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P Hufnagl

Publications and source records attributed to P Hufnagl.

At least 19 recordsLinked to original sources

Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1.

An estimated 100 million individuals suffer from birch pollen allergy. Specific immunotherapy, the only curative allergy treatment, can cause life-threatening anaphylactic side effects. Here, we report the genetic engineering of a recombinant trimer consisting of three covalently linked copies of the major birch pollen allergen, Bet v 1. The trimer exhibited profoundly reduced allergenic activity but contained similar secondary structures such as Bet v 1 wild type, Bet v 1-specific B cell and T-cell epitopes, and induced Th1 cytokine release. As immunogen, rBet v 1 trimer induced IgG antibodies, which blocked patients' IgE binding to Bet v 1 and related allergens. Thus, rBet v 1 trimer represents a novel hypoallergenic vaccine prototype for treatment of one of the most frequent allergy forms.

Allergens↗

Improvement of breast cancer prognostication using cell kinetic-based silver-stainable nucleolar organizer region quantification of the MIB-1 positive tumor cell compartment.

Recently, it was stated that the proliferative activity (P) of a cell population could be indirectly calculated by multiplying the MIB-1 immunopositivity and silver-stainable nucleolar organizer region (AgNOR) features extracted exclusively in MIB-1 positive (pos.) nuclei: P=MIB-1 x AgNOR(MIB-1pos.). To study the prognostic significance of this hypothesis, MIB- immunohistochemistry and AgNOR staining were applied on a series of 89 cases of breast cancer with an 8-year follow-up period. The mean MIB-1 immunopositivity (MIB-1mean) was evaluated immunohistometrically on paraffin sections using a TV image analysis system CM-2 (Hund, Wetzlar, Germany). Later, a combined MIB-1/AgNOR staining was applied and evaluated using a TV image analysis system AMBA (IBSB, Berlin, Germany). The AgNOR features of 150 randomly chosen tumor nuclei were investigated, irrespective of their MIB-1 status (AgNOR count, AgNOR area). Later, a second measurement was performed on 100 MIB-1 positive tumor nuclei exclusively (AgNOR countMIB-1pos., AgNOR areaMIB-1pos.). AgNOR count and AgNOR countMIB-1pos. showed a different data distribution [2.7+/-0.7 (mean+/-SD) vs 3.9+/-1.1; r=0.315, P=0.014]. Similar results were obtained for AgNOR area and AgNOR areaMIB-1pos. (5.1+/-2.1 microm2 vs 7.5+/-2.4 microm2; r=0.501, P<0.001). Kaplan-Meier survival curves revealed significant differences for MIB-1mean (P=0.0018) and AgNOR areaMIB-1pos. (P=0.0340). In Cox models, both parameters provided independent prognostic information. Using their combination, the P, three groups of patients with statistically different survival could be separated (P=0.0014). Thus, the combination of MIB-1-immunopositivity and AgNOR measurements in MIB-1 positive nuclei appears to be more useful in breast cancer prognosis than the exclusive application of one of the two methods. By this combined application, probably effects of tumor biology are represented more precisely.

Adenocarcinoma↗

Fetal autopsy: a review of recent developments.

OBJECTIVE: To analyze recent changes in fetal autopsy in response to developments in prenatal medicine. METHOD: During the period 1988 through 1997, 783 fetuses (75% induced abortions, 18% spontaneous abortions, and 7% stillbirths, all between the 12th and 40th week of gestation) with prenatally diagnosed congenital malformations and chromosomal aberrations were analyzed. We divided the autopsies into two periods: period A (1988-92, n=370) and period B (1993-97, n=413). All fetuses were analyzed before completion of 20 weeks of gestation. The malformations of the organ systems were presented according to their frequency for fetuses independent of the weeks of gestation. RESULTS: An autopsy was performed prior to the completion of 20 weeks of gestation for 24% of the fetuses in period A and 45% in period B (P<0.0001). The number of diagnosed congenital heart malformations increased from 16% in period A to 23% in period B. The number of congenital heart malformations before completion of 20 weeks of gestation was only 21% in period A as compared to 42% in period B. CONCLUSIONS: In period B, clinical questions were raised in a more concrete form and ultrasound images gave more detailed information than in period A. Access to prenatal findings prior to postmortem examination has helped investigators to develop a specific, clinically oriented, autopsy strategy based on the prenatal findings which makes it possible to diagnose very small malformations and to select sectional planes of special interest. The use of an interdisciplinary database is required for communication.

