A multicentre, clinical evaluation of a hydro-responsive wound dressing: the Glasgow experience.

OBJECTIVE: Our aim was to assess the effectiveness of hydro-responsive wound dressing (HRWD) in debridement and wound bed preparation of a variety of acute and chronic wounds that presented with devitalised tissue needing removal so that healing may proceed. METHOD: This was a non-comparative evaluation of acute and chronic wounds that required debridement as part of their normal treatment regimen. Clinicians recorded wound changes including a subjective assessment level of devitalised tissue and wound bed preparation, presence of pain, wound status (e.g., wound size) and periwound skin condition. Data was also collected from clinicians and patients to provide information on clinical performance of the dressing. RESULTS: We recruited 100 patients with a variety of wound types into the study. Over 90% of the clinicians reported removal of devitalised tissue to enable a healing response in both chronic and acute wounds. Specifically, over the course of the evaluation period, levels of devitalised tissue (necrosis and slough) reduced from 85.5% to 26.3%, and this was accompanied by an increase in wound bed granulation from 12.0% to 33.7%. Correspondingly, there was a 40% reduction in wound area, hence a clinically relevant healing response was seen upon treatment with HRWD. It is also noteworthy that this patient population included a significant proportion of chronic wounds (51.4%) that showed no signs of wound progression within <4 weeks before study inclusion. Of these chronic wounds, 93% demonstrated wound progression upon treatment with HRWD. Despite reported pain levels being low pre- and post-dressing change, overall wound pain improved (reduced) in 48% of patients. Periwound skin condition showed a tendency towards improvement, and the fluid management capabilities of the HRWD was reported as good to excellent in the majority of cases. Wound infections were reduced by at least 60% over the evaluation period. A simple cost-effective analysis demonstrated significant savings using HRWD (£6.33) over current standard practice regimens of a four-step debridement process (£8.05), larval therapy (£306.39) and mechanical pad debridement (£11.46). CONCLUSION: HRWD was well tolerated and was demonstrated to be an efficient debridement tool providing rapid, effective and pain free debridement in a variety of wound types.

Qutenza patch--our early experience.

Qutenza is a high potency capsaicin topical patch which has been recommended for the treatment of peripheral neuropathic pain. The aim of this study was to assess our selected patients' response to Qutenza application. All patients had their dynamic pain score recorded prior to application and were asked to fill in a standardised questionnaire for three months post application. Patients were also asked to document any changes to the character of their pain, changes in sleep, activities of daily living and mood as well as any changes to their medication usage. 21 patients had Qutenza applied in a 5 month period. 17 patients completed the questionnaire in a 5 month period. We found that the mean overall reduction in pain score at 3 months was 32.7%. 8 of our patients (47%) reported improved sleep, activities of daily living and mood. 6 patients (35%) reported a reduction in medication use, while 7 (41%) reported an improvement in the character of their pain.

Impact of rituximab therapy for treatment of acute humoral rejection.

INTRODUCTION: Antibody mediated rejection (AMR) is associated with a greater incidence of allograft loss because traditional approaches - pulse steroid or anti-lymphocyte antibodies are usually ineffective. This retrospective analysis documented the benefit of rituximab administration in addition to plasmapheresis (PP). METHODS: We retrospectively reviewed the data from 54 kidney transplant patients treated for AMR between 2001 and 2006, including 26 patients who received PP plus rituximab (Group A), versus 28 subjects who underwent PP without rituximab (Group B). Only patients whose serum IgG levels were below normal values received intravenous gamma globulin (IVIG). In addition to clinical and demographic variables we evaluated graft/patient survivals at two years post-diagnosis, Banff classification of rejections, serum creatinine and calculated GFR values at baseline, rejection, resolution as well as three, six, 12 and 24 months thereafter. RESULTS: The demographic features of the cohorts showed no significant differences. The two-year graft survival for patients treated with rituximab plus PP was 90%, significantly better than 60% in the PP cohort (p = 0.005). Upon multivariate analysis administration of rituximab was the most significant factor (>or= 0.009); whereas, IVIG also produced a useful effect (p = 0.05). Neither the mean (>or= 0.42) nor the slope (p = 0.25) of GFR values showed a significant difference among salvaged kidneys over 24 months after completion of AMR treatment. The rates and types of infectious complications at three and six months did not show significant differences or impact on graft survival. CONCLUSION: Addition of rituximab improved the outcomes of PP treatment of antibody mediated rejection episodes.

