Effectiveness of Hemopatch® versus Surgicel® Original to control mild and moderate liver bleeding.

BACKGROUND: Adjunct hemostats can be of use in certain surgical settings. We compared the effectiveness of two hemostats, Hemopatch® and Surgicel® Original in controlling bleeding from liver lesions in an experimental model. METHODS: Control of grades 1 (mild) and 2 (moderate) bleeding (according to the Validated Intraoperative Bleeding [VIBe] SCALE) was assessed for 10 min after Hemopatch® (n = 198) or Surgicel® Original (n = 199) application on 397 liver surface lesions. The primary endpoint was hemostatic success (reaching VIBe SCALE grade 0 at 10 min). The secondary endpoint was time to hemostasis (time to reach and maintain grade 0). A generalized linear mixed model and an accelerated failure time model were used to assess the primary and secondary endpoints, respectively. RESULTS: The overall hemostatic success rate of Hemopatch® was statistically significantly superior to that of Surgicel® Original (83.8% versus 73.4%; p = 0.0036; odds ratio [OR] 2.38, 95% confidence interval [CI] 1.33-4.27) and time to hemostasis was reduced by 15.9% (p = 0.0032; 95% CI 0.749-0.944). Grade 2 bleeds treated with Hemopatch® had statistically significantly higher hemostatic success (71.7% versus 48.5%; p = 0.0007; OR 2.97, 95% CI 1.58-5.58) and shorter time to hemostasis (49.6% reduction, p = 3.6 × 10-8); differences for grade 1 bleeds (hemostatic success rate or time to hemostasis) were not statistically significant. CONCLUSIONS: Hemopatch® provided better control of VIBe SCALE bleeding compared to Surgicel® Original for Grade 2 bleeds in this porcine model, highlighting the importance of choosing a suitable hemostat to optimize control of bleeding during surgery.

Properties of collagen-based hemostatic patch compared to oxidized cellulose-based patch.

Two self-adhering hemostatic patches, based on either PEG-coated collagen (PCC) or PEG-coated oxidized cellulose (PCOC), are compared regarding to maximum burst pressure, mechanical stability, and swelling. In addition, the induction of tissue adhesions by the materials was assessed in a rabbit liver abrasion model. Both materials showed comparable sealing efficacy in a burst pressure test (37 ± 16 vs. 35 ± 8 mmHg, P = 0.730). After incubation in human plasma, PCC retained its mechanical properties over the test period of 8 h, while PCOC showed faster degradation after the 2 h time-point. The degradation led to a significantly decreased force at break (minimum force at break 0.55 N during 8 h for PCC, 0.27 N for PCOC; p < 0.001). Further, PCC allowed significantly higher deformation before break (52% after 4 h and 50% after 8 h for PCC, 18% after 4 h and 23% after 8 h for PCOC; p = 0.003 and p < 0.001 for 4 h and 8 h, respectively) and showed less swelling in human plasma (maximum increase in thickness: ~20% PCC, ~100% PCOC). Faster degradation of PCOC was visible macroscopically and histologically in vivo after 14 days. PCC showed visible structural residues with little cellular infiltration while strong infiltration with no remaining structural material was seen with PCOC. In vivo, a higher incidence of adhesion formation after PCOC application was detected. In conclusion, PCC has more reliable mechanical properties, reduced swelling, and less adhesion formation than PCOC. PCC may offer greater clinical benefit for surgeons in procedures that have potential risk for body fluid leakage or that require prolonged mechanical stability.

Host defense mechanisms in secondary syphilitic lesions: a role for IFN-gamma-/IL-17-producing CD8+ T cells?

Cell-mediated immunity is thought to be of critical importance in antisyphilitic host defense, but the exact mechanisms are still unknown. This fact is particularly true for HIV-infected persons with a deficit in CD4+ T-cell number. We therefore obtained lesional skin samples from HIV+ and HIV- patients with secondary syphilis at different time points of lesional age to search both for causative microorganisms and to characterize the inflammatory infiltrate. By doing so, we detected Treponema pallidum spirochetes with a much greater abundance in late lesions of HIV+ individuals compared with the HIV- cohort. The dominating inflammatory cells were T cells, macrophages, and neutrophils at all stages and plasma cells in older lesions. In HIV- persons, T cells consisted of equal numbers of CD4+ and CD8+ T-cells, whereas in HIV+ patients, the majority of T cells belonged to the CD8 lineage and produced both IFN-γ and IL-17. Regulatory T cells and Langerhans cells were reduced in these patients compared with their HIV- counterparts. Because of our observations, we propose that T cells of both the CD4 and CD8 lineage are needed for an at least partial protective antisyphilitic immunity. Compensation mechanisms in HIV+ individuals, such as an increase of Tc1/17 cells as well as a reduction in immunoregulatory Langerhans cells and T cells, apparently do not overcome the deficiencies in these patients to eliminate the spirochete.

Epiplakin is dispensable for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia.

Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakin-deficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.

Efficacy of Topical Hemostatic Agents: A Comparative Evaluation of Two Gelatin/Thrombin-Based Hemostatic Matrices in a Porcine Kidney Surgical Model.

