Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

Inhibition of Endothelial Wound Repair by Dominant Negative Connexin Inhibitors

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-βGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ∼50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Inhibition of Wound-Induced Accumulation of Allene Oxide Synthase Transcripts in Flax Leaves by Aspirin and Salicylic Acid1

Allene oxide synthase (AOS) mediates the conversion of lipoxygenase-derived fatty acid hydroperoxides to unstable allene epoxides, which supply the precursors for the synthesis of the phytohormone jasmonic acid (JA). In this study the characterization of AOS gene expression in flax (Linum usitatissimum) is reported. AOS was constitutively expressed in different organs of flax plants. Additionally, AOS gene expression was enhanced after mechanical wounding in both the directly damaged leaves and in the systemic tissue located distal to the treated leaves. This wound-induced accumulation of AOS required the de novo biosynthesis of other unknown proteins involved in the signaling pathway modulating wound-induced AOS gene expression. Furthermore, the wound-induced AOS mRNA accumulation was correlated with the increase in the levels of JA. Both JA and its precursor, 12-oxo-phytodienoic acid, activated AOS gene expression in a dose-dependent manner. Thus, JA could activate its own biosynthetic pathway in flax leaves. Moreover, neither salicylic acid (SA) nor aspirin influenced AOS enzymatic activity. It is interesting that pretreatment with SA or aspirin inhibited wound-induced accumulation of AOS transcripts. These results suggest that a potent inhibition of JA biosynthetic capacity in leaves can be affected by SA or aspirin at the level of AOS gene expression.

Molecular Characterization of an Arabidopsis Gene Encoding Hydroperoxide Lyase, a Cytochrome P-450 That Is Wound Inducible1

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.

Wound Signaling in Tomato Plants1 : Evidence That ABA Is Not a Primary Signal for Defense Gene Activation

The effects of abscisic acid (ABA) on the accumulation of proteinase inhibitors I (Inh I) and II (Inh II) in young, excised tomato (Lycopersicon esculentum L.) plants were investigated. When supplied to excised plants through the cut stems, 100 μm ABA induced the activation of the ABA-responsive le4 gene. However, under the same conditions of assay, ABA at concentrations of up to 100 μm induced only low levels of proteinase-inhibitor proteins or mRNAs, compared with levels induced by systemin or jasmonic acid over the 24 h following treatment. In addition, ABA only weakly induced the accumulation of mRNAs of several other wound-response proteins. Assays of the ABA concentrations in leaves following wounding indicated that the ABA levels increased preferentially near the wound site, suggesting that ABA may have accumulated because of desiccation. The evidence suggests that ABA is not a component of the wound-inducible signal transduction pathway leading to defense gene activation but is likely involved in the general maintenance of a healthy plant physiology that facilitates a normal wound response.

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene.

On the mechanism of skin wound "contraction": a granulation tissue "knockout" with a normal phenotype.

This report explores the mechanism of spontaneous closure of full-thickness skin wounds. The domestic pig, often used as a human analogue for skin wound repair studies, closes these wounds with kinetics similar to those in the guinea pig (mobile skin), even though the porcine dermis on the back is thick and nearly immobile. In the domestic pig, as in the guinea pig, daily full-thickness excisions of the central granulation tissue up to but not including the wound edges in both back and flank wounds do not alter the rate or completeness of wound closure or the final pattern of the scar. A purse-string mechanism of closure was precluded by showing that surgical interruption of wound edge continuity does not alter closure kinetics or wound shape. We conclude that "tightness" of skin is not a key factor nor is the central granulation tissue required for normal wound closure. These data imply that in vitro models such as contraction of isolated granulation tissue or of the cell-populated collagen lattice may not be relevant for understanding the cell biology of in vivo wound closure. Implications for the mechanism for wound closure are discussed.

Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants.

