Horst-Dieter Becker

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.

Therapeutic effect of argon plasma coagulation on small malignant gastrointestinal tumors

In endoscopy, argon plasma coagulation (APC) is a new principle of non-contact electrocoagulation and has proved to be a sufficient tool for palliative endoscopic treatment of gastrointestinal neoplasms, predominantly of the oesophagus and colorectum. In a study of 67 patients suffering from histologically confirmed and endosonographic T1-staged tumours of the gastrointestinum, 10 patients were selected for endoscopic APC treatment because of the impossibility of surgical therapy. Although the application was primarily of a palliative nature, in 9 of 10 cases of minor neoplasms, no further tumour could be detected in biopsies during the observation period (9.45±2.8 months). One patient was not cured locally. In none of the patients was any serious complication noticed during the outpatient follow-up. The effective results and lack of severe complications suggest this technique as an alternative therapy in selected patients with smaller gastrointestinal tumours.

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.

Arginase acts as an alternative pathway of L-Arginine metabolism in experimental colon anastomosis

L-Arginine is the substrate for the nitric oxide synthase (NOS) pathway that is essential for gastrointestinal wound healing. L-Arginine is also the substrate for the enzyme arginase which metabolizes L-arginine to ornithine and subsequently to proline and polyamines both known to interact in cell proliferation and collagen synthesis. Two distinct isoforms of arginase exist. The temporal expression of the L-arginine metabolism in experimental colon anastomosis was investigated. Male Lewis rats underwent laparotomy. A left-sided colotomy was performed and the colon reanastomosed using 6-0 prolene. Sham operation was performed in controls. On days 2, 5, 10, 14, and 28 after the surgery the anastomosis was excised. The tissue at the anastomosis (ANAST) as well as above and below the anastomosis (PDC) and from sham colon was harvested and analyzed for distinct arginase isoform I (AI) and arginase isoform II (AII) activity, protein and mRNA expression as well as immunohistochemistry. iNOS protein and mRNA expression were investigated in parallel. A mean of 3 to 4 separate rats were analyzed per time point. Statistical analysis was performed by student’s t-test, significance was reached when P < 0.05. AI activity, protein, and mRNA expression were significantly upregulated at the anastomosis compared to sham controls and PDC colons at all time points. The maximum was achieved at days 10 to 14 after wounding, and decreased to baseline levels thereafter. Inflammatory cells stained positive for AI. AII protein was not detectable. However RT-PCR showed low baseline expression. iNOS expression was upregulated early but for a shorter time period after wounding and reverted quickly to undetectable levels. In anastomotic healing, AI upregulation suggests a prolonged metabolism of arginine via arginase to polyamines and proline to provide substrate for collagen synthesis and cell proliferation. The functional implication of this arginase pathway further needs to be elucidated.

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-γ, 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.

A functioning adrenocortical hemangioma

A 60-year-old woman with hypertension (160/90 mm. Hg) who was receiving a &blocker, amiloride and potassium supplement underwent routine abdominal ultrasound, which revealed a spherical mass on the upper pole of the right kidney. Computerized tomography showed a heterogeneous mass 8 cm. in diameter clearly demarcated from the liver and kidney (see figure). The patient had borderline hypokalemia (3.7 mmol.fl., normal 3.6 to 5.5), hypernatremia (146 mmol.fl., normal 132 to 145) and increased urinary potassium excretion (143 mmo1./24 hours, normal 20 to 90). Adrenocortimtropic hormone levels were undetectable and partially suppressed aRer stimulation with corticotropin releasing fador while serum cortisol was elevated without diurnal variation. Urinary deoxycorticosterone levels were markedly elevated, while plasma aldosterone was normal and plasma renin activity was derreased (see table). The patient underwent partial adrenalectomy. Histology demonstrated a cavernous hemangioma with blood filled lacunae, benign endothelial lining and areas with necrosis, hemor-

ISOLATING AND MAINTAINING HIGHLY POLARIZED PRIMARY EPITHELIAL CELLS FROM NORMAL HUMAN DUODENUM FOR GROWTH AS SPHEROID-LIKE VESICLES

SummaryA method is described for the three-dimensional (3-D) in vitro culture of nontransformed gastrointestinal epithelial cells from the human duodenal mucosa. Biopsies obtained from human duodenum were finely minced. The tissue fragments were suspended in culture medium supplemented with 5% fetal calf serum and the appropriate antibiotics. The suspended mucosal fragments generated spheroid-like multicellular vesicles consisting of highly prismatic absorptive and goblet cells retaining most of the histological features of the tissue in vivo. We performed immunocytochemical studies to determine the origin of the vesicles using monoclonal antibodies against EP4. The histochemistry of the vesicles showed alkaline phosphatase activity. Ultrastructural studies revealed that these cells exhibit characteristics of normal duodenal cells in vivo: apical microvilli, glycocalyx, tight junctions and desmosomes, lateral membrane interdigitations, mucous droplets, and a well-developed Golgi apparatus. An overgrowth of the vesicles by fibroblasts was not seen during cultivation. In contrast with the two-dimensional cell cultures grown on artificial supports, the vesicle cells show organization similar to that of natural epithelia. The polarization and cytoarchitecture of normal gastrointestinal epithelial cells cultured as 3-D vesicles are comparable to those known for the native tissue. This study was undertaken to provide a morphological baseline for subsequent infection experiments.

Isolation and culturing of highly polarized primary epithelial cells from normal human stomach (antrum) as spheroid-like vesicles

A novel procedure is described for the three-dimensional (3-D) in vitro culture and for maintaining of nontransformed gastric epithelial cells from the human antrum mucosa (HAEC). Biopsies obtained from the antrum were cut into small pieces and the tissue fragments were incubated in culture medium containing the appropriate antibiotics. The suspended mucosal fragments generated small, spheroid-like vesicles consisting of predominantly highly prismatic, mucus-producing cells which mimic the in vivo counterparts structurally and functionally. Electron microscopic investigations revealed a number of ultrastructural and morphological features similar to those of normal gastric cells in vivo such as apical microvilli associated with a glycocalyx, tight junctions, desmosomes, membraneous infoldings, mucous droplets, and an irregular basal lamina. In comparison to the two-dimensional (2-D) gastric cell cultures grown on plane supports, the vesicles maintain an intact epithelial organization of individual cells. The prismatic phenotype, the histophysiology as well as the cytoarchitecture of the non-transformed 3-D cultured gastric epithelial cells are comparable to those of the native tissue and therefore represent a suitable model for defined pathogen-host cell interactions.

Steigerung der Zellproliferation und Angioneogenese durch kontrollierte lokale Applikation von IGF-I bei kortison induzierten Wundheilungsstörungen

Introduction Breaking strength of incisional wounds is increased after topical application of Insulin Like Growth Factor I (IGF-I). However, for excisional wounds no benefit was demonstrated. We hypothesize that a new delivery system of IGF-I stimulates excisional wound healing.

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.