Dysregulation of the actin scavenging system and inhibition of DNase activity following severe thermal injury.

BACKGROUND: Circulating cell-free DNA (cfDNA) is not found in healthy subjects, but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The hypothesis was that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA following thermal injury. METHODS: Patients with severe burns covering at least 15 per cent of total body surface area were recruited to a prospective cohort study within 24 h of injury. Blood samples were collected from the day of injury for 12 months. RESULTS: Analysis of blood samples from 64 patients revealed a significant reduction in DNase activity on days 1-28 after injury, compared with healthy controls. DNase protein levels were not affected, suggesting the presence of an enzyme inhibitor. Further analysis revealed that actin (an inhibitor of DNase) was present in serum samples from patients but not those from controls, and concentrations of the actin scavenging proteins gelsolin and vitamin D-binding protein were significantly reduced after burn injury. In a pilot study of ten military patients with polytrauma, administration of blood products resulted in an increase in DNase activity and gelsolin levels. CONCLUSION: The results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury by which high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA-induced host tissue damage and thrombosis.

ANTECEDENTES: El ADN libre de las células circulantes (circulating cell-free DNA, cfDNA) no se encuentra en sujetos sanos, pero se detecta fácilmente después de una lesión térmica y puede contribuir al riesgo de fallo multiorgánico. La hipótesis fue que una disminución en la actividad de la proteína/enzima ADNasa tras la lesión térmica podría contribuir a la elevación del cfDNA que ocurre tras la misma. MÉTODOS: Los pacientes con quemaduras graves con una extensión ≥ 15% del área de superficie corporal total (total body surface area, TBSA) se incluyeron en un estudio prospectivo de cohortes durante las primeras 24 horas posteriores a la lesión. Se recogieron muestras de sangre desde el día de la lesión hasta los 12 meses posteriores a la misma. RESULTADOS: El análisis de muestras de sangre de 64 pacientes reveló una reducción significativa de la actividad de la ADNasa en los días 1 a 28 después de la lesión, en comparación con los controles sanos. Los niveles de proteína ADNasa no se vieron afectados, lo que sugiere la presencia de un inhibidor enzimático. Un análisis adicional reveló que la actina (un inhibidor de la ADNasa) estaba presente en las muestras de suero de los pacientes, pero no en los controles, y las concentraciones de la gelsolina, proteína que causa la disociación de la actina, y la proteína de unión a la vitamina D se redujeron significativamente después de la lesión térmica. En un estudio piloto de 10 pacientes con politrauma por lesiones militares, la administración de hemoderivados produjo un aumento en la actividad de la ADNasa y de los niveles de gelsolina. CONCLUSIÓN: Este estudio sugiere un nuevo mecanismo biológico para la acumulación de cfDNA después de una lesión térmica, por el cual los altos niveles de actina liberada por el tejido dañado causarían una reducción en la actividad de la ADNasa. La restauración del sistema eliminador de actina podría, por lo tanto, restaurar la actividad de la ADNasa y reducir el riesgo de daño tisular y trombosis en el huésped inducido por el cfDNA.

Deployed research.

The Role 3 Medical Treatment Facility (Field Hospital) in Camp Bastion (R3 Bastion) is acknowledged to be one of the busiest dedicated trauma facilities in the world. Casualties typically present with severe injuries and in physiological extremis. These casualties form a unique cohort representing the most relevant population to evaluate the effectiveness of treating battlefield injuries as academic clinicians and scientists interested in trauma seek to improve outcomes for such patients in the future. This article describes four separate but related research projects that have been undertaken in Camp Bastion, Afghanistan, over the last year. They traverse the spectrum of clinical research, ranging from data collection to a randomised control trial. The aim is to discuss some of the problems encountered and the solutions that made it possible to undertake research in a theatre of operations, thereby providing a starting point for others who may wish to initiate research in a similar environment.

Isolation and identification of angiotensin-like peptides from the plasma of the snake Bothrops jararaca.

Two distinct hypertensive peptides were purified and characterized from Bothrops jararaca (Bj) plasma incubated at pH 4, 37 degrees C, 24 hr. These peptides were active on rat and Bj blood pressure, on rat isolated uterus, on guinea pig isolated ileum and on Bj isolated duodenum. At the releasing conditions no further activities were found for kininases, angiotensinases or angiotensin converting enzymes. The peptides were purified by ethanol/ether extraction, Sephadex G-25 gel filtration, semipreparative reverse-phase (C-18) HPLC and analytical (C-18) HPLC. The amino-acid sequences of the purified peptides corresponded to (Ile5)AII and (Val5-Tyr9)AI and their molecular masses were confirmed by mass spectrometry as 1046.6 and 1348.0 respectively. The presence of those two angiotensins on Bj plasma may have some evolutionary significance since (Ile5)AII is known as a mammalian angiotensin and (Val5)AII as a non-mammalian one.