Abnormalities, Multiple↗

[Electronic communication in medicine].

Rapid developments in information and communication technology allow a fast transfer of extensive data. Geographical distances do not longer hinder an intense cooperation in diagnostics. The review shows the variety of possibilities for medical applications. Technique, routine application, experiences and the legal conditions are presented for telepathology as an example.

Humans↗

The UICC Telepathology Consultation Center. International Union Against Cancer. A global approach to improving consultation for pathologists in cancer diagnosis.

BACKGROUND: The morphologic diagnosis of tumor specimens with precise tumor typing, staging, and grading remains the basis of almost all cancer treatments. Thus, in each tumor case, a histologic diagnosis of the highest quality should be the physician's priority. In approximately 10-20% of tumor cases, diagnostic uncertainty remains to some degree, requiring a second opinion in determining the biologic behavior, the histogenesis, the grade of dedifferentiation, or any other parameter. Facilitating the communication between pathologists and the exchange of cases, telepathology gains more and more importance. To benefit from this technical development, the International Union Against Cancer (UICC) has decided to establish a Telepathology Consultation Center (UICC-TPCC) for interested pathologists around the world. METHODS: The communication and exchange of histologic images works via the Internet. To ensure constant documentation, a case-based data base and image archive is used. Special TPCC software handles all requests to the TPCC and controls the interaction among requesting pathologists, TPCC, and UICC experts (transferring, reading, answering, logging, storing, etc.). All necessary data for controlling the telepathology service are stored in a customized SQL data base. The necessary equipment for the requesting pathologist is a personal computer; a digital or television camera/frame grabber, which is attached to a microscope; and access to the Internet. The requesting party contacts the TPCC's World Wide Web server and uploads the images and the clinical data of their case. To ensure uninterrupted functioning, the hardware will be part of a high-level communication center, which is connected via ATM (asynchronous transfer mode, 155 megabits per second) to the Internet. RESULTS: The UICC has decided to establish the TPCC at the Institute of Pathology at the Charité, Humboldt-University, Berlin, Germany. The TPCC will not make the diagnoses itself but will involve an affiliated specialized expert pathologist. He or she will be on the panel of UICC experts who will constitute the "diagnostic backbone" of the TPCC. The center will function as follows: If a pathologist anywhere on the globe is confronted with the diagnosis of a difficult tumor case, he takes digitized histologic images (5-40 in number) and sends them along with sufficient clinical data to the server of the UICC-TPCC, asking for a second opinion. The center checks the case and transfers it to one of the UICC experts. This expert makes his or her diagnostic suggestion, which is then transferred back to the requesting pathologist via the UICC-TPCC. CONCLUSIONS: The UICC-TPCC will be able to provide rapid and inexpensive diagnostic aid to pathologists all over the world, offering the possibility of a second opinion in accordance with the UICC-TNM and World Health Organization (WHO) standards. During the first and second year, the UICC-TPCC will be financed by sponsors. Telepathology makes the distribution of new developments of diagnostic standards, e.g., of the TNM system, WHO terminology, new tumor classifications, and updated information on actual technologies, globally accessible in a direct and rapid way. It also enables a high quality of education and teaching.

Computers↗

[Autopsy techniques in congenital heart defects. Influence of prenatal diagnosis on the planning and carrying out of autopsies].

