = 0.013). No statistical correlation could be found between disability and sex, blunt versus penetrating injury, days to diagnosis, or number of embolization attempts. CONCLUSION: Patients sustaining head trauma with basilar skull fractures and presenting with the described signs and symptoms should be evaluated for PTCCF. Risk of disability does not appear to be influenced by number of attempts at embolization or time to diagnosis. However, age may have a significant impact on outcome.

Mucosal immunogenicity of polysaccharides conjugated to a peptide or multiple-antigen peptide containing T- and B-cell epitopes.

In this study we investigated the mucosal and systemic responses to two T-cell-independent polysaccharides, a serogroup f polysaccharide (formed of rhamnose glucose polymers [RGPs]) from Streptococcus mutans OMZ 175 and a mannan from Saccharomyces cerevisiae, covalently conjugated either to a linear peptide (peptide 3) or to a multiple-antigen peptide (MAP), both derived from S. mutans protein SR, an adhesin of the I/II protein antigen family of oral streptococci. Peptide 3 and MAP, which contained at least one B- and one T-cell epitope, were tested as carriers for the polysaccharides and as protective immunogens. Intragastric intubation of rats with the conjugates (RGPs-peptide 3, RGPs-MAP, mannan-peptide 3, and mannan-MAP) associated with liposomes produced salivary immunoglobulin A (IgA) antibodies which reacted with RGPs or mannan, peptide 3 or MAP, protein SR, and S. mutans or S. cerevisiae cells. Administration of conjugate boosters to the animals showed that both carriers conjugated to the polysaccharides were able to induce, in immunized animals, a salivary antipolysaccharide IgA memory. In contrast, animals primed and challenged with unconjugated polysaccharide showed no anamnestic response. Rats orally immunized with the conjugates also developed systemic primary antipolysaccharide and antipeptide IgM antibody responses which were characterized by a switch from IgM to IgG during the course of the secondary response. Data presented here demonstrated that both peptide 3 and the MAP construct can act as good carriers for orally administered polysaccharides. Unexpectedly, the use of a MAP did not further improve the immunogenicity of polysaccharides at the mucosal level; nevertheless, such a construct should be of great interest in overcoming the problem of genetic restriction induced by linear peptides.

Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-alpha release.

The present work was initiated to define mechanisms that account for the binding on human monocytes of streptococcal cell wall polysaccharides formed by rhamnose glucose polymers (RGPs), and subsequent stimulatory activities. We show here that RGPs bind to and stimulate human monocytes to produce TNF-alpha in a dose-dependent manner. To detect cell surface RGPs binding proteins, intact monocytes were biotinylated before lysis with Nonidet P-40 and solubilized proteins were incubated with RGPs Affi-Prep beads. One major membrane protein of 55 kDa was specifically detected and identified as CD14 because it reacted with anti-CD14 mAbs. Furthermore, anti-CD14 mAbs were able to perform a dose-dependent inhibition of RGPs binding, and suppressed TNF-alpha release from RGPs-stimulated monocytes. Moreover, we demonstrated that RGPs also bind to CD11b; however, this binding is not implicated in synthesis of TNF-alpha. Interestingly, RGPs binding to monocytes was enhanced by human normal serum (HNS) whereas HNS inhibits the TNF-alpha-stimulating activity of RGPs. Western blotting analysis of HNS proteins purified on RGPs Affi-prep beads revealed three specific bands of 75, 55, and 32 kDa reactive with anti-C3 Abs, anti-CD14 mAbs (TUK4), and anti-human mannan binding protein (hMBP)-derived peptide IgG, respectively. These results suggest that C3, soluble CD14, and hMBP form complexes that are probably active in enhancing the binding of RGPs to monocytes. Additional studies have shown that hMBP that recognizes RGPs prevents, unlike the LPS binding protein, TNF-alpha release by inhibiting the binding of RGPs to CD14 Ag. By incubating cells with a constant amount of RGPs-hMBP complexes in the presence or absence of increasing concentrations of C1q, we also demonstrated that C1q receptor mediates the binding and probably the uptake of RGPs-hMBP complexes by human monocytes.

Immunogenicity of polysaccharides conjugated to peptides containing T- and B-cell epitopes.

