Actin-dependent deposition of putative endosomes and endoplasmic reticulum during early stages of wound healing in characean internodal cells.

We investigated the behaviour of organelles stained with FM1-43 (putative endosomes) and/or LysoTracker Red (LTred; acidic compartments) and of the endoplasmic reticulum (ER) during healing of puncture and UV-induced wounds in internodal cells of Nitella flexilis and Chara corallina. Immediately after puncture, wounds were passively sealed with a plug of solid vacuolar inclusions, onto which a bipartite wound wall was actively deposited. The outer, callose-containing amorphous layer consisted of remnants of FM1-43- and LTred-labelled organelles, ER cisternae and polysaccharide-containing secretory vesicles, which became deposited in the absence of membrane retrieval (compound exocytosis). During formation of the inner cellulosic layer, exocytosis of secretory vesicles with the newly formed plasma membrane is coupled to endocytosis via coated vesicles. Migration of FM1-43- and LTred-stained organelles, ER and secretory vesicles towards the cell cortex and deposition of a bipartite wound wall could also be induced by spot-like irradiation with ultraviolet light. Cytochalasin D reversibly inhibited the accumulation and deposition of organelles. Our study indicates that active actin-dependent deposition of putative recycling endosomes is required for wound healing (plasma membrane repair) and supports the hypothesis that deposition of ER cisternae helps to restore wounding-disturbed Ca(2+) metabolism.

Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus.

In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones. Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus. We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg). Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles. Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans. More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed. Although HIV-1 gp 160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro. These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented. Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component.

Restricted antigenic variability of the epitope recognized by the neutralizing gp41 antibody 2F5.

OBJECTIVE: To investigate whether variations of the conserved gp41 amino-acid sequence ELDKWA affect its binding or neutralization by monoclonal antibody (MAb) 2F5. DESIGN AND METHODS: Neutralization assays were performed with primary isolates from different HIV-1 subtypes and the sequences corresponding to the 2F5 epitope region were analysed. Studies of MAb 2F5 peptide reactivity were performed by spot analysis, using peptides immobilized on cellulose. The frequency of emergence of neutralization-resistant virus variants was determined by immune selection experiments in the presence of MAb 2F5. RESULTS: Primary isolates from clades A, B and E were neutralized by MAb 2F5. Neutralization sensitivity correlated with the presence of the LDKW motif. A K-to-N change in the core sequence was identified in a neutralization-resistant patient isolate. Neutralization resistant virus variants that were selected in the presence of MAb 2F5 were found to contain D-to-N, D-to-E, or K-to-N changes within the LDKW sequence. Neither in natural isolates nor in variants obtained under immune selection conditions in the laboratory were changes in the L and W positions observed. Studies of MAb 2F5 binding to variations of the ELDKWA peptide confirmed that the changes at the first and last positions did not significantly reduce binding capacity, whereas amino-acid changes from D to N, D to E, and K to N almost completely abrogated binding of MAb 2F5. CONCLUSION: Sequence analysis of a variety of primary isolates suggests that the major determinant of MAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif.

Mucosal model of immunization against human immunodeficiency virus type 1 with a chimeric influenza virus.

Previously, we constructed a chimeric influenza virus that expresses the highly conserved amino acid sequence ELDKWA of gp41 of human immunodeficiency virus type 1 (HIV-1). Antisera elicited in mice by infection with this chimeric virus showed neutralizing activity against distantly related HIV-1 isolates (T. Muster, R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger, J. Virol. 68:4031-4034, 1994). In the present study, we demonstrated that intranasal immunizations with this chimeric virus are also able to induce a humoral immune response at the mucosal level. The immunized mice had ELDKWA-specific immunoglobulins A in respiratory, intestinal, and vaginal secretions. Sustained levels of these secretory immunoglobulins A were detectable for more than 1 year after immunization. The results show that influenza virus can be used to efficiently induce secretory antibodies against antigens from foreign pathogens. Since long-lasting mucosal immunity in the genital and intestinal tracts might be essential for protective immunity against HIV-1, influenza virus appears to be a promising vector for HIV-1-derived immunogens.

