CD8+ T-cell response against MUC1-derived peptides in gastrointestinal cancer survivors.

The tumor-associated antigens CEA, MUC1 and Her2/neu are broadly expressed in gastrointestinal tumors, and are attractive candidates for targeting by T-cell-based immunotherapy. However, little is known about the natural cytotoxic T-cell response of patients suffering from colorectal or gastric carcinoma against these three as well as other antigens. Using a quantitative reverse transcription-polymerase chain reaction-based assay for IFN-gamma, we analyzed the CD8+ T-cell repertoire present in the blood of HLA-A2+ gastrointestinal tumor survivors against five known epitopes derived from CEA, MUC1 and Her2/neu. The results show that most of the patients (16 from 22 tested) have detectable, peripheral CD8+ T cells directed against at least one of these three proteins. Interestingly, the majority of these patients reacts to the two MUC1-derived HLA-A*0201 epitopes tested (14 from 16), demonstrating that this protein represents one dominant target for CD8+ T cells in gastrointestinal cancer.

Documentation of 7051 chronic wounds using a new computerized system within a network of wound care centers.

HYPOTHESIS: Wound care can be prospectively recorded on a large scale if a standardized wound documentation system is established. Based on such a documentation system, a network of specialized wound care centers can generate a large and valid database of wound characteristics, wound-healing dynamics, and wound care. DESIGN: A clinical prospective analysis. SETTING: Ten German specialized surgical wound care centers. PATIENTS AND METHODS: In a 2-year pilot phase, 4175 patients with 7051 chronic nonhealing wounds were documented using a new computerized system and treated following defined standards. RESULTS: A total of 1761 diabetic, 1349 venous, 1146 ischemic, 1079 pressure, and 759 postoperative nonhealing wounds and 957 ulcers with other causes were prospectively documented. Chronicity of ulceration was shown by the long wound duration of 433 days (range, 14-1867 days). Wound documentation was well integrated into daily practice, as shown by a mean +/- SD documentation time of 5.7 +/- 2.2 minutes per visit. A multivariate analysis of factors known to interfere with wound healing revealed significant effects of patient compliance, grading of wound depth, and patient age (P<.001 for all). CONCLUSIONS: The German Wound Net achieved, for the first time, centralized and prospective documentation of more than 7000 chronic wounds treated according to defined guidelines. This new concept of a network of specialized wound care centers working with standardized treatment plans and prospective documentation of chronic wounds may open a new dimension for wound-healing studies and may represent an optimal platform for multicenter trials.

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.

Subcapsular liver hematoma in HELLP syndrome: Evaluation of diagnostic and therapeutic options--a unicenter study.

OBJECTIVE: Subcapsular liver hematoma formation has been reported in less than 2% of pregnancies complicated by HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome. The purpose of this study was to identify the main diagnostic and therapeutic options for management of these patients. STUDY DESIGN: In this 10-year retrospective review, we performed a computer-directed search of all cases of confirmed HELLP syndrome with hepatic hematoma treated in the surgical department of our tertiary care referral medical center. RESULTS: Five patients with subcapsular liver hematoma in HELLP syndrome could be identified. All patients received transabdominal ultrasound, computed tomography, or magnetic resonance imaging. Conservative treatment was successful in three patients. Emergency surgical intervention was necessary in two patients, including one liver transplantation. CONCLUSION: The case series shows the full diagnostic spectrum with transabdominal ultrasound, computed tomography, and magnetic resonance imaging, as well as the different therapeutic options varying from conservative therapy to operative management, including liver transplantation.

Age-dependency of insulin-like growth factors, insulin-like growth factor-binding proteins, and acid labile subunit in plasma and wounds of surgical patients.

Wound problems are common in the elderly. We hypothesized that age-related decrements in blood levels of components of the insulin-like growth factor (IGF) system are reflected in the wound environment. In this prospective, observational study IGF-I, IGF-II, IGF-binding protein-2, IGF-binding protein-3, and acid labile subunit were measured by immunoassays in the wound fluid and plasma of young (23.5 +/- 3.3 years) and elderly (78.9 +/- 6.2 years) patients before and daily for 4 days after elective surgery. IGFs, IGFBP-3, and acid labile subunit in plasma were significantly lower in the elderly group (p < 0.0001). The decrements of these proteins in plasma were reflected in corresponding decrements of 25-70% in the wound fluid of elderly patients (p < 0.0001). Additionally, bioavailability of IGF-I was less in the aged. The IGF parameters in the wound displayed a constant ratio with those of blood, suggesting that blood contributes a major share of the IGF that enters the wound during the initial phase of healing. The current data adds to accumulating evidence that a decline in the IGF system in aged patients contributes to the healing deficits observed in the elderly.

Differential expression of inflammatory mediators in radiation-impaired wound healing.

