Deleterious effects of plasma-derived cellular debris in a porcine model of hemorrhagic shock.

BACKGROUND: Recent studies identify large quantities of inflammatory cellular debris within Fresh Frozen Plasma (FFP). As FFP is a mainstay of hemorrhagic shock resuscitation, we used a porcine model of hemorrhagic shock and ischemia/reperfusion to investigate the inflammatory potential of plasma-derived cellular debris administered during resuscitation. METHODS: The porcine model of hemorrhagic shock included laparotomy with 35 % hemorrhage (Hem), 45 min of ischemia from supraceliac aortic occlusion with subsequent clamp release (IR), followed by protocolized resuscitation for 6 h. Cellular debris (Debris) was added to the resuscitation phase in three groups. The four groups consisted of Hem + IR (n = 4), Hem + IR + Debris (n = 3), Hem + Debris (n = 3), and IR + Debris (n = 3). A battery of laboratory, physiologic, cytokine, and outcome data were compared between groups. RESULTS: As expected, the Hem + IR group showed severe time dependent decrements in organ function and physiologic parameters. All animals that included both IR and Debris (Hem + IR + Debris or IR + Debris) died prior to the six-hour end point, while all animals in the Hem + IR and Hem + Debris survived. Cytokines measured at 30-60 min after initiation of resuscitation revealed significant differences in IL-18 and IL-1ß between all groups. CONCLUSIONS: Ischemia and reperfusion appear to prime the immune system to the deleterious effects of plasma-derived cellular debris. In the presence of ischemia and reperfusion, this model showed the equivalency of 100 % lethality when resuscitation included quantities of cellular debris at levels routinely administered to trauma patients during transfusion of FFP. A deeper understanding of the immunobiology of FFP-derived cellular debris is critical to optimize resuscitation for hemorrhagic shock.

Cryoablation for Chest Wall Trauma: A Brief Report.

Respiratory failure secondary to rib fractures is a major source of morbidity and mortality in trauma patients, particularly in older populations. Management of pain in these patients is complex due to the nature of the injuries. We present 3 patients who underwent a video-assisted thoracoscopic cryoablation of intercostal nerves for pain control after chest trauma. None of the patients developed post-operative complications related to poor respiratory status such as pneumonia or atelectasis. At one-month clinic follow-up, all patients reported no chest pain and were not using opiate analgesics. In patients for whom there is a contraindication to rib fixation in the setting of unstable rib fractures, cryoablation may be a method by which to improve respiratory status and decrease ventilator dependency due to pain. Cryoablation of intercostal nerves may provide a more durable and clinically feasible solution to aid in the healing process of these patients.

The Relationship between Serum Sodium, Serum Osmolality, and Intracranial Pressure in Patients with Traumatic Brain Injury Treated with Hyperosmolar Therapy.

BACKGROUND: Treatment of elevated intracranial pressure (ICP) in traumatic brain injury (TBI) is controversial. Hyperosmolar therapy is used to prevent cerebral edema in these patients. Many intensivists measure direct correlates of these agents-serum sodium and osmolality. We seek to provide context on the utility of using these measures to estimate ICP in TBI patients. MATERIALS AND METHODS: Patients admitted with TBI who required ICP monitoring from 2008 to 2012 were included. Intracranial pressure, serum sodium, and serum osmolality were assessed prior to hyperosmotic therapy then at 6, 12, 18, 24, 48, and 72 hours after admission. A linear regression was performed on sodium, osmolality, and ICP at baseline and serum sodium and osmolality that corresponded with ICP for 6-72-hour time points. RESULTS: 136 patients were identified. Patients with initial measures were included in the baseline analysis (n = 29). Patients who underwent a craniectomy were excluded from the 6-72-hour analysis (n = 53). Initial ICP and serum sodium were not significantly correlated (R2 .00367, P = .696). Initial ICP and serum osmolality were not significantly correlated (R2 .00734, P = .665). Intracranial pressure and serum sodium 6-72 hours after presentation were poorly correlated (R2 .104, P < .0001), as were ICP and serum osmolality at 6-72 hours after presentation (R2 .116, P < .0001). DISCUSSION: Our results indicate initial ICP is not correlated with serum sodium or osmolality suggesting these are not useful initial clinical markers for ICP estimation. The association between ICP and serum sodium and osmolality after hyperosmolar therapy was poor, thus may not be useful as surrogates for direct ICP measurements.