As a result of the quality of prenatal ultrasound and the expanded experience of prenatal diagnosticians, it is possible to observe congenital heart malformations in increasingly greater detail and at an ever earlier stage of gestation [4]. Since it is on the basis of ultrasound findings that decisions to terminate pregnancies are made, it is of cardinal importance that after termination monitoring and confirmation of the prenatal diagnosis be carried out. This need can only be adequately met by autopsy. There are different methods for carrying out autopsies when there is suspicion of a congenital heart defect: a) the Anderson sequential segmental analysis as modified according to the Berlin method; b) use of a special autoptic method corresponding to the ultrasound findings, based on defining a preferred sectional plane; c) stereomicroscopically; or d) microscopically after embedding and preparation of serial microscopic sections. For the pathologist the consequence is that he has to adapt his autopsy method to the ultrasound findings and the age of the fetus. This enables him to determine an optimal, case-based autopsy strategy for each type of cardiac defect, which is essential for monitoring of the prenatal diagnosis. The present paper discusses the various autoptic methods used in cases of congenital heart malformations and the consequences for the pathologist of the continuing improvements in prenatal diagnostics.

Autopsy↗

Comparison of different telepathology solutions for primary frozen section diagnostic.

In a retrospective study on a set of 125 cases we compared the following three telepathology solutions for primary frozen section diagnosis: ATM-TP (connection via ATM), TPS 1.0 (connection via LAN) and TELEMIC (connection via Internet), which represent different concepts of telepathological procedures. A set of 125 routine frozen sections (breast) was selected from the Charité cases of the year 1999. Four experienced pathologists diagnosed retrospectively all of these cases using the ATM-TP and TPS systems and 53 of them with the TELEMIC system. Using the ATM-TP we recorded no false positive (0%), 4 false negative (3.2%) and 4 deferred (3.2%) cases. Using the TPS we recorded no false positive (0%), 4 false negative (3.2%) and 4 deferred (3.2%) cases. Using the TELEMIC we recorded in 53 cases no false positive (0%), no false negative (0%) and 16 deferred (30.2%) cases. The average time of 2.2 minutes per case using ATM-TP is also short enough for routine frozen section diagnostic. This is also true for the TPS system with 7.2 minutes per case.

Breast Neoplasms↗

Technique and feasibility of a dual staining method for estrogen receptors and AgNORs.

A new staining method for dual demonstration of Estrogen receptors (ER) and argyrophilc Nucleolus-Organizer Regions (AgNORs) was developed. To rule out possible reciprocal effects, serial slides of 10 invasive ductale breast cancers were stained with either the single staining method or the simultaneous ER/AgNOR-staining method and investigated comparatively. By measuring the slides with the image analysis system AMBA, reciprocal effects could be excluded. It was proven that dual staining of both markers results in a reproducible and specific staining result. We concluded that it is justified to measure AgNORs in immunohistochemically stained cells.

Biomarkers, Tumor↗

Diagnosis of congenital heart malformations--possibilities for the employment of telepathology.

GOAL: In a study of 10 autopsy cases with congenital cardiac malformations we investigated whether obtaining a second opinion by means of telepathology could satisfy quality standards for the diagnosis of cardiac malformations and what the advantages and disadvantages of such a procedure might be. MATERIAL: The investigatory samples were 10 formalin-fixed hearts with complex malformations from 9 fetuses and one newborn on which autopsies had been performed at the Pathological Institute of the Charité Hospital. The requests for a second opinion, which included text and image data, were sent in the form of Microsoft PowerPoint presentations to 5 experts in 4 countries. Per case the number of images that were sent was between 3 and 7. The size of the files was between 439 and 942 kb. The time required for preparation of the cases for sending them to the specialists was between 1 and 2 hours: this encompassed the time for putting the notation on the images, compressing them, creating a file that included both the images and the clinical data and then sending the case file. RESULTS: All 10 cardiac malformations were correctly identified. In 8 of the 10 cases at least one expert had questions. After these questions had been answered and further images had been sent final correct diagnoses were made in all cases. All experts said that the quality of the images was very good. Use of a standardized findings questionnaire, which also included the marking of anatomic structures and of pathological findings in the images, proved useful. Standardized findings forms facilitate orientation during interpretation of the cases and should be used generally to avoid misunderstandings in telepathological communication. CONCLUSIONS: In general it is possible to obtain an effective and reliable diagnosis of congenital heart malformations by means of telepathology. It is far quicker to get a second opinion by this means than by conventional means.

