A cytoplasmic component of pyridine nucleotide fluorescence in rat diaphragm: evidence from comparisons with flavoprotein fluorescence

Pyridine nucleotide (PN) and flavoprotein (Fp) fluorescence were monitored in the isolated intact rat diaphragm. A substantial increase in PN fluorescence occurred when N₂ replaced O₂ in glucose medium. This response was much reduced in pyruvate medium and/or by pretreatment with iodoacetic acid (IAA). The anaerobic levels of Fp fluorescence were less affected by substrate and IAA. Substitution of glucose by pyruvate did not alter the PN fluorescence of the resting aerobic tissue, but increased Fp fluorescence. After a tetanus with glucose present the PN of the anaerobic muscle, but not the Fp underwent a substantial transient oxidation. This oxidation was absent in pyruvate medium. It is concluded that a cytoplasmic component of the PN fluorescence is present in skeletal muscle. The levels of Fp fluorescence in the resting and contracting aerobic tissue supplied with pyruvate suggest that the resting tissue respiration was ADP limited. On this basis the level of PN fluorescence in the aerobic resting state was less than expected; the source of the PN fluorescence was both mitochondrial and cytoplasmic.     

Retroviral gene therapy of collagen-induced arthritis by local delivery of IL-4

Rheumatoid arthritis (RA) is an autoimmune arthritis, for which treatment options remain limited. This study investigated the potential role of adoptive cellular gene therapy as a novel means for treating the RA animal model collagen-induced arthritis (CIA). Adoptive transfer of antigen-specific T-cell hybridomas retrovirally transduced to express IL-4 1 day before booster immunization significantly reduced the number of inflamed joints. Cell transfer after clinical onset of disease had no therapeutic effect. Bioluminescence imaging showed that the hybridomas migrated to the inflamed joints, thus delivering the regulatory protein locally at the site of inflammation. The homing was, at least in part, due to chemotaxis in response to proinflammatory chemokines that are expressed in inflamed joints. There were no significant changes in the cytokine milieu of the draining lymph nodes, nor in the systemic levels of anti-collagen antibodies in treated mice. We conclude that the beneficial clinical effects observed in our model were most likely based on the local action(s) of IL-4 in the inflamed joints and that the local delivery (and effects) of regulatory cytokines, like IL-4, constitutes a novel and effective method of preventing organ-specific autoimmune disease and of minimizing systemic adverse effects.     

Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance.

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS-PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (K(d)(app)) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (k(off)=1.2 x 10(-2)s(-1)) and the association rate constant (k(on)=3.5 x 10(6)M(-1)s(-1)).     

Animal and human dentin microstructure and elemental composition

Animal teeth are a common model in studies on dentin adhesive materials. The aim of this study was to compare microstructural parameters (density and diameter of dentinal tubules (DT), peritubular dentin (PTD) thickness, PTD and intertubular dentin (ITD) surface area) and chemical characteristics of canine, porcine, equine, and human root dentin. The middle layers of dentin were harvested just below a cemento-enamel junction from incisors and investigated by means of scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDXS). SEM evaluation of the specimens revealed, that porcine dentin shared most similarities with human dentin. When comparing the density of DTs, canine dentin was also found to be similar to human dentin. Elemental composition of the root dentin did not differ significantly in porcine, equine and human dentin, but in canine dentin higher magnesium value in PTD compared to ITD was found. It is known that microstructural and chemical characteristics affect the strength of the adhesive bonds created among restorative materials and dentin. According to the results of this study, porcine dentin seems to be the most appropriate model to study dental materials to be used in human restorative dentistry.     

Aerosolized clindamycin is superior to aerosolized dexamethasone or clindamycin-dexamethasone combination in the treatment of severe Porphyromonas gingivalis aspiration pneumonia in an experimental murine model

Adjunctive corticosteroid treatment to reduce excessive local inflammatory response in pneumonia is controversial. To study the effects of an early local adjunct dexamethasone treatment on the course of pneumonia and inflammatory/cytokine response, mice were intratracheally inoculated with live Porphyromonas gingivalis and treated with either clindamycin (C), dexamethasone (D), C+D combination, or were not treated (Pg). Six mice from each group were euthanized at 6, 24, 72, and 168 hours after inoculation. Levels of tumor necrosis factor (TNF)-α, soluble TNF-α receptors (sTNFR1 and sTNFR2), interleukin (IL)-1β, and IL-6 in the serum and lung-homogenate supernatant were determined. Lung samples were histopathologically assessed and all findings compared to those found in 24 sham-inoculated mice (phosphate-buffered saline [PBS]). Severe P. gingivalis-induced bronchopneumonia progressed from 24 hours, peaked at 72 hours, and resolved after 168 hours with changes in local and systemic cytokine levels. Clindamycin-treated mice developed only mild bronchopneumonia that resolved fast (72 hours) with an early (6-24 hours) normalization of local and systemic cytokine levels. Similar course of pneumonia and cytokine level changes were observed in mice treated with C+D, but later. Early (6-24 hours) local elevation of sTNFRs was observed in C and C+D groups of mice, whereas nontreated (Pg) mice had increased systemic sTNFRs. Severe bronchopneumonia with delayed resolution was observed in D-group mice, with an early local and systemic decrease in sTNFR1 and persistent elevation of local TNF-α. Clindamycin or a clindamycin-dexamethasone combination treatment significantly improves the course of P. gingivalis-aspiration pneumonia, but more so if clindamycin alone is used. A favorable course of pneumonia seems to be associated with an early elevation of sTNFRs and normalization of TNF-α.     

Quadriceps keystone island flap for radical inguinal lymphadenectomy: a reliable locoregional island flap for large groin defects

Background: Radical inguinal lymphadenectomy (RIL) for bulky metastatic melanoma and non‐melanoma skin cancers of the inguinal region, while shown to improve morbidity and survival oncologically, can result in substantial morbidity from wound complications. Skin defects cannot be closed primarily and the substantial dead space predisposes to seroma, wound dehiscence and infection. Despite the clear need for reconstructive options, extended series describing reconstruction of large inguinal defects in this setting have not been reported. Methods: A prospectively entered, retrospectively reviewed study of 20 consecutive patients undergoing quadriceps keystone island flaps (QKIF) for the closure of complicated inguinal defects is described. Results: There was 100% flap survival, with no partial or complete flap losses. A reduction in wound breakdown/dehiscence from reported rates was seen, with four patients (20%) having wound breakdown, compared to double that rate in reported series. Other wound complications comprised six patients (30%) with mild wound infections, seven patients (35%) with seromas and two patients (10%) with haematomas. Conclusion: The QKIF is an effective means of reconstructing inguinal defects after RIL, particularly in high‐risk patients, and is technically simpler than other reconstructive techniques advocated for this purpose. Furthermore, the QKIF offers patients with advanced disease (where management is primarily palliative) a potentially improved quality of life with reduced operative morbidity.