Campath-1H as rescue therapy for the treatment of acute rejection in kidney transplant patients.

INTRODUCTION: Kidney transplant patients with acute rejection episodes refractory to antilymphocyte preparations require aggressive treatment to salvage renal function and reduce the progression of chronic allograft nephropathy. PATIENTS AND METHODS: During a 6-month period, we administered Campath-1H as salvage therapy to five patients who had been previously treated with thymoglobulin and/or OKT3. In addition to measurements of the serum creatinine and BUN levels, we estimated creatinine clearance and glomerular filtration rates (GFR) using the Cockcroft-Gault and the MDRD equations at the time of initiation of therapy as well as at 2 weeks and 2 months thereafter. RESULT: Four of the five patients responded to Campath-1H therapy; kidney function improved to nearly the level before the rejection episode. The estimated creatinine clearance increased approximately threefold and the GFR approximately fourfold higher than the values before Campath-1H administration. The adverse events were mild and self-limited. CONCLUSION: Salvage of refractory acute rejection episodes may be possible in selected patients using Campath-1H.

Effects of endotoxin on serum chemokines in man.

AIM: Endotoxin is known to be a primary initiator of sepsis and septic shock. Migration of immunocompetent cells due to chemotactic attraction plays a central role in the initiation of the immune response. Two major groups of chemokines can be distinguished: C-x-C chemokines like Interleukin-8 attract mainly neutrophils, C-C chemokines (e.g. RANTES) attract monocytes and T-cells. The aim of this study was to get further insight into chemokine profiles after a single endotoxin bolus in man. MATERIALS AND METHODS: We investigated the effect of systemically administered endotoxin (4ng/kg BW i.v.) in 8 healthy volunteers. Clinical data (heart rate, mean arterial pressure, temperature), serum levels of IL-8, and RANTES, as well as white blood cell count were obtained before and hourly for five hours after endotoxin administration. RESULTS: Heart rate and MAP showed significant changes (p<0.05) after 2-3 hours. All volunteers presented with low-grade fever after 2 hours. WBC was elevated 43% and 63% after 4 and 5 hours, respectively. Both chemokines were significantly different from baseline two hours after endotoxin challenge: While IL-8 was significantly increased RANTES serum levels were diminished. CONCLUSION: From our data we conclude that this endotoxin model was effective to mimic the clinical appearance of sepsis. Chemokines like IL-8 and RANTES are integrated in the early immune response to endotoxin challenge in man.

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1.

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA.

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.

Rev-mediated nuclear export of RNA is dominant over nuclear retention and is coupled to the Ran-GTPase cycle.

The human immunodeficiency virus type-1 Rev protein induces the nuclear export of intron-containing viral mRNAs that harbor its binding site, the Rev response element (RRE). A leucine-rich region of Rev, the activation domain, is essential for function and has been shown to be a nuclear export signal (NES). Although Rev exports viral RNAs that resemble cellular mRNAs, competition studies performed using microinjected Xenopus laevis oocytes have previously indicated that Rev utilizes a non-mRNA export pathway. Here, we show that Rev is able to induce the export of both spliceable and non-spliceable RRE-containing pre-mRNAs and that this activity is not dependent on the location of the RRE within the RNA. Importantly, even RNA molecules of different classes, such as U3 snoRNA and U6 snRNA, which are retained in the nucleus by non-pre-mRNA mechanisms, are exported to the cytoplasm in response to Rev. Consistent with the notion that Rev-mediated export of RRE-containing RNA is mechanistically distinct from the export of processed cellular mRNA, a chimeric Rev protein in which its NES is replaced by the NES of hnRNP A1 does not induce the export of a Rev-responsive mRNA. Finally, we demonstrate that Rev/RRE-activated RNA export is, like other nuclear export pathways, linked to the Ran-GTPase cycle.

Kinetoplastid RNA-editing-associated protein 1 (REAP-1): a novel editing complex protein with repetitive domains.

