Langerhans cell histiocytosis as differential diagnosis of a mediastinal tumor

We describe the case of a 55-year-old man who presented with parasternal swelling. The chest CT scan showed a large tumor of the chest wall infiltrating the subcutaneous tissue. To assume histologic diagnosis an open biopsy was performed. Between the myofibrils a coarse, white tumor with infiltrative growth was noted. Histopathologic examination revealed expanded atrophic skeletal muscle that was infiltrated by histiocytic cells. Numerous eosinophilic granulocytes and lymphocytes CD20 and CD3 positive could be detected and immunohistochemical staining was also positive for S-100 proteins and CD1a. Histologic findings were characteristic of Langerhans cell histiocytosis (LCH). To the best of our knowledge a LCH originating from the mediastinum in an adult as presented has not been previously described.

In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test

BACKGROUND Patients with Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative. OBJECTIVE As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug. METHODS Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3(+) CD4(+), CD3(+) CD8(+) and NKp46(+) cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated. RESULTS Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8-55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12-62%). The expression of granulysin was significantly induced in NKp46(+) and CD3(+) CD4(+) cells (sensitivity 40%, CI: 16-68% and 53%, CI: 27-79% respectively). Cytokine production could be demonstrated in 38%, CI: 14-68% and 43%, CI: 18-71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23-83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80-99%)-100% (CI: 90-100%). CONCLUSIONS AND CLINICAL RELEVANCE Granulysin expression in CD3(+) CD4(+) , Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52-96%) and specificity of 95% (80-99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.

IMMUNOGLOBULINS IN PERIODONTAL DISEASE

Periodontal disease is the major cause of tooth loss in man. The initial histological picture of the inflamed gingiva is characteristic of local inflammatory reaction involving polymorphonuclear leukocytes, vasculitis and localized tissue loss. Subsequent clinical stages of periodontal disease (mild gingivitis) show histological evidence of the involvement of the immune response with initial accumulation of macrophages, and lymphocytes devoid of surface staining immunoglobulins (presumably T cells). As the disease progresses, a predominance of surface and cytoplasmic staining lymphocytes and plasma cells are seen (severe gingivitis and periodontitis). Whether the occurrence of the immunoglobulin positive lymphocytes and the concurrent loss of collagen and resorption of alveolar bone seen in periodontitis is indicative of a direct cause and effect relationship has been a controversy.^ The majority of investigations in the periodontal field have involved the use of peripheral blood lymphocytes or serum. Blastogenic responses of peripheral blood lymphocytes and serum antibody titers from periodontal patients to a variety of oral bacteria have not shown any correlation between response and the severity of disease. The need to study the local immune response in inflamed gingiva is apparent. Since there are no baseline studies on the functional capabilities of the lymphoid cells present in gingiva from periodontitis patients, an in depth study involving the role of the immunoglobulin positive lymphocytes was investigated.^ Inflamed gingiva from four clinically defined periodontal disease states (mild gingivitis, severe gingivitis, periodontitis and severe periodontitis) were placed in gingival organ cultures. Class specific immunoglobulins were quantitated in gingival organ culture supernatants using an indirect sandwich technique. A significant difference in mean levels of IgA and IgG was seen between mild gingivitis and periodontitis (P < .00l, P = .001), as well as in IgG levels between periodontitis and severe periodontitis (P = .001). The predominance of IgG in gingival organ culture supernatants and the statistically significant findings that the overall mean levels of IgG between mild gingivitis and periodontitis (P = .014) and between severe periodontitis and periodontitis (P = .001) suggested a possible indicator of periodontal disease. The presence of IgG in gingival organ culture supernatants was shown to be a product of actively secreting plasma cells. The incorporation of radiolabelled amino acids into IgG was noted over a seven-day period with a peak response at day 4-5. The inhibition of IgG synthesis by cyclohexamide confirmed the contention that IgG was a product of de novo synthesis and not serum derived.^ The specificity of immunoglobulins derived from gingival organ cultures were studied using a whole bacterial agglutination test. Oral bacteria frequently cultured from periodontal patients were assessed for their ability to be agglutinated by gingival organ culture supernatants. A positive correlation of antibody titer and severity of disease was seen with five strains of Actinomyces viscosus, two of Actinomyces naeslundii and one Actinomyces israelii. The agglutination of bacteria was shown to be due to the specific interaction of immunoglobulin and cell-wall antigen. ^