To develop a general model of polysaccharide-peptide vaccine, we have investigated the efficiency of linear peptides derived from protein SR, and adhesin of the I/II protein antigen family of oral streptococci, to act as carriers for two T cell-independent polysaccharides: serogroup f polysaccharide from Streptococcus mutans OMZ 175 (poly f) and Saccharomyces cerevisiae mannan. Peptide 3 (YEKEPTPPTRTPDQ) and peptide 6 (TPEDPTDPTDPQDPSS), accessible on the native SR protein as demonstrated by their reactivity in enzyme-linked immunosorbent assays with rat antisera raised against protein SR, correspond to immunodominant regions of SR. Peptide 3 contains at least one B- and one T-cell epitope, as demonstrated by its ability to induce peptide- and SR-specific antibody responses without any carrier and to stimulate the proliferation of rat lymph node cells primed either with free peptide or native SR, whereas peptide 6 contains only B-cell epitope(s). Peptide 3 was then covalently coupled though reductive amination to either poly f or mannan, and peptide 6 was coupled to poly f. Subcutaneous immunizations of rats with poly f-peptide 3 or mannan-peptide 3 conjugates produced a systemic immunoglobulin M (IgM) and IgG antibody response, and the elicited antibodies reacted with free poly f or mannan, peptide 3, protein SR, and S. mutans or S. cerevisiae whole cells. Rats immunized with poly f-peptide 6 did not develop any antipeptide or anti-SR response. Furthermore, a booster immunization of animals with poly f-peptide 3 or mannan-peptide 3 conjugates induced high titers of anti-peptide 3, anti-poly f, and antimannan antibodies, which occurred quickly. The response is anamnestic for the peptide and the polysaccharides and is characterized by an Ig switch from IgM to IgG. The data presented here confirm that the presence of B- and T-cell epitopes is necessary to induce an anamnestic antipeptide response and that a peptide containing relevant B- and T-cell epitopes can act as a good carrier in improving an antipolysaccharide anamnestic immune response.

Augmentation of the failed pharyngeal flap.

Pharyngeal flaps are frequently used with good results to eliminate hypernasality and/or nasal escape. In a small percentage of patients, however, cicatricial contracture of the flap occurs to such a degree that velopharyngeal incompetence returns. The authors have devised a method of augmenting the scarred flap with small, superiorly based flaps lateral and adjacent to the original flap. Four cases are presented in which speech analysis was conducted prior to the procedure and then for 9 to 24 months postoperatively. The postoperative speech results are all within normal limits.

Epitope mapping of Streptococcus mutans SR protein and human IgG cross-reactive determinants, by using recombinant proteins and synthetic peptides.

alpha-Hemolytic oral streptococci are known to possess a family of cell surface cross-reactive proteins termed Ag I/II, having a molecular mass of approximately 180 to 210 kDa. These proteins are implicated in bacterial adherence to various oral tissues, and we showed recently that the SR protein, an I/II Ag-related protein, from Streptococcus mutans OMZ 175 serogroup f possesses Ag mimicry with human IgG. In this study, regions of the SR protein encoding the cross-reactive epitope(s) were analyzed by expressing selected restriction fragments from the cloned sr gene. The three SR-derived polypeptides reacted in ELISA with anti-SR rabbit IgG, whereas only the two polypeptides located along the carboxyl-terminal two thirds of the SR protein reacted with anti-human IgG rabbit IgG. In order to locate more precisely the human IgG-cross-reactive region, we synthesized six peptides, on the basis of the recently determined complete nucleotide sequence of the sr gene. Among these peptides, peptide 2, corresponding to the alanine-rich repeating amino-terminal region, peptide 3, located in the three tandem proline-rich regions, and peptide 6, located near the cell wall-spanning region, were the most interesting in term of antigenicity and immunogenicity. Anti-peptide 2, 3, and 6 rabbit IgG reacted with free SR and with cell wall-associated SR. Peptide 1, located near the amino terminus, was poorly immunogenic. Peptides 4 and 5, located in the putative human IgG-cross-reactive region, were immunogenic; however, anti-peptide 4 rabbit IgG reacted only weakly with SR or human IgG, whereas anti-peptide 5 rabbit IgG reacted strongly with SR and human IgG, and peptide 5 was recognized by anti-SR and anti-human IgG rabbit IgG. These results confirm the cell surface accessibility of this epitope and its potential participation in eliciting, in rabbits, anti-SR IgG cross-reactive with human IgG.

Optimal detergent activation of rat liver microsomal UDP-glucuronosyl transferases toward morphine and 1-naphthol: contribution to induction and latency studies.

The detergent-activation profiles of UDP-glucuronosyl transferases (UGTs, EC 2.4.1.17) toward 1-naphthol and toward morphine have been determined: three non-ionic detergents, Triton X-100, Brij 58 and Lubrol Px and one zwitterion detergent, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonic acid (CHAPS) were studied. The results showed that marked inhibition of 1-naphthol-UGT and morphine UGT activities occurred with high concentrations of Triton X-100. Lubrol Px, at high concentrations, inhibited 1-naphthol-UGT but not morphine-UGT. It appeared that the detergent/protein ratio suitable for optimal activation of both isoenzymes was limited to 0.2 for these detergents. In contrast, Brij 58 and CHAPS displayed optimal activation of the two enzymes for a large range of detergent/microsomal protein ratios (respectively from 0.2 to 1 and from 0.4 to 1), making them the most suitable for induction and/or latency studies of both isoenzymes. The influence of maximal activation status on the effect of 3-methylcholanthrene and phenobarbital treatment on morphine-UGT and 1-naphthol-UGT activity has also been evaluated. The findings provided evidence that detergent-activation profiles and optimal detergent-activated versus "native" UGT activity determination give crucial informations about the characteristics of a given isoenzymic form of UGT, i.e. its sensitivity to specific alterations of the phospholipid environment, its latency and its inducibility.