Long-Pulsed Neodymium-Doped Yttrium Aluminum Garnet Laser for Glomuvenous Malformations in Adolescents.

Currently there exist few reported cases where lasers are used successfully to treat glomuvenous malformations in adolescents. In the two cases described here, we provide evidence that the long-pulsed neodymium-doped yttrium aluminum garnet laser is an effective and safe alternative treatment for these lesions. Our case series is unique because it focuses on adolescents, the population that most often seeks treatment for this dermatologic condition.

Calcipotriol plus betamethasone dipropionate gel compared with its active components in the same vehicle and the vehicle alone in the treatment of psoriasis vulgaris: a randomised, parallel group, double-blind, exploratory study.

A two-compound ointment containing calcipotriol plus betamethasone dipropionate is an effective treatment for psoriasis vulgaris. The same active ingredients have now been combined in a gel formulation. Our objective was to compare the efficacy and safety of once daily treatment of the two-compound gel with the single components in the same gel vehicle and the gel vehicle alone, in patients with psoriasis vulgaris on the trunk and/or limbs. 364 patients received once daily treatment for up to 8 weeks with either the two-compound gel, the single components in the gel vehicle or the gel vehicle alone. The percentage of patients whose disease was clear or very mild and who had at least a two-step improvement in the Investigator's Global Assessment of disease severity at week 8, was significantly higher with calcipotriol plus betamethasone dipropionate (27.2%) than with betamethasone dipropionate (16.9%, p = 0.027), calcipotriol (11.4%, p = 0.006) or gel vehicle (0.0%, p < 0.001). This exploratory study showed that the two-compound gel was safe and more efficacious than its individual ingredients in the treatment of psoriasis vulgaris.

Calcipotriol plus betamethasone dipropionate scalp formulation is effective and well tolerated in the treatment of scalp psoriasis: a phase II study.

BACKGROUND: There is a need for more effective therapy for scalp psoriasis. OBJECTIVE: To assess the efficacy and safety of a 2-compound scalp formulation including calcipotriol and betamethasone dipropionate in the treatment of scalp psoriasis. METHODS: Patients (n = 218) with scalp psoriasis were randomized to treatment with the 2-compound scalp formulation (n = 108) or betamethasone dipropionate in the same vehicle (n = 110). The treatments were applied once daily on the scalp for up to 8 weeks. RESULTS: The 2-compound scalp formulation showed a significantly higher efficacy than betamethasone dipropionate on the total sign score at the end of treatment (p = 0.042) and after 2 weeks (p = 0.005). CONCLUSION: The calcipotriol plus betamethasone dipropionate scalp formulation was superior to betamethasone dipropionate in the same vehicle when used once daily for up to 8 weeks in the treatment of scalp psoriasis.

Identification of mitogen-activated protein kinase docking sites in enzymes that metabolize phosphatidylinositols and inositol phosphates.

BACKGROUND: Reversible interactions between the components of cellular signaling pathways allow for the formation and dissociation of multimolecular complexes with spatial and temporal resolution and, thus, are an important means of integrating multiple signals into a coordinated cellular response. Several mechanisms that underlie these interactions have been identified, including the recognition of specific docking sites, termed a D-domain and FXFP motif, on proteins that bind mitogen-activated protein kinases (MAPKs). We recently found that phosphatidylinositol-specific phospholipase C-gamma1 (PLC-gamma1) directly binds to extracellular signal-regulated kinase 2 (ERK2), a MAPK, via a D-domain-dependent mechanism. In addition, we identified D-domain sequences in several other PLC isozymes. In the present studies we sought to determine whether MAPK docking sequences could be recognized in other enzymes that metabolize phosphatidylinositols (PIs), as well as in enzymes that metabolize inositol phosphates (IPs). RESULTS: We found that several, but not all, of these enzymes contain identifiable D-domain sequences. Further, we found a high degree of conservation of these sequences and their location in human and mouse proteins; notable exceptions were PI 3-kinase C2-gamma, PI 4-kinase type IIbeta, and inositol polyphosphate 1-phosphatase. CONCLUSION: The results indicate that there may be extensive crosstalk between MAPK signaling and signaling pathways that are regulated by cellular levels of PIs or IPs.

Fear conditioning is associated with altered integration of PLC and ERK signaling in the hippocampus.

