Hautklinik Heidelberg

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis. An integrated transcriptome, genome, methylome and proteome analysis of over 200 lung adenocarcinomas reveals high rates of somatic mutations, 18 statistically significantly mutated genes including RIT1 and MGA, splicing changes, and alterations in MAPK and PI(3)K pathway activity. This report from The Cancer Genome Atlas Research Network presents molecular profiling of 230 resected untreated lung adenocarcinomas. Integrated analyses of transcriptome, genome, methylome and proteome collectively identify high rates of somatic mutation, significantly mutated genes including RIT1 and MGA, splicing alterations driven by somatic genomic changes, and point to as yet unidentified lesions that alter MAPK and PI(3)K pathway activity. These data establish a foundation for classification and further investigations of the leading cause of cancer death worldwide.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

32P-labelled DNA of HPV 16 which has been isolated and molecularly cloned from a cervical carcinoma (Dürst et al., 1983) was used to screen the cellular DNAs obtained from 20 different biopsies of Morbus Bowen or Bowenoid papulosis, respectively, by Southern blot analysis. Under conditions of differing stringency for the hybridization, HPV 16 DNA or related sequences were identified in 6 out of 10 cases of Morbus Bowen (4 out of 5 from a genital localization) and in 8 out of 10 biopsies from Bowenoid papulosis. One additional case of the latter disease contained DNA sequences of an HPV type not yet classified. There is evidence for the presence of another HPV DNA in two of the HPV-16-positive tumors. A large number of normal genital tissue samples were negative for HPV DNA.

In 2011, the U.S. National Lung Cancer Screening Trial (NLST) reported a 20% reduction of lung cancer mortality after regular screening by low‐dose computed tomography (LDCT), as compared to X‐ray screening. The introduction of lung cancer screening programs in Europe awaits confirmation of these first findings from European trials that started in parallel with the NLST. The German Lung cancer Screening Intervention (LUSI) is a randomized trial among 4,052 long‐term smokers, 50–69 years of age, recruited from the general population, comparing five annual rounds of LDCT screening (screening arm; n = 2,029 participants) with a control arm ( n = 2,023) followed by annual postal questionnaire inquiries. Data on lung cancer incidence and mortality and vital status were collected from hospitals or office‐based physicians, cancer registries, population registers and health offices. Over an average observation time of 8.8 years after randomization, the hazard ratio for lung cancer mortality was 0.74 (95% CI: 0.46–1.19; p = 0.21) among men and women combined. Modeling by sex, however showed a statistically significant reduction in lung cancer mortality among women (HR = 0.31 [95% CI: 0.10–0.96], p = 0.04), but not among men (HR = 0.94 [95% CI: 0.54–1.61], p = 0.81) screened by LDCT ( p heterogeneity = 0.09). Findings from LUSI are in line with those from other trials, including NLST, that suggest a stronger reduction of lung cancer mortality after LDCT screening among women as compared to men. This heterogeneity could be the result of different relative counts of lung tumor subtypes occurring in men and women.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) can sample enlarged mediastinal lymph nodes in patients with nonsmall cell lung cancer (NSCLC). To date, EBUS-TBNA has only been used to sample nodes visible on computed tomography (CT). The aim of the present study was to determine the accuracy of EBUS-TBNA in sampling nodes <or=1 cm in diameter. NSCLC patients with CT scans showing no enlarged lymph nodes (no node >1 cm) in the mediastinum underwent EBUS-TBNA. Identifiable lymph nodes at locations 2r, 2l, 4r, 4l, 7, 10r, 10l, 11r and 11l were aspirated. All patients underwent subsequent surgical staging. Diagnoses based on aspiration results were compared with those based on surgical results. In 100 patients (mean age 58.9 yrs; 68 males), 119 lymph nodes ranging 5-10 mm in size were detected and sampled. Malignancy was detected in 19 patients but missed in two; all diagnoses were confirmed by surgical findings. The mean diameter of the punctured lymph nodes was 8.1 mm. The sensitivity of EBUS-TBNA for detecting malignancy was 92.3%, specificity was 100%, and the negative predictive value was 96.3%. No complications occurred. In conclusion, endobronchial ultrasound-guided transbronchial needle aspiration can accurately sample even small mediastinal nodes, therefore avoiding unnecessary surgical exploration in one out of six patients who have no computed tomography evidence of mediastinal disease. Potentially operable patients with no signs of mediastinal involvement on computed tomography may benefit from pre-surgical endobronchial ultrasound-guided transbronchial needle aspiration and staging.