Purpose: Topical hemostatic agents are an important means of controlling or preventing bleeding. This study was performed to compare gelatin-thrombin matrix with smooth particles (SmGM) versus gelatin-thrombin matrix with stellate particles (StGM) in a porcine kidney bleeding model. Materials and methods: In male pigs, reproducible lesions (diameter and depth ∼10 mm) were created in the renal cortex. Each lesion was treated topically using either SmGM or StGM. Blood loss was quantified before and 2, 5 and 10 minutes after treatment. Dry mass, ultrastructural and histologic analyses were also performed. Results: Thirty-two lesions were treated with SmGM and 32 with StGM; median initial bleeding rates were 27.6 and 29.1 mL/min, respectively. Two minutes post-application, SmGM was associated with significantly less bleeding than StGM (0.574 vs 0.920 mL/min; p < .0001). This difference stemmed principally from lesions with initial blood loss >29 mL/min, where bleeding rates at 2 minutes were ∼3-fold higher with StGM (1.636 vs 0.567 mL/min; p ≥ 0.040). Dry mass per unit volume of hemostatic agent was significantly higher with SmGM versus StGM. SmGM formed discrete, smooth particles, while StGM particles were stellate and tended to coalesce. Histologic analysis showed more solid mass, larger particles and less intervening space with SmGM versus StGM. Conclusions: In a severe, high-volume bleeding model, residual bleeding at 2 minutes was significantly lower with SmGM versus StGM, and SmGM showed greater consistency across bleeding intensities. These findings may be attributable to dry mass per unit volume and/or ultrastructural differences between the two agents.

Controlled release of fibrin matrix-conjugated platelet derived growth factor improves ischemic tissue regeneration by functional angiogenesis.

Sustained, local, low dose growth factor stimulus of target tissues/cells is believed to be of imminent importance in tissue regeneration and engineering. Recently, a technology was developed to bind growth factors to a fibrin matrix using the transglutaminase (TG) activity of factor XIIIa, thus allowing prolonged release through enzymatic cleavage. In this study we aimed to determine whether TG-PDGF.AB in fibrin could improve tissue regeneration in a standard ischemic flap model. In vitro determination of binding and release kinetics of TG-PDGF.AB allowed proof of concept of the developed binding technology. A single spray application of TG-PDGF.AB in fibrin matrix at a concentration of 10 and 100ng/ml significantly reduced ischemia-induced flap tissue necrosis in vivo on day 7 after ischemic impact compared to controls. TG-PDGF.AB at a concentration of 100ng/ml fibrin induced distinct angiogenesis as reflected by significantly improved tissue perfusion assessed by laser Doppler imaging as well as enhanced von Willebrand factor (vWF) protein expression determined by immunohistochemical means. In addition, significantly more mature microvessels were observed with 100ng/ml TG-PDGF.AB in fibrin compared to control and vehicle groups as evidenced by an improved smooth muscle actin (sma)/vWF protein ratio. In conclusion, PDGF.AB in a conjugated fibrin matrix effectively reduced ischemia-induced tissue necrosis, increased tissue perfusion and induced the growth of a mature and functional neovasculature. The sealing properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by the TG-hook binding technology may present an innovative and suitable tool in tissue regeneration. STATEMENT OF SIGNIFICANCE: In our experimental study we elucidated recombinant platelet derived growth factor (PDGF) as a potential candidate in inducing angiogenesis. To avoid preterm growth factor degradation in vivo PDGF.AB was covalently linked to a fibrin scaffold using a bi-domain functionalized peptide (FXIII substrate site and plasmin cleavage site). This allowed PDGF binding to fibrin during spray application to the donor site and subsequent prolonged release via endogenous plasmin. This resulted in a mature vascular network thus enhancing tissue perfusion and consequently improved clinical outcome. With our present work we could certainly provide researchers and clinicians with an innovative versatile and reproducible technology not only to induce functional vascularity but also to improve attempts in tissue engineering in general by e.g. using different growth factors. Hence, we believe that this approach studied in the present work may provide a valuable input in an effort to drive the aim forward bringing experimental work in tissue engineering to clinic by using a clinically well characterized and used fibrin scaffold in combination with a human recombinant growth factor (fibrin scaffold linked with the specific binding technology).

Plectin-isoform-specific rescue of hemidesmosomal defects in plectin (-/-) keratinocytes.

The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause skin blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.

Swelling, sealing, and hemostatic ability of a novel biomaterial: A polyethylene glycol-coated collagen pad.