Oberflächenveränderung verschiedener zahnmedizinischer Werk-stoffe nach in-vitro Alterung

Susceptibility of different restorative materials to toothbrush abrasion and coffee staining Objective: The aim of this study was to evaluate the susceptibility of different restorative materials to surface alterations after an aging simulation. Methods: Specimens (n=15 per material) of five different restorative materials (CER: ceramic/Vita Mark II; EMP: composite/Empress Direct; LAV: CAD/CAM composite/Lava Ultimate; COM: prefabricated composite/Componeer; VEN: prefabricated composite/Venear) were produced. Whereas CER was glazed, EMP and LAV were polished with silicon polishers, and COM and VEN were left untreated. Mean roughness (Ra and Rz) and colorimetric parameters (L*a*b*), expressed as colour change (E), were measured. The specimens underwent an artificial aging procedure. After baseline measurements (M1), the specimens were successively immersed for 24 hours in coffee (M2), abraded in a toothbrushing simulator (M3), immersed in coffee (M4), abraded (M5) and repeatedly abraded (M6). After each aging procedure (M2-M6), surface roughness and colorimetric parameters were recorded. Differences between the materials regarding Ra/Rz and E were analysed with a nonparametric ANOVA analysis. The level of significance was set at α=0.05. Results: The lowest roughness values were obtained for CER. A significant increase in Ra was detected for EMP, COM and VEN compared to CER. The Ra/Rz values were found to be highly significantly different for the materials and measuring times (M) (p<0.0001). Regarding E most alterations were found for EMP and COM, whereas CER and LAV remained mostly stable. The E values were significantly different for the materials and M (p<0.0001). Conclusion: The ceramic and the CAD/CAM composite were the most stable materials with regard to roughness and colour change and the only materials that resulted in Ra values below 0.2 μm (the clinically relevant threshold). Venears and Componeers were more inert than the direct composite material and thus might be an alternative for extensive restorations in the aesthetic zone.

Ultrasound-guided proximal paravertebral anaesthesia in cattle

OBJECTIVE To develop and evaluate a method for ultrasound-guidance in performing the proximal paravertebral block for flank anaesthesia in cattle through a cadaveric study, followed by clinical application. STUDY DESIGN prospective experimental cadaveric study and clinical series. ANIMALS Previously frozen lumbar sections of cows without known spinal abnormalities were used. The clinical case group comprised of ten animals for which a right flank laparotomy was indicated. METHODS Twenty cow cadavers were used to perform ultrasound-guided bilateral injections of 1.0 mL dye (1.0 mL 1% Toluidine Blue in 1% Borax) at the intervertebral foramen at the level of T13, L1 and L2 spinal nerves. Distance and depth of injection, staining of the dorsal and ventral nerve branches, and deviation from the target were evaluated. The investigator's confidence as to visualisation and expected success at staining the nerve was assessed. Ten clinical cases received the ultrasound-guided proximal paravertebral anaesthesia. Analgesic success was evaluated using a 4-grade scoring system at 10 minutes after the injection and during surgery, respectively. Categorical variables were described using frequencies and proportions. RESULTS Both dorsal and ventral branches of the spinal nerves T13, L1 or L2 were at least partially stained in 41% of injections, while in 77% of injections one of the branches was stained. Five out of ten clinical cases had a satisfactory anaesthesia. There was no significant association between confidence at injection and either staining or analgesic success. CONCLUSION Results from the cadaveric and clinical study suggest no significant improvement using ultrasound guidance to perform proximal paravertebral block in cows compared to our previous clinical experience and to references in the literature using the blind method. CLINICAL RELEVANCE Further research should be conducted to improve the ultrasound-guided technique described in this study.

HLA class II antigen expression in colorectal carcinoma tumors as a favorable prognostic marker.

The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes.

In vitro analysis of osteogenic inhibition in non-union spinal fusion caused by nucleus pulposus cells.