Kinetoplastid RNA editing consists of the addition or deletion of uridines at specific sites within mitochondrial mRNAs. This unusual RNA processing event is catalyzed by a ribonucleoprotein (RNP) complex that includes editing site-specific endoribonuclease, RNA ligase and terminal uridylnucleotidyl transferase (Tutase) among its essential enzymatic activities. To identify the components of this RNP, monoclonal antibodies were raised against partially purified editing complexes. One antibody reacts with a mitochondrially located 45 kDa polypeptide (p45) which contains a conserved repetitive amino acid domain. p45 co-purifies with RNA ligase and Tutase in a large ( approximately 700 kDa) RNP, and anti-p45 antibody inhibits in vitro RNA editing. Thus, p45 is the first kinetoplastid RNA-editing-associated protein (REAP-1) that has been cloned and identified as a protein component of a functional editing complex.

The HIV-1 Rev protein.

The nuclear export of intron-containing HIV-1 RNA is critically dependent on the activity of Rev, a virally encoded sequence-specific RNA-binding protein. Rev shuttles between the nucleus and the cytoplasm and harbors both a nuclear localization signal and a nuclear export signal. These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export. HIV-1 Rev therefore represents an excellent system with which to study aspects of transport across the nuclear envelope.

Cerebral blood flow during experimental endotoxemia in volunteers.

OBJECTIVE: To measure cerebral blood flow, cerebral metabolic rate for oxygen, cerebral oxygen delivery, and cerebral vascular resistance during experimental endotoxemia in volunteers. DESIGN: Experimental, prospective study. SETTING: University general clinical research center. SUBJECTS: Healthy volunteers (six male, four female, 30.1 +/- 1.9 yrs of age). INTERVENTIONS: Volunteers had radial, pulmonary arterial, and jugular venous bulb catheters inserted. All volunteers received a bolus of Escherichia coli endotoxin (4 ng/kg). Cerebral blood flow was measured, using the Kety-Schmidt technique. MEASUREMENTS AND MAIN RESULTS: Cerebral and systemic hemodynamics and oxygenation variables were measured at baseline and hourly for 5 hrs after endotoxin administration. A systemic hyperdynamic response characterized by an increase in body temperature (97.9 +/- 0.02, 100.2 +/- 0.02, and 99.7 +/- 0.02 degrees F [36.6 +/- 0.01, 37.9 +/- 0.1, and 37.6 +/- 0.1 degrees C] at baseline, 3, and 5 hrs, respectively), cardiac index (3.7 +/- 0.2, 6.2 +/- 0.2, and 5.7 +/- 0.2 L/min/m2 at baseline, 3, and 5 hrs), and heart rate (70 +/- 2.6, 96 +/- 2.6, and 93 +/- 2.9 beats/min at baseline, 3, and 5 hrs), and a decrease in mean arterial pressure (99.3 +/- 2.2, 84.4 +/- 2.8, and 84 +/- 3.4 mm Hg at baseline, 3, and 5 hrs) and systemic vascular resistance (1498 +/- 53, 788 +/- 37, 849 +/- 36 dyne.sec/cm5.m2 at baseline, 3, and 5 hrs) followed the endotoxin bolus. Cerebral blood flow (65.4 +/- 4.3, 57.7 +/- 3.1, and 58.6 +/- 3.0 mL/100 g/min at baseline, 3, and 5 hrs), cerebral oxygen delivery (11.6 +/- 0.7, 9.8 +/- 0.6, and 9.5 +/- 0.6 mL/100 g/min at baseline, 3, and 5 hrs), cerebral metabolic rate for oxygen (3.8 +/- 0.4, 3.3 +/- 0.3, and 3.0 +/- 0.3 mL/100 g/min at baseline, 3, and 5 hrs), and cerebral vascular resistance (1.4 +/- 0.2, 1.4 +/- 0.2, and 1.3 +/- 0.2 mm Hg/mL/100 g/min at baseline, 3, and 5 hrs) were unchanged throughout the 5-hr study period. Signs of cerebral dysfunction were not apparent, although the volunteers appeared drowsy during the latter part of the study. CONCLUSION: A dose of endotoxin sufficient to induce systemic vasodilation in healthy subjects does not influence cerebral blood flow or the cerebral metabolic rate for oxygen.

Acute alterations in growth hormone-insulin-like growth factor axis in humans injected with endotoxin.