Trends in the development of hemostatic agents are towards self-adhering pads. This study investigates a novel biomaterial made of a polyethylene glycol-coated collagen pad (PCC). The swelling and adherence of PCC were investigated in vitro, and the hemostatic and sealing ability was investigated in vivo. In vitro, the maximum swell of PCC submerged in human plasma for 24 h is 65%. The greatest swell was in thickness, averaging 24% to a mean thickness of 2.5 ± 0.19 mm (mean±SD) (N = 20). PCC withstood clinically relevant pressures when applied to a collagen casing washed with bile, lymph, urine, saline, and cerebrospinal fluid mixed at 33% and 67% with blood. In vivo, PCC provided complete hemostasis when applied to severe, arterial bleeds of actively ventilated pulmonary parenchyma at 3, 5, 8, and 10 min after application in a heparinized porcine pulmonary segmentectomy model. The mean rate of bleeding was 17.7 ± 8.6 ml/min. The lungs were ventilated at 15 ± 4 breaths per min and an airway pressure of 19 ± 2 cm H2O. PCC had no incidence of hematoma and an 11% incidence of intraoperative air leak (N = 36). These data are promising for future clinical application of a new versatile, self-adhering hemostatic sealing pad consisting of a polyethylene glycol-coated collagen.

Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption.

Epiplakin is a large (>725 kDa) cytoskeletal protein exclusively expressed in epithelial tissues. It has a unique structure, consisting entirely of plakin repeat domains (PRDs), one of the hallmarks of spectraplakin protein family members. Previous studies, including the phenotypic analyses of knockout mice, failed to reveal the biological function of epiplakin. Using in vitro binding assays, we show here that all but one of the 16 PRDs of mouse epiplakin bind to keratins of basal keratinocytes. Nevertheless, in primary keratinocyte cell cultures, epiplakin only partially colocalized with keratin intermediate filament networks. However, upon application of cellular stress in the form of keratin hyperphosphorylation, osmotic shock or UV irradiation, the entire cytoplasmic epiplakin pool became associated with keratin. In response to such types of stress, epiplakin initially translocated to the still-intact keratin filament network and remained associated with keratin after its disruption and transformation into granular aggregates. Time-course experiments revealed that serine/threonine (okadaic acid) and tyrosine (orthovanadate) phosphatase inhibitor-induced filament disruption in differentiated keratinocytes proceeded faster in epiplakin-deficient cells compared with wild-type cells. Our data suggest that epiplakin plays a role in keratin filament reorganization in response to stress, probably by protecting keratin filaments against disruption in a chaperone-like fashion.

Plectin rodless isoform expression and its detection in mouse brain.

The widely expressed cytolinker protein plectin shows extensive isoform diversity both at the N-terminus and in the central part of the molecule. Judged on mRNA data, plectin variants lacking the central rod domain are expressed at a approximately 20-fold lower level than full-length proteins and their detection on the protein level can be difficult. Here we present data on the expression of plectin rodless isoforms in mouse brain and in rat glioma C6 cells on RNA and protein levels. Our data indicate that among the rodless variants expressed in neuronal tissues, those starting with exon 1c (plectin 1c) seem to be the most prominent ones. In addition, we show that similar to other monoclonal antibodies reported in the literature, the widely used mAb 7A8 recognizes an epitope within plectin's rod domain and therefore is unsuited to detect rodless variants of plectin.

Plectin 5'-transcript diversity: short alternative sequences determine stability of gene products, initiation of translation and subcellular localization of isoforms.

Plectin is a large cytoskeletal linker protein expressed as several different isoforms from a highly complex gene. This transcript diversity is mainly caused by short 5'-sequences contained in alternative first exons. To elucidate the influence of these sequence differences and to determine potential differential functionality of the resulting protein forms, we conducted a systematic investigation of plectin isoforms on transcript and protein levels. Isoform expression was highly dependent on the different 5' ends, largely due to effects of the 5'-untranslated regions. Initiation of translation downstream of the expected start site led to loss of actin- and integrin beta4-binding in some isoforms. The small alternative N-terminal sequences (5-180 residues) profoundly affected the subcelluar localization of this >500 kDa protein. Specifically, plectin 1f was concentrated at focal adhesion contacts and plectin 1b was exclusively targeted to mitochondria, providing a connection of these organelles to intermediate filaments. Thus, with plectin as a model, we demonstrate a role for 5'-untranslated regions and alternative 5'-splicing as an important regulatory mechanism of protein expression and protein function.

Epiplakin gene analysis in mouse reveals a single exon encoding a 725-kDa protein with expression restricted to epithelial tissues.

Based on cDNA cloning and sequencing, human epiplakin has been classified as a member of the plakin protein family of cytolinkers. We report here the characterization of the mouse epiplakin gene locus and the isolation of full-length mouse epiplakin cDNA using BAC vectors. We found that the protein is encoded by a single remarkably large exon (>20 kb) that consists of a series of 0.8-1.5-kb-long DNA repeats, eight of which are virtually identical. Consequently, mouse epiplakin contains 16 plakin repeat domains, three more than reported for the human protein and eight more than predicted for the mouse protein based on the contig characterized by the Mouse Genome Sequencing Consortium. Using antibodies raised to a highly conserved repeating epiplakin sequence domain, we show that the protein in cells is expressed in its full length (725 kDa), and we provide evidence that the size of human epiplakin previously may have been underestimated. In addition we show on transcript and protein levels that epiplakin is restricted to epithelial tissues and that its gene maps to mouse chromosome 15 (human chromosome 8). This study lays the groundwork for future genetic approaches aimed at defining the biological role of this unique protein.