BACKGROUND: Radiation impairs healing, although the underlying mechanisms are not clearly defined. Normal healing requires a fine balance of promoting and inhibiting factors. We hypothesize that there may be a down-regulation of promoting factors (nitric oxide) and, in turn, an up-regulation of healing inhibiting factors (TNF-alpha and IFN-gamma) in the wound after radiation. MATERIAL AND METHODS: Groups of 10 rats were irradiated using single dose 12 or 24 Gy electron radiation at the dorsal skin. Control rats were sham-irradiated. On Day 5 a skin incision in the irradiated area was performed and polyvinyl alcohol sponges were inserted subcutaneously. Rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitrite and nitrate (index of NO synthesis), TNF-alpha, and IFN-gamma were measured within the wound fluid. Expression of the inducible NO-synthase (iNOS) was investigated by immunohistochemistry. Wound-derived fibroblasts were tested in vitro for NO and collagen synthesis. RESULTS: Irradiation significantly reduced wound collagen deposition and wound-breaking strength (P < 0.05). Impaired healing was reflected in diminished wound NO synthesis and iNOS expression (P < 0.01). TNF-alpha and IFN-gamma were increased in irradiated wounds (P < 0.05). Ex vivo, NO synthesis and collagen deposition by fibroblasts from irradiated rats were decreased (P < 0.01). In vitro irradiation of fibroblasts from nonirradiated rats decreased both NO and collagen production (P < 0.01). CONCLUSION: Radiation-impaired healing is reflected in an imbalance of promoting and inhibiting factors, leading to increased levels of TNF-alpha and IFN-gamma and decreased NO expression in the wound.

Upregulation of arginase expression in wound-derived fibroblasts.

BACKGROUND: Wound-derived fibroblasts (WFBs) are phenotypically different from normal dermal fibroblasts (NFBs). We have previously shown that the wound phenotype correlates with expression of the inducible isoform of nitric oxide synthase (iNOS) in fibroblasts. l-Arginine is the sole substrate for iNOS. Arginase is an alternative pathway of l-arginine metabolism in wounds. To clarify the role of l-arginine in wound healing, we investigated arginase expression and activity in WFB. METHODS: Male Lewis rats underwent dorsal skin incisions and subcutaneous PVA sponge implantation. WFBs were harvested from sponges retrieved at different days postimplantation. Normal fibroblasts were obtained from uninjured skin by an explant technique. Arginase activity was measured by newly formed urea (nmol/min/mg protein) and protein expression was detected by Western blotting using specific antibodies for type I (AI) and type II (AII). The effect of transforming growth factor beta1 (TGF-beta1), interleukin-4 (IL-4), lipopolysaccharide, and wound fluid on arginase activity was also investigated. RESULTS: WFB arginase activity was significantly elevated compared with NFB activity at all times postwounding. This was paralleled by increased AI protein expression by Western blotting. AII was not detectable. TGF-beta and IL-4 significantly increased arginase activity and protein expression whereas lipopolysaccharide and wound fluid did not affect it. CONCLUSIONS: The upregulation of the arginase expression in WFB underlines the distinct regulation of l-arginine metabolism in WFBs. Further work is needed to elucidate the functional implications.

Intracellular Ca2+ regulation in hepatocytes under experimental transplantation conditions.

Under transplant conditions excessive accumulation of intracellular calcium ([Ca2+](i)) is considered to be a mediator of cell injury during ischaemia and re-oxygenation. To clarify this consideration as well as the necessity of calcium-free preservation solutions, we used a well-known in-vitro model. Furthermore, a new application to mimic clinical situation was established, and we evaluated the correlation between [Ca2+](i) change and cell survival in monolayers of isolated rat hepatocytes undergoing cold hypoxia in defined solutions and during re-oxygenation. [Ca2+](i) was measured in single cells by ratio imaging of Fura-2 fluorescence after various periods of cold hypoxia (ischaemia phase) in different preservation solutions [UW, HTK, EC and Krebs-Henseleit buffer (KH)] and following warm normoxic reperfusion (re-oxygenation phase) with KH. Cell survival was measured simultaneously by trypan-blue exclusion. Cell survival decreased, depending on preservation solution and preservation time. The partially tremendous [Ca2+](i) change under cold hypoxia did not correlate with the change in cell survival. For example, UW-stored cells showed a [Ca2+](i) loss from 280 nM to 56 nM, compared with KH-stored cells with a [Ca2+](i) increase of up to 445 nM. Our results indicate that [Ca2+](i) plays only a minor role in the pathomechanisms of hypoxic and re-oxygenation hepatocellular injury.

Models to study ischemia in chronic wounds.

Wound healing is a complex immune response designed to achieve tissue repair following injury. Imbalance of stimulating and inhibiting factors cause failure of healing. Ischemia is a major cause of wound repair dysregulation and may be limb and life threatening. Investigating ischemic wound healing using animal models minimizes the complex accompanying factors that are usually present in humans, such as age or diabetes. This paper presents a limited review on normal physiological healing and on models that are used to study compromised healing under ischemic conditions.

Skin grafting of venous ulcers: a review of its current role.

As a therapeutic option, grafting of venous ulcers has not been very successfully received despite the different types of grafting methods. Currently, there are only a few controlled randomized trials offering clear guidance to clinicians. The development of artificially bioengineered skin constructs has led to a renewed interest in wound bed preparation, and preliminary successes suggest that the role of skin grafting could be studied in the current context.