The extracellular signal-regulated protein kinases (ERKs) are proline-directed, serine/threonine kinases that regulate a variety of cellular functions, including proliferation, differentiation, and plasticity. In the present report, we provide evidence that ERK2 and phosphatidylinositol-specific phospholipase C (PLC)-beta and -gamma isozymes interact in the rat hippocampal formation. We found that anti-PLC-beta1a, -beta2, -beta4, -gamma1 and -gamma2, but not -beta3, immune complexes isolated from rat hippocampal formation postnuclear fractions contain anti-ERK2 immunoreactivity. Further, we show that PLC catalytic activity is associated with anti-ERK2 immunoprecipitates isolated from the hippocampal formation, and that the amount of enzyme activity is significantly increased following fear-conditioned learning. The observed interactions may be mediated by consensus sequences conforming to an ERK2 docking site, termed a D-domain, that we identified in PLC-beta1a, -beta2, -beta4 -gamma1 and -gamma2. Based on these results, we propose that PLC-beta and PLC-gamma isozymes form signaling complexes with ERK2 in rat brain, and these complexes play critical roles in learning and memory, as well as a variety of other neuronal functions.

Identification of phospholipase C-gamma1 as a mitogen-activated protein kinase substrate.

The discovery of sequence motifs that mediate protein-protein interactions, coupled with the availability of protein amino acid sequence data, allows for the identification of putative protein binding pairs. The present studies were based on our identification of an amino acid sequence in phosphatidylinositol-specific phospholipase C-gamma1 (PLC-gamma1) that fits the consensus sequence for a mitogen-activated protein kinase (MAPK) binding site, termed the D-domain. Extracellular signal-regulated kinase 2 (ERK2), an MAPK, and phospho-ERK2 were bound by an immobilized peptide sequence containing the identified PLC-gamma1 D-domain. Furthermore, a peptide containing the PLC-gamma1 D-domain was able to competitively inhibit the in vitro phosphorylation of recombinant PLC-gamma1 by recombinant phospho-ERK2, whereas a control peptide derived from a distant region of PLC-gamma1 was ineffective. Similarly, the peptide containing the PLC-gamma1 D-domain, but not the control peptide, competitively inhibited the in vitro phosphorylation of Elk-1 and c-Jun catalyzed by recombinant phospho-ERK2 and phospho-c-Jun N-terminal kinase 3 (phospho-JNK3), another type of MAPK, respectively. Incubation of anti-PLC-gamma1 immunocomplexes isolated from rat brain with recombinant phospho-ERK2 opposed the increase in PLC-gamma1-catalyzed hydrolysis of phosphatidylinositol 4,5-P(2) (PtdIns(4,5)P(2)), which was produced by a tyrosine kinase associated with the immunocomplexes, whereas in vitro phosphorylation of recombinant PLC-gamma1 by recombinant phospho-ERK2 did not alter PLC-gamma1-catalyzed PtdIns(4,5)P(2) hydrolysis. These studies have uncovered a previously unidentified mechanism for the integration of PLC-gamma1- and ERK2-dependent signaling.

Two-layer antibody capture of enzymes on the surface of microtiter plates: application to the study of the regulation of phospholipase C-gamma1 catalytic activity.

In vitro quantification of the catalytic activity of an enzyme isoform requires the availability of selective agents that allow for either measurements in the presence of the other enzyme isoforms or purification of the isoform and subsequent performance of these measurements on the purified enzyme. Isozyme-specific antibodies are useful tools for these types of analyses. In the present report, we detail a method for the measurement of phospholipase C-gamma1 enzyme activity employing native enzyme that is immobilized on microtiter plates. The method uses biotinylated antiglobulin bound to streptavidin-coated microtiter plates to immobilize antiphospholipase C-gamma1 antibody and subsequently capture phospholipase C-gamma1 from brain tissue lysates. This method avoids biotinylation of the primary (antiphospholipase C-gamma1) antibody, making it less labor intensive than previously described methods for using streptavidin-coated plates; in addition, it is highly reproducible and sensitive and allows for quantification of enzyme activity. We employ the technique to show that one or more tyrosine kinases copurify with rat brain phospholipase C-gamma1. The method is applicable to the study of any enzyme isoform for which antibodies that capture the native form of the enzyme are available and could easily be employed in high-throughput procedures.