Autopsy↗

Different proliferation patterns in breast cancer: AgNOR measurements in ER-negative and ER-positive tumor cells.

The relation between estrogen receptors (ER) and argyrophilic nucleolar organizer regions (AgNORs) in situ within human breast cancer cells was analyzed. For AgNOR measurements in 49 invasive breast carcinomas, a new reproducible staining method for dual demonstration of ER and AgNORs was applied. Quantitative AgNOR variables were determined in ER-positive and ER-negative tumor cells by digital image analysis. The relationships between AgNOR parameters of ER-positive and ER-negative cells and other prognostic factors of breast cancer [Bloom-Richardson-Grading and growth fraction (Ki-67 index)] were investigated. A higher AgNOR content in ER-negative cells and a special clustering phenomenon in ER-positive tumor cells were found. Correlation with other criteria of malignant potential could be exclusively demonstrated for ER-negative cells. ER-negative cells of breast cancer can be characterized as the more malignant and possibly prognosis-dictating cell fraction. Thus, ER-negative cells probably contribute more to the progression of the tumor disease and furthermore to the prognosis than ER-positive cells. We recommend measurement AgNORs exclusively in ER-negative cells of breast cancer.

Adult↗

AgNOR analysis of atypical ductal hyperplasia and intraductal carcinoma of the breast.

OBJECTIVE: To determine the diagnostic and prognostic value of argyrophilic nucleolar organizer regions (AgNORs) in atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and microinvasive ductal carcinoma (MDCA) of the breast. STUDY DESIGN: Image analysis of histologic sections from biopsies of 46 breast ADH and DCIS and 18 cases of MDCA. Determination of morphometric features of cell nuclei and nucleolar organizer regions by using AMBA software system. Data were compared with the estrogen receptor/progesterone receptor (ER/PR) content as well as with the growth fraction, determined immunohistochemically. RESULTS: AgNOR number and total AgNOR area increased from ADH to DCIS. The highest values were recorded in cases of DCIS with microinvasion. Differences between ADH and intraductal or microinvasive ductal carcinoma were statistically significant. Within the group of intraductal carcinomas, the lowest values were measured in the solid type and highest values in the comedo type. A correlation was found between AgNOR features and growth fraction but not between these features and ER/PR status. CONCLUSION: Selected AgNOR features are relevant for differentiation between ADH and DCIS as well as between low and high grade DCIS and microinvasive ductal carcinoma. Therefore, objective and reproducible data obtained by AgNOR analysis may allow better evaluation of the prognostic significance of these lesions.

Biomarkers, Tumor↗

Comparison of various imaging methods with particular evaluation of color Doppler sonography for planning surgery for breast tumors.

OBJECTIVE: Color Doppler sonography (CD) was compared with other diagnostic imaging methods [mammography (MG), breast ultrasound (US) and magnetic resonance imaging (MRI)] in the planning of surgery for breast tumors. MATERIALS AND METHODS: 99 patients with breast cancer and 101 with ultimately benign breast lesions were examined preoperatively. The specificity and sensitivity were calculated, as well as the predictive values. Various qualitative and semiquantitative CD parameters were also analysed for their diagnostic value. RESULTS: The sensitivity/specificity of the various methods (in %) was: MG 85/77; US 95/80; CD 82/75; MRI 90/63. The positive predictive value (ppv)/negative predictive value (npv) (in %) was: MG 79/83; US 81/94; CD 72/84; MRI 79/63. The median maximum systolic flow velocity and the resistance index (RI) were significantly higher in breast cancer vessels than in benign lesions. The number of pulsating color pixels detected by CD was significantly higher for breast cancer. In cases of breast cancer significantly more blood flows were detected in the body of the tumor than at its periphery. CONCLUSION: Color Doppler sonography was not superior to other diagnostic methods for preoperative assessment of a breast lesion. The combination of all diagnostic procedures gave a correct classification rate of 93.3% and is much better than the correct classification of any single diagnostic imaging procedure.

Aged↗

Characterization of IgE-reactive autoantigens in atopic dermatitis. 1. Subcellular distribution and tissue-specific expression.