Chemical and physical signals have been reported to mediate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Among the chemical signals, phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and the peptide systemin represent the best characterized systems. Furthermore, electrical and hydraulic mechanisms have also been postulated as putative Pin2-inducing systemic signals. Most of the chemical agents are able to induce Pin2 gene expression without any mechanical wounding. Thus, ABA, JA, and systemin initiate Pin2 mRNA accumulation in the directly treated leaves and in the nontreated leaves (systemic) that are located distal to the treated ones. ABA-deficient tomato and potato plants do not respond to wounding by accumulation of Pin2 mRNA, therefore providing a suitable model system for analysis of the signal transduction pathway involved in wound-induced gene activation. It was demonstrated that the site of action of JA is located downstream to the site of action of ABA. Moreover, systemin represents one of the initial steps in the signal transduction pathway regulating the wound response. Recently, it was reported that heat treatment and mechanical injury generate electrical signals, which propagate throughout the plant. These signals are capable of inducing Pin2 gene expression in the nontreated leaves of wounded plants. Furthermore, electrical current application to tomato leaves leads to an accumulation of Pin2 mRNA in local and systemic tissues. Examination of photosynthetic parameters (assimilation and transpiration rate) on several types of stimuli suggests that heat-induced Pin2 gene expression is regulated by an alternative pathway from that mediating the electrical current and mechanical wound response.

Estudio ATHAC 04-05: estudio observacional sobre el uso de apósitos neutros o impregnados en agente antibacteriano de una innovadora tecnología patentada: la tecnología lípido coloidal (TLC) para el tratamiento de heridas agudas y/o crónicas

Introducción: El estudio ATHAC recoge datos sobre heridas agudas y crónicas que son candidatas a un tratamiento a base de apósitos grasos neutros como la gama URGOTUL. Objetivos: Describir las características de las heridas, describir los tratamiento aplicados a estas heridas y explorar las opiniones de los profesionales de enfermería y los pacientes sobre los tratamientos en términos de aplicabilidad, adaptabilidad y confort para el paciente. Material y métodos: 1.500 pacientes fueron incluidos en el estudio de acuerdo al tipo de herida y a los tratamientos en uso. Se recogieron datos a partir de dos cuestionarios: uno para el paciente y otro para la enfermera responsable de sus cuidados. Las enfermeras recogieron los datos en el primer día de inclusión y los pacientes respondían al cuestionario 1 mes más tarde o antes si la herida había cicatrizado. Las variables recogidas por la enfermera fueron: datos sociodemográficos, etiología de las lesiones, características y localización de las heridas, aspectos y opiniones sobre el tratamiento. A los pacientes se les preguntó por la duración del tratamiento, el estado de la lesión en el momento de contestar y desde su punto de vista, así como su opinión sobre el dolor, satisfacción general y aceptabilidad. Se llevaron a cabo análisis descriptivos uni y bivariados. Para cada paciente, si tenía más de una lesión, se recogieron datos de la lesión de mayor tamaño. Resultados: Finalmente, se estudiaron 1.432 pacientes con una o más lesiones (420 tenían más de una lesión). El 60,4% eran mujeres y la edad media fue de 66 ± 19 años. En el caso de las heridas crónicas (657 lesiones) predominaron las úlceras venosas (47%) y las úlceras por presión (23%). En las heridas agudas (775 lesiones), la mayoría fueron traumáticas (41%) y quemaduras (32,5%). La principal localización en todas las lesiones fueron los miembros inferiores (57,4% en heridas crónicas y 39% en agudas). El 84,4% de los casos indicó presentar algún tipo de dolor previo al comienzo de este estudio. Al finalizar el estudio, un porcentaje menor del 20% indicó presentar algún tipo de dolor. El 72% de las heridas agudas y el 35% de las crónicas, como refieren los pacientes, había cicatrizado al finalizar el estudio (en un tiempo medio de entre 20-40 días). El 54% de las heridas crónicas evoluciona favorablemente y el 26% de las agudas. Más del 80% de las heridas estudiadas fueron tratadas con la gama URGOTUL. Conclusión: URGOTUL es una buena opción para el tratamiento de este tipo de heridas, especialmente para las heridas agudas, en relación a su carácter atraumático y a su capacidad de cicatrización, así como la buena aceptación y satisfacción de los pacientes.