Theta waves in the Papez circuit during alertness and dreaming in the rat.

An electro-oscillographic study of the hippocampus, thalamic anterior nuclei and cingulate and pericingulate cortical areas was performed in the rat during the wakefulness-sleep cycle. High voltage (usually from 100 to 200 microV) theta waves, oscillating at regular frequencies from 6 to 10 Hz, were found to occur simultaneously in all of these components of the Papez circuit during attentive behavior and dreaming.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells.

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 +/- 1.9 mV) compared to NWR (-69 +/- 1.4 mV), NWR 1K-1C (-60.8 +/- 1.6 mV), WKY (-67.1 +/- 3.2 mV) and WKY 1K-1C (-56.9 +/- 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 +/- 0.86) when compared to arteries of all the other groups (NWR 0.93 +/- 0.65, NWR 1K-1C 1.24 +/- 0.45, WKY 1.22 +/- 0.32, WKY 1K-1C 1.15 +/- 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

Role of smooth muscle cell membrane potential in neointima formation in arteries of spontaneously hypertensive rats.

Based on observations that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) have altered resting potentials as well as abnormal cell proliferation rates, neointima formation after controlled balloon injury was compared in arteries from SHR and Wistar Kyoto rats (WKY). SHR aortic VSMC showed hyperpolarized resting membrane potentials (-93+/-8 mV) when compared to those from WKY (-61+/-6 mV). Histomorphometric analysis of cross sections from aortic segments submitted to balloon injury showed reduced neointima formation in SHR (neointima/media ratio: 0.04+/-0.03) as compared to WKY (0.2+/-0.1). On the other hand, in injured carotid arteries, neointima formation was more extensive in SHR (neointima/media ratio 5.0+/-0.9) than in WKY (0.8+/-0.7), leading in most cases to luminal occlusion. Measurements of VSMC resting potential showed that carotid artery cells from SHR were depolarized with respect to those from WKY (-46+/-4 vs. -69+/-5 mV, respectively). The results demonstrate an inverse relationship between VSMC membrane polarization and neointima formation in SHR arteries, suggesting that genetic modifications in SHR determine a dysfunctional cellular physiology that may influence cell proliferation subsequent to injury.

Oestrous cycle and pregnancy alter the reactivity of the rat uterine vasculature.

Isolated uterine vascular beds from virgin and pregnant rats were used to assess vascular reactivity and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. One uterine horn from female rats in each oestrous cycle day and gestation day 17 was removed and perfused with physiological saline solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to acetylcholine (ACh) generated. Responsiveness to ACh was tested in the presence of N-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit NO synthase, cyclo-oxygenase and K+ channels respectively. Cirazoline-induced tone was smaller in the pregnant compared with the proestrous group. Sensitivity to ACh was cycle day and pregnancy dependent with pregnant > dioestrous day-1 > dioestrous day-2 > proestrous and oestrous. L-NA shifted the curve to the right in all groups except dioestrous day-1. IBU inhibited the ACh response in the pregnant group only. TBA virtually abolished the response in all groups. These results suggest that in the uterine vascular bed from pregnant rats, EDHF, along with NO and a dilator prostanoid mediate ACh-induced dilatation. In contrast, in the dioestrous day-1 group, only EDHF seems to be released by ACh in this vascular bed. In the oestrous, dioestrous day-2 and proestrous groups, ACh releases both EDHF and NO.

Influence of oestrous cycle and pregnancy on the reactivity of the rat mesenteric vascular bed.

In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an alpha(1)-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to ACh generated. Responsiveness to ACh was tested in the presence of N(omega)-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo-oxygenase and K(+) channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 + or - 1.9 mV) compared to NWR (-69 + or - 1.4 mV), NWR 1K-1C (-60.8 + or - 1.6 mV), WKY (-67.1 + or - 3.2 mV) and WKY 1K-1C (-56.9 + or - 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 + or - 0.86) when compared to arteries of all the other groups (NWR 0.93 + or - 0.65, NWR 1K-1C 1.24 + or - 0.45, WKY 1.22 + or - 0.32, WKY 1K-1C 1.15 + or - 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state

Theta waves in the papez circuit during alertness and dreaming in the rat

An electro-oscillographic study of the hippocampus, thalamic anterior nuclei and cingulate and pericingulate cortical areas was performed in the rat during the wakefulness-sleep cycle. High voltage (usually from 100 to 200 uV) theta waves, oscvillating at regular frequencies from 6 to 10 Hz, were found to occur simultaneously in all of these components of the Papez circuit during attentive behavior and dreaming