ABSTRACT Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain‐like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL‐deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes–both of ectodermal origin–are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL‐deficient mice is reminiscent of the spontaneous mouse mutant furless ( fs ). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.—Roth, W., Deussing, J., Botchkarev, V. A., Pauly‐Evers, M., Saftig, P., Hafner, A., Schmidt, P., Schmahl, W., Scherer, J., Anton‐Lamprecht, I., von Figura, K., Paus, R., Peters, C. Cathepsin L deficiency as molecular defect of furless : hyperproliferation of keratinocytes and pertubation of hair follicle cycling. FASEB J. 14, 2075–2086 (2000)

BACKGROUND: Nonmelanoma carcinomas of the skin represent the most frequent cancers among the Caucasian population worldwide. They occur with high frequency in renal allograft recipient patients after prolonged immunosuppression. PURPOSE: We analyzed tumors obtained from both immunosuppressed and nonimmunosuppressed patients for human papillomavirus (HPV) DNA. METHODS: Twenty-nine specimens of nonmelanoma carcinomas of the skin were obtained from 19 renal allograft recipient patients; these included 20 specimens of squamous cell carcinoma (SCC) from 11 patients, five specimens of basal cell carcinoma (BCC) from four patients, and four specimens of carcinoma in situ (CIS) from four patients. Forty-one specimens of nonmelanoma carcinomas of the skin were obtained from 32 nonimmunosuppressed patients; these included 26 SCC specimens from 19 patients, 11 BCC specimens from nine patients, and four keratoacanthoma (benign epithelial tumor) specimens from four patients. A polymerase chain reaction method involving use of degenerate oligonucleotide primers, in which the conserved region of the open reading frame of the HPV L1 (major capsid protein) gene is amplified, was used to amplify total cellular DNA purified from individual tumors. The DNA of each specimen was subjected to 16 different amplification reactions; different primer combinations were used in order to increase the sensitivity and specificity of HPV detection. Resulting products were probed with a radioactively labeled, degenerate oligonucleotide. HPV-specific DNA was either sequenced directly after elution from the gel or amplified with semi-nested, degenerate primers, after which the products were cloned and sequenced. Sequences were compared with all known papillomavirus sequences. RESULTS: Thirteen (65%) of the 20 SCC specimens and three of the five BCC specimens from immunosuppressed (renal allograft recipient) patients contained identifiable HPV-related sequences, among them 13 putative novel HPV genomes. In addition, all other malignant tumor specimens from this patient group revealed faint signals upon amplification and hybridization; the origin of these signals has not been identified in the present study. In nonimmunosuppressed patients, eight (31%) of 26 SCC specimens and four (36%) of 11 BCC specimens contained sequences of HPV types. Two putative novel HPV sequences could be identified in this group. Faint signals of yet undetermined origin were observed in eight of the SCC specimens and in two of the BCC specimens. Two of four keratoacanthoma specimens contained sequences of known HPV type. (Keratoacanthoma is a nonmalignant lesion for which the natural history has not been defined.) The spectrum of HPV types in both groups of patients differed substantially. CONCLUSIONS: These data point to the frequent presence of HPV sequences in SCCs and BCCs of the skin. The etiologic relationship of these infections to the respective malignant tumors remains to be evaluated. IMPLICATIONS: The presence of HPV DNA in a large percentage of specimens of nonmelanoma carcinomas of the skin from immunosuppressed patients, as well as from nonimmmunosuppressed patients, renders a papillomavirus infection as a possible factor in the etiology of this disease.

Seven biopsies from different carcinomas of the tongue were analyzed for human papilloma virus (HPV) sequences by Southern blot analysis. After hybridization with various types of human papilloma viruses, 3 tumors were found to be positive. Whereas DNA from one tumor hybridized with HPV 2 DNA under conditions of high stringency, the 2 other positive biopsies contained HPV 16 DNA. All positive tumors revealed high copy numbers of the respective DNA. The cleavage pattern of the HPV-2-positive tumor differed from the established HPV 2 prototypes. Minor differences from the HPV 16 prototype were also noted in one of the HPV-16-positive tongue carcinomas.