El uso de apósitos hidrocelulares de la gama Allevyn® en heridas agudas: resultados a partir del estudio AURIGA-04 en Atención Primaria

Aunque los apósitos de cura en ambiente (CAH) húmedo se han utilizado predominantemente en heridas crónicas, ello no es óbice para que su uso en heridas agudas permita solucionar algunos problemas, como el conseguir un ambiente óptimo que facilite la migración epitelial, así como una adecuada protección de las heridas y una correcta gestión del exudado. Es por ello que, dentro del marco del estudio AURIGA-04, nos planteamos la realización de un estudio prospectivo observacional, abierto y multicéntrico, de medidas repetidas en una cohorte de pacientes que presentan heridas agudas de diversa etiología en el que se incluyeron pacientes con heridas traumáticas, quirúrgicas o quemaduras tratados por profesionales de Atención Primaria, con el objetivo de generar evidencias acerca de la utilización de apósitos de CAH, en concreto, de la gama de apósitos hidrocelulares Allevyn®, en el tratamiento de heridas agudas. Se consideraron como criterios de exclusión heridas con signos clínicos de infección. Solo se incluyó una lesión por paciente. En el caso de los pacientes con heridas agudas, la muestra a estudio quedó compuesta por 61 pacientes con una edad media de 71,1 años; 36 casos corresponden a mujeres (60%). El estado general de salud de la muestra era bueno en un 49,1% de los casos y prácticamente la totalidad de los pacientes presentaba pluripatología. Un 10% de los pacientes consumía fármacos que podían interferir en la cicatrización y un 6% presentaba malnutrición. El 67,2% de las lesiones eran heridas traumáticas, el 24,6% quirúrgicas y un 8,2% quemaduras. Un 37% de las lesiones fueron clasificadas como superficiales y el 63% restante como profundas. Presentaban 64 días de evolución previa a su inclusión en el estudio y una superficie media de 23,34 cm2. Los pacientes permanecieron en el estudio un promedio de 43,6 días, con una cadencia de cambios de apósito cada 2,7 días. Durante el estudio cicatrizaron el 63,9% de las heridas, en un promedio de 40,9 días. La superficie inicial de las lesiones mostró una reducción significativa a lo largo del seguimiento (inicial: 23,34 cm2, final: 4,27 cm2, p ≤ 0,001). En los modelos de regresión de Cox, sólo resultó estadísticamente significativa la edad del paciente, en el grupo de lesiones traumáticas (RR: –0,03, p= 0,039). Durante el período de seguimiento, un 80,3% de los pacientes fue tratado en combinación con Intrasite Conformable®, Iruxol Mono® o la combinación de ambos productos. Los profesionales que realizaron las curas con Allevyn® valoraron muy favorablemente su manejo durante todo el estudio; así, en más del 96% de los cambios se consideró fácil de colocar, en el 100% indoloro y también en el 100% de fácil retirada. A la vista de los resultados, los apósitos de CAH y, en concreto, la gama de apósitos Allevyn®, tienen una clara aplicación en el caso de las heridas agudas, permitiendo un óptimo abordaje local de las lesiones, proporcionando unos niveles óptimos de humedad, pH y temperatura para las células implicadas en el proceso de cicatrización. Así, se promueve la formación de tejido de granulación y facilita la epitelización, además de proteger la herida ante traumatismos y gérmenes.

A Wormian Bone, Mimicking an Entry Gunshot Wound of the Skull, in an Anthropological Specimen

Wormian bones (WB) are irregular small cranial ossicles found along suture lines and fontanels. In Brazil, gunshot wounds to the skull are quite common in young individuals. Nevertheless, as far as we know, this is the first report of a WB giving an erroneous aspect of gunshot entrance due to its displacement position. The present manuscript describes the case of a Brazilian young man who died due to ballistic trauma, where a gaping bony defect on the right side of the skull was thought to be the exit wound of an injury related to the destruction found on the left side, highly suggestive of firearm injury. Thus, this case study has brought to light similarities between a traumatic lesion and an orifice of a WB, with emphasis on differential diagnosis during routine anthropological examinations.