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1.

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.

[The viral and allergic origin of nasal polyposis]. / Untersuchungen zur viralen und allergischen Genese der Polyposis nasi.

In spite of recent advances in basic science, the etiology and pathogenesis of nasal and paranasal polyps have still not been clarified. Well-defined causal factors are hereditary diseases such as cystic fibrosis and primary ciliary dyskinesia. Allergic and non-allergic processes of the airways, particularly intrinsic asthma and intolerance to non-steroidal antiinflammatory drugs, are implicated in the development of nasal polyposis. In the 1930s nasal polyps were strongly considered to be a manifestation of allergy. The theory was challenged in the 1970s. Many patients with nasal polyps have no identifiable allergic disease. The cause of nasal polyps is speculative. 221 patients were included in this study, 154 suffering from chronic rhinosinusitis and another 46 from nasal polyps. The incidences of aspirin intolerance and of allergy were of no statistic relevance. The data indicate that preoperative cortisone application does improve the disease-free interval.

Multimodality treatment in stage IV squamous cell carcinoma of the head and neck. A long-term follow-up.

We treated 114 patients with advanced inoperable head and neck cancer with a combined-modality protocol that included two cycles of chemotherapy followed by radiotherapy or three chemotherapy and in 18 patients with a radiosensitizing agent. At the beginning of the treatment all but one patient presented with a stage IV cancer. With a follow-up of 42-58 months, four patients are alive (three from the radiosensitizing group and one of the chemotherapy group). Complete response after the radiosensitizing agent correlated with superior prolonged disease-free survival in comparison to complete responses after chemotherapy at the level of p less than 0.009.

[Possibilities of treating nasal turbinates in the framework of endonasal microsurgery]. / Möglichkeiten der Nasenmuschelbehandlung im Rahmen der endonasalen Mikrochirurgie.

Pathologic alterations of the turbinates are common in infectious diseases of the nose and paranasal sinuses. Over 300 patients were treated for paranasal sinuses disease from 1986 to 1989 by endonasal microsurgery. One-third of the patients were found intraoperatively to have alterations of the middle and inferior turbinate causing obstruction and functional disorders of the nose and sinuses. Our concept comprises a step-by-step microsurgical approach to the turbinates. The technique and the results are presented.

[Computerized tomography as routine examination procedure in the diagnosis of paranasal sinus diseases from the otorhinolaryngologist's viewpoint. Results]. / Die Computertomographie als Standarduntersuchungs-verfahren zur Nasennebenhöhlendiagnostik aus der Sicht des Hals-Nasen-Ohren-Arztes. Ergebnisse.

The renaissance of endonasal microsurgery is not only based on a deeper knowledge of the mucociliar clearance and better operation instruments but depends as well as on the improved radiological diagnosis provided by computed tomography. Since 1986, high-resolution computed tomography in two plains has been part of the authors' preoperative routine diagnostic program for paranasal sinus diseases at their clinic. One hundred and fifty-eight computed tomograms of patients with chronic rhinosinusitis, complicated acute rhinosinusitis, and polyposis nasi were reexamined. The involvement of the paranasal sinuses and abnormalities of the lateral nasal wall were analyzed. The anterior ethmoid is the most frequently affected area in all of the diseases. The computed tomogram is most important for the rhinosurgeon. The ethmoid cell system can be visualized without superimposing structures. Therefore even circumscript pathologic mucosa alterations and the extent of the disease can be defined. All this enables the surgeon to put forward an exact diagnosis and to plan a safe operation as regards important physiological structures of the nose and paranasal sinuses.

[Preoperative chemotherapy in head-neck tumors. Chances and risks of this therapeutic procedure]. / Pröperative Chemotherapie bei Kopf-Hals-Tumoren. Chancen und Risiken dieses therapeutischen Vorgehens.