The purpose of the present study was to characterize the acute changes in the insulin-like growth factor (IGF) system in humans after administration of endotoxin (lipopolysaccharide; LPS). Escherichia coli LPS (4 ng/kg) was injected intravenously into healthy adults, and serial blood samples were collected for the next 5 h; subjects injected with saline served as time-matched controls. LPS administration resulted in a gradual decrease in the total extractable IGF-I concentration, which was reduced by approximately 20% over the final 2 h of the experiment; levels of free IGF-I were not significantly altered. LPS also produced a marked but transient elevation in growth hormone (GH) concentration. IGF-binding protein (BP)-1 levels were elevated more than fivefold 2 h after LPS injection, and thereafter levels gradually returned toward baseline. IGFBP-2 concentration also increased after LPS injection, but the maximal increase (approximately 50% above basal) was observed during the final 2 h of the protocol. In contrast, IGFBP-3 levels did not vary over the period examined in response to LPS, and there was no apparent increase in number of BP-3 proteolytic fragments. Cortisol levels were increased early and remained two- to threefold above baseline throughout the protocol. No significant alterations in serum concentration of glucose or insulin were noted. LPS also produced an early elevation in tumor necrosis factor and a later increase in interleukin-6. These data indicate that the acute changes in the GH-IGF axis in humans in response to LPS are comparable with those observed in humans in other traumatic conditions and in animal models of endotoxemia and infection.

Transportin: nuclear transport receptor of a novel nuclear protein import pathway.

Many nuclear proteins are imported into the cell nucleus by the "classical" nuclear localization signal (NLS)-mediated import pathway. In this pathway, a sequence rich in basic residues in the protein interacts with a heterodimeric complex termed importin and this, along with the GTPase Ran, mediates nuclear import of the NLS-bearing protein. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein contains a novel nuclear localization sequence, termed M9, that does not contain any clusters of basic residues. Very recently, we showed that M9 directs import into the nucleus by a novel protein import pathway distinct from the classical NLS pathway. A 90-kilodalton protein termed transportin was identified as a protein that specifically interacts with wild-type M9 but not transport-defective M9 mutants. Transportin and an ATP-regenerating system were found to be necessary and sufficient for import of M9-containing proteins in an in vitro import assay. In this report, we provide additional evidence that transportin can interact directly with M9-containing proteins and also show that it can mediate import of full-length hnRNP A1. In addition, Ran, or a Ran-binding protein, is identified as a second protein component of this novel nuclear import pathway. Transportin relatives from Saccharomyces cerevisiae which likely serve as additional nuclear transport receptors are described.

The 1996 Moyer Award. Effects of endotoxin on the Th1/Th2 response in humans.

Monocyte/T-cell interactions play a critical role in the systemic response to infection. Distinct patterns of cytokines are produced by two different types of T-helper cells (Th). Th1 cells secrete interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13. In volunteers systemic endotoxin administration initiates many features of gram-negative sepsis including cytokine release, but the patterns (i.e., Th1/Th2 patterns) have not yet been studied. In this institutional review board-approved study we investigated the effect of an intravenous bolus of endotoxin from Escherichia coli (4 ng/kg body weight) on the Th1/Th2 response in four female and four male volunteers (mean age 27.1 +/- 0.8 years). Plasma cytokine levels for IL-2, IL-4, IL-10, IL-12, and IFN-gamma and heart rate, mean arterial pressure, temperature, white blood cell, and differential blood count were determined before and hourly for 5 hours after endotoxin administration. All volunteers had tachycardia, decreased mean arterial pressure, fever, and leukocytosis. IL-10 was significantly (p < 0.05) elevated (9.4 +/- 3.9 pg/ml vs 60.9 +/- 19.3 pg/ml) 3 hours after endotoxin was administered, whereas IL-2 levels were decreased (69 +/- 26 U/ml vs 30.6 +/- 14.9 U/ml). IL-4 and IFN-gamma were not detectable in plasma. No changes were seen in the plasma levels of IL-12. Systemic responses did not correlate with changes in cytokine levels. Cytokine patterns found in this study suggest that after low-dose endotoxin administration the T-cell immune response is shifted towards the Th2 cell type response. This early shift towards a Th2 cell response may contribute to the depressed cell-mediated immune response associated with sepsis.