A monoclonal antibody is described that has been generated in the mouse against cultured human blood monocytes/macrophages. The antibody, designated 25F9, belongs to the IgG1 subclass, detects antigens of m.w. 86,000, and does not react with freshly isolated blood monocytes but reacts with monocytes after 3 days of culture. The expression of the 25F9 antigen on macrophages increases with culture time. Furthermore, the antibody is negative on platelets, granulocytes, lymphocytes, and a large number of human cell lines except the two melanoma lines MeWo and Mel 57. In cryostat sections of normal human tissue (skin, lung, liver, thymus) and of inflammatory or neoplastic tissue (cutaneous lymphoma, eczema, BCG-granuloma, and melanoma), the antibody reacts with scattered macrophages in the dermis but not with epidermal Langerhans cells, with alveolar macrophages, with liver Kupffer cells, and with scattered macrophages in the cortex and medulla of thymus. In eczema, BCG-granuloma, and cutaneous lymphoma, only a few infiltrating macrophages were stained. On the other hand, a large number of macrophages and melanophages reacted positively in melanoma. In some cases melanoma cells also stained weakly positive. Thus, the antibody detects a differentiation antigen preferentially expressed on mature, tissue-fixed macrophages and absent from blood monocytes.

We describe a newly developed multi-function video image analysis system for the computer-aided evaluation of capillaroscopic findings in microcirculation research. The Cap image analysis system comprises an IBM-compatible PC with a Matrox image processing card and real-time video tape digitalization. The video recorder is driven by a personal computer to which it is connected via an RS-232 interface. In contrast to currently available systems, the program presented here makes it possible to select any of several integrated image analysis functions, depending on the quality of the video image. Some examples of the analysis functions available are measurements of erythrocyte flow velocity using the line shift diagram method, the spatial correlation method, and the auto flying spot method. The standard features of the new program include a number of special functions and automatic movement correction. The system thus makes it possible not only to measure numerous morphological parameters such as capillary diameter, length, torquation index and capillary density, but also to perform video densitometric analysis, for example using fluorescent dyes.

Although known for almost 80 years, the physiological role of plasmalogens (PLs), the major mammalian ether lipids (ELs), is still enigmatic. Humans that lack ELs suffer from rhizomelic chondrodysplasia punctata (RCDP), a peroxisomal disorder usually resulting in death in early childhood. In order to learn more about the functions of ELs, we generated a mouse model for RCDP by a targeted disruption of the dihydroxyacetonephosphate acyltransferase gene. The mutant mice revealed multiple abnormalities, such as male infertility, defects in eye development, cataract and optic nerve hypoplasia, some of which were also observed in RCDP. Mass spectroscopic analysis demonstrated the presence of highly unsaturated fatty acids including docosahexaenoic acid (DHA) in brain PLs and the occurrence of PLs in lipid raft microdomains (LRMs) isolated from brain myelin. In mutants, PLs were completely absent and the concentration of brain DHA was reduced. The marker proteins flotillin-1 and F3/contactin were found in brain LRMs in reduced concentrations. In addition, the gap junctional protein connexin 43, known to be recruited to LRMs and essential for lens development and spermatogenesis, was down-regulated in embryonic fibroblasts of the EL-deficient mice. Free cholesterol, an important constituent of LRMs, was found in these fibroblasts to be accumulated in a perinuclear compartment. These data suggest that the EL-deficient mice allow the identification of new phenotypes not related so far to EL-deficiency (male sterility, defects in myelination and optic nerve hypoplasia) and indicate that PLs are required for the correct assembly and function of LRMs.

BACKGROUND: The purpose of this clinical trial was to compare efficacy and safety of the Boswellia serrata extract H15 with mesalazine for the treatment of active Crohn's disease. PATIENTS AND METHODS: Randomised, double-blind, verum-controlled, parallel group comparison for which 102 Patients were randomised. The per protocol population included 44 patients treated with H15 and 39 patients treated with mesalazine. As primary outcome measure the change of the Crohn Disease Activity Index (CDAI) between the status of enrolment and end of therapy was chosen. H 15 was tested on non-inferiority compared to standard treatment with mesalazine. RESULTS: The CDAI between the status of enrolment and end of therapy after treatment with H15 was reduced by 90 and after therapy with mesalazine by 53 scores in the mean. In this non-inferiority-trial the test hypothesis was confirmed by the statistical analysis. The difference between both treatments could not be proven to be statistically significant in favor to H15 for the primary outcome measure. The secondary efficacy endpoints confirm the assessment of the comparison of H15 and mesalazine. The proven tolerability of H15 completes the results of the shown clinical efficacy. CONCLUSIONS: The study confirms that therapy with H15 is not inferior to mesalazine. This can be interpreted as evidence for the efficacy of H15 according to the state of art in the treatment of active Crohn's disease with Boswellia serrata extract, since the efficacy of mesalazine for this indication has been approved by the health authorities. Considering both safety and efficacy of Boswellia serrata extract H15 it appears to be superior over mesalazine in terms of a benefit-risk-evaluation.