We submit our present work dealing with stage III and IV squamous cell carcinomas of the head and neck referred for preoperative chemotherapy. Our results are based on a histological serial section investigation of the tumour tissues. In at least 50% inadequate tumour resection was observed with a tendency to increase if preoperative chemotherapy was conducted.

Sudden sensorineural hearing loss influence of hemodynamical and hemorheological factors on spontaneous recovery and therapy results.

The effect of a hemodilution therapy for sudden sensorineural hearing loss was analysed comparing the results of a prospective randomised double blind study and a retrospective study at the Frankfurt university. The prospective study consists of a treatment group receiving infusions with hydroxyethyl starch-pentoxifyllin and a control group with infusions with normal saline. Another patient group receiving hydroxyethyl starch-pentoxifyllin-naftidrofuryl infusions was analysed retrospectively. The overall therapy results are in the same range for all 3 groups. However patients with a hemoglobin value above 14 mg % in the control group had a poorer result compared to the other groups. The patient group with systolic blood pressure above 130 mm Hg treated with hydroxyethyl starch-pentoxifyllin had a significant hearing improvement over normal saline and hydroxyethyl starch-pentoxifyllin-naftidrofuryl. With increasing systolic blood pressure, patients in the control and hydroxyethyl starch-pentoxifyllin-naftidrofuryl groups had a statistically significant lower therapy result.

Radiosensitizing with cis-platin in advanced head and neck cancer. Results and problems.

Treatment consisting of surgery and/or radiation therapy for patients with squamous cell carcinoma of the head and neck has frequently been successful in earlier stages of disease. Advanced and non-resectable tumor stages have a very poor cure rate. We initiated this trail to assess the role of the potentiation between cis-PDD and radiation previously reported in advanced head and neck tumors. Eighteen patients were investigated in this study. The treatment consisted of cis-PDD and hyperfractionated radiotherapy. Seventeen (94%) of the patients responded to the treatment regimen with either a complete regression (5/18 = 33%) or a partial regression (11/18 = 61%) of the tumor. Median survival was short and lasted 12+ months among complete responders and 8+ months among partial responders. However all patients did experience an increased and not tolerable incidence of delayed radiation toxicity such as mucositis combined with necrotic stomatitis. Both complications limited the compliance to the therapy. Because of these complications we had to stop the ongoing study.

Proliferation-associated monoclonal antibodies: a useful tool for prognosis in squamous cell carcinomas of head and neck?

Three different proliferation-associated monoclonal antibodies have been studied immunohistochemically to evaluate their prognostic value in patients with squamous cell carcinomas of head and neck. The monoclonal antibody Ki 67 shows a heterogeneous distribution pattern, whereas epidermal growth factor receptor and transferrin receptor expression show a homogeneous one. Concluding from our results the chances to use the described monoclonal antibodies as prognostic markers are limited.

[Pathohistologic studies of serial sections of squamous cell cancer of the oral cavity and oropharynx following cytostatic induction therapy with bleomycin and cisplatin]. / Pathohistologische Serienschnittuntersuchungen bei Plattenepithelkarzinomen der Mundhöhle und des oropharynx nach zytostatischer Induktionstherapie mit Bleomycin und cis-platin.

43 patients with advanced squamous cell carcinoma of the oral cavity and oropharynx were treated preoperatively by a combined chemotherapy with bleomycin and cisplatinum. After surgery histological serial sections of resection specimens were examined. In no case did the tumour tissues show a complete response. After induction chemotherapy 35 patients had histologically partial response - shrinkage by more than 50%. In these cases histological evaluation revealed tumour areas with pseudocysts containing on the one hand necrotic material and showing keratinisation on the other. A tumour was also found histologically under regrown epithelium in 10 cases where complete tumour remission was postulated clinically. Eight patients showed histologically a minor response - shrinkage below 50% with only little necrotic areas. Exophytic tumours responded better to chemotherapy than endophytic tumours. Endophytic tumours showed small isolated tumour areas in the periphery of the infiltrated tissues.