Discectomy and spinal fusion is the gold standard for spinal surgery to relieve pain. However, fusion can be hindered for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. Clinical observations indicate that presence of residual intervertebral disc (IVD) tissue might hinder the ossification. We hypothesize that BMP-antagonists are constantly secreted by IVD cells and potentially prevent the ossification process. Furthermore, L51P, the engineered BMP2 variant, stimulates osseo-induction of bone marrow-derived mesenchymal stem cells (MSC) by antagonizing BMP-inhibitors. Human MSCs, primary nucleus pulposus (NPC) and annulus pulposus cells (AFC) were isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4Mio/mL) and separated by culture inserts from MSCs. MSCs were kept in 1:control medium, 2:osteogenic medium±alginate beads, 3:osteogenic medium+NPC (±L51P) and 4:osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related genes, alkaline phosphatase assay and histological staining were performed. Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC. No such inhibition was observed if co-cultured with alginate only or in the presence of AFC. The results were confirmed on the RNA and protein level. Addition of L51Pto the co- cultures, however, induced mineralization of MSCs in presence of NPC. We demonstrated that NPC secrete BMP-antagonists that prevent osteogenesis of MSCs and L51P can antagonize BMP-antagonists and induce bone formation.

Investigation of bone inhibition in non-union spinal fusion caused by intervertebral disc cells in vitro

Introduction: Discectomy and spinal fusion is the gold standard for spinal surgery to relieve pain. However, fusion can be hindered for yet unknown reasons that lead to non-fusions with pseudo-arthrose. It is hence appealing to develop biomaterials that can enhance bone formation. Clinical observations indicate that presence of residual intervertebral disc (IVD) tissue might hinder the ossification. We hypothesize that BMP-antagonists are constantly secreted by IVD cells and potentially prevent the ossification process. Furthermore, L51P, the engineered BMP2 variant, stimulates osteoinduction of bone marrow-derived mesenchymal stem cells (MSC) by antagonizing BMP-inhibitors. Methods: Human MSCs, primary nucleus pulposus (NPC) and annulus pulposus cells (AFC) were isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4Mio/mL) and separated by culture inserts from MSCs in a co-culture set-up. MSCs were kept in 1:control medium, 2:osteogenic medium+alginate control, 3:osteogenic medium+NPC (±L51P) and 4:osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related genes, Alkaline Phosphatase (ALP) assay and histological staining were performed. Results: Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC. No such inhibition was observed if co-cultured with alginate only or in the presence of AFC. The results were confirmed on the RNA and protein level. Addition of L51P to the co-cultures induced mineralization of MSCs, however a reduced ALP was observed. Conclusion: We demonstrated that NPC secrete BMP-antagonists that prevent osteogenesis of MSCs and L51P can antagonize BMP-antagonists and induce bone formation.

Monitoring of industrial exposure for chloracne

This study (1) established comedogenicity dose response curves for the pure compounds of 3,3$\sp\prime$,4,4$\sp\prime$-tetrachloroazobenzene (TCAB) and 3,3$\sp\prime$,4,4$\sp\prime$-tetrachloroazoxybenzene (TCAOB) individually and as a couple-compound using a rabbit ear model; (2) used a rabbit ear model to establish comedogenicity potential for TCAB and TCAOB as they existed in a given industrial herbicide manufacture process; (3) evaluated actual environmental contamination in a herbicide industrial setting by air monitoring and wipe sampling; (4) biologically monitored potentially exposed workers for alterations in follicular orifice size as an index of actual exposure to chloracnegenic compounds; and (5) biologically monitored potentially exposed workers for changes in weight, cholesterol, triglycerides and blood sugar.^ A silastic monomer mold (an objective measure) was used to measure change in follicular orifice size over time. This required taking impressions of (1) skin of the forehead and right and left malar crescents of workers and (2) the skin of the external ear of the rabbit. Molds were stained using a solution of hematoxylin and digitized using a Nikon UFX microscope (magnification 300 X), a drawing tube and a digitizing tablet attached to an IBM Personal Computer. Comedogenicity assays were used to establish dose-response curves for TCAB, TCAOB and the couple-compound TCAB + TCAOB.^ No evidence of chloracne or toxicity was observed in any of the workers. Nor, was there a statistically significant increase in size of follicular orifice means measured over time. This was attributed to extensive personal and environmental hygiene programs along with teaching the workers about chloracne, its cause and its prevention. These programs may have been the greatest factor in preventing the development of chloracne in this group of workers. Monitoring of the plant environment showed relatively high concentrations of the couple-compound (TCAB + TCAOB). Comedogenicity assays showed a linear dose-response relationship over time for TCAB, TCAOB and the couple-compound. An antagonistic action was found for the TCAB/TCAOB of the couple-compound; such action may provide some protection to workers in this type of setting. It is speculated that the observed antagonistic action may be due to the difference in binding affinities of TCAB/TCAOB for receptor sites. ^