Bartonella henselae causes vasculoproliferative disorders in humans. We identified a nonfimbrial adhesin of B. henselae designated as Bartonella adhesin A (BadA). BadA is a 340-kD outer membrane protein encoded by the 9.3-kb badA gene. It has a modular structure and contains domains homologous to the Yersinia enterocolitica nonfimbrial adhesin (Yersinia adhesin A). Expression of BadA was restored in a BadA-deficient transposon mutant by complementation in trans. BadA mediates the binding of B. henselae to extracellular matrix proteins and to endothelial cells, possibly via beta1 integrins, but prevents phagocytosis. Expression of BadA is crucial for activation of hypoxia-inducible factor 1 in host cells by B. henselae and secretion of proangiogenic cytokines (e.g., vascular endothelial growth factor). BadA is immunodominant in B. henselae-infected patients and rodents, indicating that it is expressed during Bartonella infections. Our results suggest that BadA, the largest characterized bacterial protein thus far, is a major pathogenicity factor of B. henselae with a potential role in the induction of vasculoproliferative disorders.

BACKGROUND: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses. METHODS: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61). RESULTS: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions. CONCLUSION: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01915524 .

BACKGROUND: Hereditary angioedema (HAE) is an autosomal dominant disease (Mendelian Inheritance in Man 106100) caused by an inherited deficiency of C1 inhibitor (C1-INH) function. The clinical symptoms include skin swelling, abdominal pain, and life-threatening episodes of upper airway obstruction. We evaluated the efficacy of C1-INH concentrate for treating sudden airway compromise. METHODS: A series of 95 patients with HAE and a functional deficiency of C1-INH belonging to 59 families underwent screening for laryngeal edema. Double-blind treatment of randomized patients was not justifiable because of the life-threatening nature of this condition. Efficacy was evaluated by determining the interval from injection of C1-INH concentrate to the beginning of resolution of symptoms. The mean duration of episodes of laryngeal edema was compared in treated and untreated patients. Clinical information was obtained from emergency department physicians, the hospitals involved, reports of the general practitioners, and patients and their relatives. RESULTS: Forty-two patients had 517 episodes of laryngeal edema. Eighteen patients received 500- or 1000-U injections of C1-INH concentrate in 193 episodes. The C1-INH concentrate was effective in all laryngeal edemas. The interval from injection to interruption in progress of symptoms ranged from 10 minutes to 4 hours (mean +/- SD, 42.2 +/- 19.9 minutes). The mean +/- SD duration of laryngeal edema was 15.3 +/- 9.3 hours in patients who received C1-INH concentrate and 100.8 +/- 26.2 hours in those who did not. CONCLUSIONS: Injected C1-INH concentrate is highly and rapidly effective in the treatment of laryngeal edema of HAE. Relief and resolution of symptoms begins 30 to 60 minutes after injection, and duration of the upper airway obstruction is substantially reduced.

The objective of this study was to determine the frequency of reactivity to a series of commonly used fragrances in dermatological patients. A total of 48 fragrances (FF) were chosen, based on the publication of Fenn in 1989 in which the top 25 constituents of 3 types (1. perfumes, 2. household products, 3. soaps) of 400 commercial products on the US market had been determined. In a pilot study on a total of 1069 patients in 11 centres, the appropriate test concentration and vehicle were examined. For most fragrances, 1% and 5% were chosen, and petrolatum proved to be the best vehicle in comparison to isopropyl myristate and diethyl phthalate. In the main study, a set of 5 to 10 fragrances at 2 concentrations was patch tested in each centre on a minimum of 100 consecutive patients seen in the patch test clinic. These patients were also patch tested to a standard series with the 8% fragrance mix (FM) and its 8 constituents. In patients with a positive reaction to any of the 48 FF, a careful history with regard to past or present reactions to perfumed products was taken. A total of 1323 patients were tested in 11 centres. The 8% FM was positive in 89 patients (8.3% of 1072 patients). Allergic reactions to the constituents were most frequent to oak moss (24), isoeugenol (20), eugenol (13), cinnamic aldehyde (10) and geraniol (8). Reactions read as allergic on day 3/4 were observed only 10X to 7 materials of the new series (Iso E Super (2), Lyral (3), Cyclacet (1), DMBCA (1), Vertofix (1), citronellol (1) and amyl salicylate (1)). The remaining 41 fragrances were negative. 28 irritant or doubtful reactions on day 3/4 were observed to a total of 19 FF materials (more than 1 reaction: 5% citronellol (2), 1% amyl salicylate (2), 1% isononyl acetate (3), 0.1% musk xylol (2), 1% citral (2), and 1% ionone beta (2)). Clinical relevance of positive reactions to any of the FF series was not proved in a single case. This included the 4 reactions in patients who were negative to the 8% FM. In conclusion, the top 25 fragrances commonly found in various products caused few reactions in dermatological patients and these few appeared to be clinically irrelevant, with the possible exception of Lyral. However, this data should be interpreted in the light of the relatively small number of patients tested (only 100 in most centres).