[Pathohistologic serial section studies in patients with complete tumor remissions after completed cytostatic induction therapy]. / Pathohistologische Serienschnittuntersuchungen bei Patienten mit kompletter Tumorremission nach durchgeführter zytostatischer Induktionstherapie.

Initial combination drug regimen containing cis-platin in patients with head and neck cancer produced a high percentage of clinical response. Despite numerous trials showing high tumor response rates, a survival advantage resulting from the use of chemotherapy has yet not been demonstrated conclusively. This trial was initiated to study the cytotoxic effect of chemotherapeutic agents on squamous cell carcinomas. 31 patients with oropharyngeal carcinomas received prior to surgical resection a polychemotherapy consisting of cis-platin and bleomycin. After surgical tumor resection the histopathologic examination based on the serial sections of the tumor tissues and neck dissection blocks showed no case of complete tumor response. Even in 9 patients with complete clinical tumor response the histopathological investigation revealed tumor cells.

Adjuvant chemotherapy in stage III and IV squamous cell carcinoma of the head and neck.

Initial combination drug regimen containing cisplatin in patients with stage III and IV head and neck cancer produced a high percentage of clinical response. This trial was initiated to assess the role of multimodality treatment (chemotherapy plus radiotherapy) versus chemotherapy alone. Ninety-six patients entered into this study; 80 patients were evaluable at time of analysis (Table I). Patients were randomized between chemotherapy and radiotherapy (group I) and chemotherapy alone (group II). The chemotherapy administered consisted of cisplatin, bleomycin and methotrexate and was given in 2 cycles over 35 days. Local radiotherapy followed. In group II 3 cycles of chemotherapy were given without radiotherapy. The overall tumour response after chemotherapy rose up to 75 per cent. After radiotherapy in group II the response rate sank to 59 per cent. In both regimes the remission duration was very short. Patients receiving only two cycles of chemotherapy do not have a statistically shorter survival than patients, who were treated by chemotherapy plus radiotherapy, or by a 3rd cycle of chemotherapy.

Laryngoplasty: results and problems after the creation of a neoglottis.

Voice rehabilitation in the laryngectomy patient has been a major goal of reconstructive laryngeal surgery. 36 patients underwent laryngectomy with the creation of a phonatory neoglottis as outlined by Staffieri. In 13 patients good voice rehabilitation was achieved. In 23 patients who failed to develop voice production, altered swallowing function was the major problem.

[Measuring cis-dichlorodiamino platinum(II) concentrations in human blood and mouth mucosa]. / Messungen von Cis-Dichlorodiamino-Platin (II) Konzentrationen in Plasma und Mundschleimhaut des Menschen.

Using the method of atomic absorption spectroscopy, platinum (DDP) concentrations were determined in tissue samples and plasma from 10 patients who suffered from a squamous cell carcinoma and were receiving cis-platinum. The drug was administered by a rapid infusion (60 mg/m2). Tissue samples and plasma were collected 5 h after infusion. Platinum concentrations found in squamous cells of the oral cavity varied from 3 to 20 micrograms/g with a median concentration of 7.5 micrograms/g. The corresponding plasma concentrations were 1.5-2.5 micrograms/ml. The data suggest that within 5 h after infusion an enrichment of cis-platinum takes place in human tissue.

[Effectiveness of the radiosensitizer cisplatin. A pilot study]. / Zur Wirksamkeit des Radiosensitizers Cis-Platin. Eine Pilotstudie.

18 patients with advanced squamous cell carcinoma of the head and neck region were treated via chemotherapy and radio therapy. In this regimen cisplatinum was used as a radiosensitizer. The total remission rate was 94% while remission duration and survival could not be calculated. Severe clinical local toxicity occurred as a side effect.