Determinación de la actividad de la óxido nítrico sintetasa endotelial y del factor NF-kB, en la piel del paciente con pseudoangiomatosis eruptiva : informe preliminar

La pseudoangiomatosis eruptiva se caracteriza por la aparición brusca de múltiples pápulas eritematosas, asintomáticas, rodeadas de un halo blanquecino, con remisión espontánea. Histológicamente se observa dilatación vascular con escaso infiltrado inflamatorio. Su etiología permanece incierta a pesar de ser relacionada con virus o picaduras de insectos. Basados en el compromiso vascular, el objetivo del trabajo fue investigar la actividad de la enzima endotelial oxido nítrico sintetasa (eNOS) y la expresión del factor NF-kB por inmunohistoquimica en un intento de esclarecer su patogenia. Material y métodos: Se estudiaron diez pacientes con diagnóstico clínico de pseudoangiomatosis eruptiva (PAE) que presentaron la dermatosis en forma epidémica. Se realizaron biopsias teñidas con Hematoxilina-Eosina y Tricrómico de Masson. Se efectuó estudio virológico de los pacientes Nª 4, 9 y 10 mediante determinaciones serológicas para echovirus, enterovirus, citomegalovirus, parvovirus B19 y hepatitis A, B y C. En cinco pacientes se obtuvo material para determinación de eNOS y NF-kB. Resultados: Todos los pacientes, 5 hombres y 5 mujeres presentaron pápulas eritematosas rodeadas por un halo blanquecino, especialmente en las extremidades, alrededor de las rodillas. Histológicamente mostraron vasos dilatados y células endoteliales prominentes con un infiltrado discreto perivascular. Todos los estudios serológicos fueron negativos. La actividad de eNOS fue significativamente menor comparada con la piel normal (p= 0,002) y la expresión de NF- ĸB fue fuertemente positiva en los vasos de la dermis papilar y reticular. Conclusiones: Todos los pacientes fueron afectados en verano, por lo que la picadura del mosquito debe ser considerada como un factor etiológico. La baja expresión de eNOS está relacionada con la vasodilatación y la expresión aumentada de NF-ĸB confirma que el proceso es de tipo inflamatorio.

Responses of the tropical gorgonian coral Eunicea fusca to ocean acidification conditions

Ocean acidification can have negative repercussions from the organism to ecosystem levels. Octocorals deposit high-magnesium calcite in their skeletons, and according to different models, they could be more susceptible to the depletion of carbonate ions than either calcite or aragonite-depositing organisms. This study investigated the response of the gorgonian coral Eunicea fusca to a range of CO2 concentrations from 285 to 4,568 ppm (pH range 8.1-7.1) over a 4-week period. Gorgonian growth and calcification were measured at each level of CO2 as linear extension rate and percent change in buoyant weight and calcein incorporation in individual sclerites, respectively. There was a significant negative relationship for calcification and CO2 concentration that was well explained by a linear model regression analysis for both buoyant weight and calcein staining. In general, growth and calcification did not stop in any of the concentrations of pCO2; however, some of the octocoral fragments experienced negative calcification at undersaturated levels of calcium carbonate (>4,500 ppm) suggesting possible dissolution effects. These results highlight the susceptibility of the gorgonian coral E. fusca to elevated levels of carbon dioxide but suggest that E. fusca could still survive well in mid-term ocean acidification conditions expected by the end of this century, which provides important information on the effects of ocean acidification on the dynamics of coral reef communities. Gorgonian corals can be expected to diversify and thrive in the Atlantic-Eastern Pacific; as scleractinian corals decline, it is likely to expect a shift in these reef communities from scleractinian coral dominated to octocoral/soft coral dominated under a "business as usual" scenario of CO2 emissions.

Assembly of Lampbrush Chromosomes from Sperm Chromatin

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24–48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific “landmarks.” Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.

A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules >20 Å and that transcytosis is an N-ethylmaleimide–sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein–lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.