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of approximately 5 x 10(-3) s(-1), corresponding to 8-9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at approximately 1,500+/-700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.

Preservatives are biologically reactive substances, and their allergenic potential has been known for a long time. This study examined the role of different preservatives in a large number of patients with suspected allergic contact dermatitis. Patch test data and data from the patients' history were collected from the 24 departments participating in the Information Network of Departments of Dermatology from 1 January 1990 to 31 December 1994. Patch test data from 28,349 patients tested with preservatives of the standard series (SS), from 11,485 patients tested additionally with a preservative series (PS), and from 1787 patients tested with an industrial biocide tray (IB) were evaluated. Sensitization rates (standardized) of the SS preservatives were all > 1%, with thiomersal rating highest (5.3%), the parabens lowest (1.6%), and the remainder (chlormethylisothiazolinone/methylisothiazolinone, formaldehyde and methyldibromoglutaronitrile/phenoxyethanol (MDBGN/PE)) in the range of 2%. The most important allergens of the PS were, in women, alkylaminobenzoate (contained in milking fat) (2.5%), MDBGN/PE (2.2%), benzalkonium chloride (1.8%), chloracetamide (1.4%), diazolidinyl urea (1.3%), octylgallate (1.2%) and Bronopol (1.1%). In men rates differed only with regard to alkylaminobenzoate (0.9%). Patients tested with the IB series reacted most often to methylene-bis-thiocyanate (5%), but with a reaction index of -0.7, many reactions were most probably false positives. A further seven preservatives, mostly formaldehyde-releasers used in cutting fluids, gave sensitization rates of between 1% and 3%. Glutaraldehyde, not contained in the series but often tested additionally, showed a remarkable increase in sensitization during the study period. Health care personnel were frequently affected. Altogether, this study identified areas of concern within the different groups of preservatives. The overall impact of most of the preservatives on public health seems to be low, but for diagnostic reasons preservatives must be included in patch test series.

The efficacy and safety of zanolimumab in patients with refractory cutaneous T-cell lymphoma (CTCL) have been assessed in two phase 2, multicenter, prospective, open-label, uncontrolled clinical studies. Patients with treatment refractory CD4(+) CTCL (mycosis fungoides [MF], n = 38; Sézary syndrome [SS], n = 9) received 17 weekly infusions of zanolimumab (early-stage patients, 280 and 560 mg; advanced-stage patients, 280 and 980 mg). The primary end point was objective response (OR) as assessed by composite assessment of index lesion disease activity score. Secondary end points included physician's global assessment (PGA), time to response, response duration, and time to progression. ORs were recorded for patients in both CTCL types (MF, 13 ORs; SS, 2 ORs). In the high-dose groups (560 and 980 mg dose groups), a response rate of 56% was obtained with a median response of 81 weeks. Adverse events reported most frequently included low-grade infections and eczematous dermatitis. Zanolimumab showed marked clinical efficacy in the treatment of patients with refractory MF, with early onset of response, high response rate, and durable responses. The treatment was well tolerated with no dose-related toxicity other than the targeted depletion of peripheral T cells. A pivotal study has been initiated based on these findings.

A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at Mr 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-gamma (IFN-gamma), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) but not with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphocytes, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG granulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr. From this it is concluded that the antibody 27E10 detects an antigen expressed by a subset of peripheral blood monocytes. In situ the antigen is found only in inflammatory tissues and is absent from normal resident mononuclear phagocytes.