A novel receptor-mediated nuclear protein import pathway.

Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.

Validation in volunteers of a near-infrared spectroscope for monitoring brain oxygenation in vivo.

Cerebral oximeters based on near-infrared spectroscopy may provide a continuous, noninvasive assessment of cerebral oxygenation. We evaluated a prototype cerebral oximeter (Invos 3100; Somanetics, Troy, MI) in 22 conscious, healthy volunteers breathing hypoxic gas mixtures. Using the first 12 subjects (training group), we developed an algorithm based on the mathematic relationship that converts detected light from the field surveyed by the probe to cerebral hemoglobin oxygen saturation (CSfO2). To develop the algorithm, we correlated the oximeter result with the estimated combined brain hemoglobin oxygen saturation (CScombO2, where CScombO2 = SaO2 x 0.25 + SjO2 x 0.75 and SjO2 = jugular venous saturation). We then validated the algorithm in the remaining 10 volunteers (validation group). A close association (r2 = 0.798-0.987 for individuals in the training group and r2 = 0.794-0.992 for individuals in the validation group) existed between CSfO2 and CScombO2. We conclude that continuous monitoring with cerebral oximetry may accurately recognize decreasing cerebral hemoglobin oxygen saturation produced by systemic hypoxemia.

The influence of carbon dioxide and body position on near-infrared spectroscopic assessment of cerebral hemoglobin oxygen saturation.

Near-infrared spectroscopy may allow continuous and noninvasive monitoring of regional brain hemoglobin oxygen saturation by measuring the differential absorption of infrared light by oxyhemoglobin and deoxyhemoglobin. We have previously examined the correlation between the spectroscopic signal generated by a prototype cerebral oximeter (Invos 3100; Somanetics, Troy, MI), and global brain hemoglobin oxygen saturation calculated from arterial and jugular venous bulb oxygen saturations. Because the technology does not distinguish between arterial and venous hemoglobin saturation, changes in the proportion of cerebral arterial and venous blood volume, which may result from changes in blood flow or venous distending pressure, may confound measurements. In eight conscious volunteers breathing hypoxic oxygen mixtures, we examined the influence of supine, 20 degrees Trendelenburg, and 20 degrees reverse Trendelenburg positions on the correlation of the spectroscopic measurement of cerebral oxygen saturation in the field assessed by the probe (CSfO2) and the calculated brain hemoglobin oxygen saturation (CScombO2), estimated as 0.25 x arterial saturation plus 0.75 x jugular venous bulb oxygen saturation. We found that changes in position did not influence the association between CSfO2 and CScombO2 (r2 = 0.69-0.885) during hypoxic challenge. In a second set of eight volunteers, we studied the influence of hypercapnia and hypocapnia and body position on the association between CSfO2 and CScombO2, and found that they were less well correlated (r2 = 0.366-0.976) in individual patients. Because changes in body position and Paco2 confound the relationship between CSfO2 and CScombO2, changes in CSfO2 can best be assessed if position and Paco2 are constant.

Cytogenetics of an intrachromosomal transposition in Neurospora.

Knowledge of intrachromosomal transpositions has until now been primarily cytological and has been limited to Drosophila and to humans, in both of which segmental shifts can be recognized by altered banding patterns. There has been little genetic information. In this study, we describe the genetic and cytogenetic properties of a transposition in Neurospora crassa. In Tp(IR-->IL)T54M94, a 20 map unit segment of linkage group I has been excised from its normal position and inserted near the centromere in the opposite arm, in inverted order. In crosses heterozygous for the transposition, about one-fifth of surviving progeny are duplications carrying the transposed segment in both positions. These result from crossing over in the interstitial region. There is no corresponding class of progeny duplicated for the interstitial segment. The duplication strains are barren in test crosses. A complementary deficiency class is represented by unpigmented, inviable ascospores. Extent of the duplication was determined by duplication-coverage tests. Orientation of the transposed segment was determined using Tp x Tp crosses heterozygous for markers inside and outside the transposed segment, and position of the insertion relative to the centromere was established using quasi-ordered half-tetrads from crosses x Spore killer. Quelling was observed in the primary transformants that were used to introduce a critical marker into the transposed segment by repeat-induced point mutation (RIP).