We have previously reported that patients suffering from atopic dermatitis (AD) frequently display IgE autoantibody reactivity to human proteins expressed in cell lines of different histogenetic origins. Molecular cloning and in situ staining experiments revealed that certain IgE-reactive autoantigens were expressed preferentially in target organs of atopy while others could be detected in various tissues and cell types. Here we use serum IgE from AD patients to investigate the distribution of autoantigens in subcellular fractions of the human epithelial cell line A431 and in human tissue specimens. Results obtained showed that IgE-reactive autoantigens can be detected in the nuclear > microsomal > mitochondrial > cytoplasmic fraction of A431 cells as well as in a variety of human tissue specimens (brain, bone, intestine, liver, lung, muscle, skin, uterus) and effector cells of atopy (basophils, mast cells, T cells). If IgE autoreactivity plays a pathogenetic role in severe forms of atopy, organ-specific manifestations (e.g. AD) may result from the transport to and deposition of IgE-reactive autoantigens in certain target organs (skin) rather than from a preferential expression of the autoantigens in the affected tissues.

Adult↗

Characterization of IgE-reactive autoantigens in atopic dermatitis. 2. A pilot study on IgE versus IgG subclass response and seasonal variation of IgE autoreactivity.

Previously we reported that patients with severe forms of atopy (e.g. atopic dermatitis, AD) frequently display IgE reactivity against autoantigens. Here we investigated the effects of periodate treatment and reducing versus nonreducing conditions on IgE recognition of nitrocellulose-blotted human cell extracts. IgE and IgG subclass reactivities of AD patients to blotted human cellular extracts as well as to ELISA plate-bound purified endogenous (myosin, histones) antigens and an environmental allergen (timothy grass pollen allergen, Phl p 5) were compared. Serum samples were collected over a period of 12 months from 3 autoreactive AD patients with pollen allergy and tested for IgE reactivity to nitrocellulose-blotted human epithelial and endothelial cell extracts as well as to birch and timothy grass pollen allergens. Results obtained indicate that (1) IgE autoantibodies may be directed primarily against protein and not carbohydrate epitopes, (2) reducing conditions seem to expose or better extract epitopes recognized by IgE autoantibodies, (3) IgE and IgG1-4 autoantibody responses were poorly associated and (4) IgE responses to autoallergens may reflect skin manifestations and may be boosted by seasonal exposure to pollen allergens. Our results thus indicate that IgE autoreactivity may represent a true form of autoimmunity directed against partly denatured peptide epitopes which may be boosted by exogenous allergen contact.

Adult↗

Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.

Recently we demonstrated that a high percentage of atopic dermatitis (AD) patients displayed specific immunoglobulin E reactivity to human proteins. Here we show that IgE autoreactivity is found predominantly in AD patients with severe skin manifestations and reveal the molecular nature of four IgE autoantigens. An expression cDNA library constructed from a human epithelial cell line (A 431) was screened with serum IgE from two AD patients. DNA sequence analysis of three IgE-reactive clones identified the alpha-chain of the nascent polypeptide-associated complex, cytokeratin type II, and the BCL7B oncogen as atopy-related IgE autoantigens (ara). The fourth cDNA coded for an IgE autoantigen containing a typical calcium binding motif that occurred in histogenetically different cells and tissues (keratinocytes, muscle, brain). Recombinant Escherichia coli-expressed IgE autoantigens bound IgE from AD but not from patients with other immunologically mediated disorders (graft vs. host disease, systemic lupus erythematosus) and elicited immediate type skin reactions in AD patients. In serum samples collected from an AD patient over a period of 5 years, IgE anti-ara NAC antibody levels peaked during disease exacerbation. Our finding that ara BCL7B was detected in serum bound to IgE antibodies suggests that intracellular IgE autoantigens can become released after tissue damage and may occur as IgE immune complexes. Via binding to antigen presenting cells as well as to effector cells, IgE autoantigen immune complexes may contribute to exacerbation and/or perpetuation of severe atopic diseases even in the absence of exogenous allergens.

Amino Acid Sequence↗