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D Spehner

Publications and source records attributed to D Spehner.

At least 37 records · Page 2Linked to original sources
PubMed1996

Examination of cutaneous macroglobulinosis by immunoelectron microscopy.

Cutaneous macroglobulinosis is a rare cutaneous manifestation of Waldenström's disease. Lesions are though to result from accumulation of macroglobulin in the dermis and are therefore called IgM storage papules. Ultrastructural findings in the previously reported cases were contradictory and the nature of the deposits was not established by electron microscopy. The purpose of this study was to analyse such deposits by the use of immunoelectron microscopy. A 60-year-old woman had multiple erythematous papules for 1 year. The histopathological changes consisted of plasmocytic infiltration of the dermis and eosinophilic deposits. The skin changes and other investigations led to a diagnosis of Waldenström's disease. Samples from normal and diseased skin were analysed by electron microscopy and by immunogold labelling with anti-IgM antibodies, after Lowicryl K4M embedding. An extracellular electron dense granular and filamentous material was observed in the mid- and upper dermis, between and within the collagen bundles. No periodicity was noted and no deposits were seen at the dermoepidermal junction. Immunoelectron microscopy showed a positive labelling located only on these deposits, in both normal-appearing skin and in lesions. In this patient, immunoelectron microscopy clearly demonstrated the presence of large amounts of IgM in the dermis, which were found in the lesions of cutaneous macroglobulinosis and in normal skin. These results suggest that the IgM storage papules result from a greater density of deposits rather than a site-specific accumulation.

Female↗
PubMed1995

The vaccinia virus J5L open reading frame encodes a polypeptide expressed late during infection and required for viral multiplication.

A number of open reading frames (ORFs) are found in the vaccinia virus (VV) genome whose activities in the viral life cycle have not yet been determined. This report examines one such ORF, designated J5L, which was demonstrated to be essential for viral multiplication. Stable inactivation of the J5L ORF by insertion of a lacZ ORF was impossible unless another copy of the J5L ORF was present in the VV genome. Fusion genes between the J5L ORF and either the lacZ gene or the VV K1L gene were employed to study its temporal expression as well as its protein product. These experiments showed that J5L is transcribed late in infection and gives rise to a protein product which migrates by SDS-PAGE with the expected molecular weight (16 kDa). Numerous unsuccessful attempts to establish a stable cell line expressing J5L suggest that the J5L gene product could be cytotoxic.

Animals↗
PubMed1995

Role of B cell receptor Ig alpha and Ig beta subunits in MHC class II-restricted antigen presentation.

The ability of the B cell antigen receptors (BCRs) to enhance MHC class II-restricted antigen presentation was ascribed to mig-associated Ig alpha/Ig beta heterodimers. The relative role of Ig alpha and Ig beta subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Ig alpha and Ig beta chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Ig alpha targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Ig beta targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response.

Animals↗
PubMed1994

Respiratory syncytial virus nucleocapsid protein (N) expressed in insect cells forms nucleocapsid-like structures.

The gene coding for the N protein of RSV strain Long has been cloned and sequenced. It was introduced behind the polyhedrin promoter of the shuttle vector pVL941 and baculoviruses containing the N gene were constructed by homologous recombination. Infection of Spodoptera frugiperda 9 cells resulted in the production of large amounts of a protein similar in size and antigenicity to the authentic N protein. The baculovirus expressed N protein was concentrated in the cytoplasm of the insect cells and could be extracted at low salt concentration. Nucleocapsid structures similar to those purified from RSV-infected cells could be observed by electron microscopy after negative staining of cellular extracts.

Animals↗
PubMed1994

Localization of viral protein X in simian immunodeficiency virus macaque strain and analysis of its packaging requirements.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) encode the accessory viral protein X (Vpx) known to be incorporated into virions in amounts comparable to those of the Gag proteins. The localization of Vpx within SIVmac-infected HUT-78 cells and SIVmac virions was studied by immunoelectron microscopy. Vpx appeared to be associated with extracellular virions as well as budding viral particles at the surface of infected cells. Immunolabelling of purified viral cores suggested that Vpx was a component of the amorphous material surrounding the core structure. Furthermore, a detergent insoluble fraction containing SIV core proteins was devoid of Vpx. To investigate the protein requirement for packaging of Vpx, BHK-21 cells were co-infected with vaccinia virus recombinants encoding Vpx and other SIV proteins able to assemble into virus-like particles. Analysis by immunoprecipitation of the extracellular particulate material as well as immunoelectron microscopy demonstrated that co-expression of Vpx with the Pr56gag polyprotein was sufficient for the formation of pseudo-virions containing Vpx. Virus-like particles that appeared upon expression of p16gag did not contain Vpx. The results suggest that Vpx is packaged into viral particles through its binding to the Gag polyprotein. The precise positioning of Vpx within the space separating the viral envelope from the core structure is postulated to result from the reorganization of viral proteins that occurs upon Gag polyprotein cleavage and budding.

Animals↗
PubMed1994

Recombinant MUC 1 vaccinia virus: a potential vector for immunotherapy of breast cancer.

Breast cancer is considered as the major cause of mortality by cancer for women. Even if chemotherapy, radiotherapy and surgery have improved the life expectancy of patients bearing tumours, breast cancer is responsible for the death of 42,000 women per year in USA and 25,000 women in France. In this context, cancer vaccines may add an attractive alternative therapeutic strategy to the current existing treatments. We describe here the construction of recombinant vaccinia viruses co-expressing a tumour associated antigen (MUC 1) and an "adjuvant" cytokine, which have potential applications in the active immunotherapy of breast cancer. Indeed, recombinant vaccinia viruses have been extensively used during the past decade to induce a protective response against a whole variety of pathogens, and has proven to be of great value in the elicitation of a cellular immune response leading to the rejection of tumour grafts in mouse models.

Animals↗
PubMed1993

In vitro and in vivo study of the ectromelia virus homolog of the vaccinia virus K1L host range gene.

Ectromelia virus encodes a protein which is homologous to the product of the vaccinia virus host range gene, K1L, except for eight conservative and two non-conservative substitutions and an additional threonine residue at the carboxyl terminus. Unlike the vaccinia virus gene, the ectromelia virus homolog failed to support optimal virus replication in RK-13 cells and appeared to be expressed 20-fold less efficiently. This lower level of expression was not due to the genetic background of the virus, K1L RNA transcription, sequence of the K1L RNA leader, or stability of K1L RNA or protein. Infections of RK-13 cells with ectromelia or vaccinia virus mutants lacking an intact K1L gene resulted in transient expression of early genes followed by a rapid and irreversible cessation of both virus and host protein synthesis. Infections of the disease-susceptible ANCR or -resistant C57BL/6 mice with the K1L-lacking ectromelia virus yielded a pathogenesis pattern indistinguishable from wild-type, suggesting that the ectromelia virus homolog of vaccinia virus K1L is not important for ectromelia virus in vivo replication and spread.

Animals↗
PubMed1993

Vaccination of mice against canine distemper virus-induced encephalitis with vaccinia virus recombinants encoding measles or canine distemper virus antigens.

Measles and canine distemper are caused by serologically related viruses. Although dogs immunized with measles virus (MV) do not elicit canine distemper virus (CDV) neutralizing antibodies, they are protected against the fatal disease. To investigate the potential role of the MV antigens in protection against CDV, we have immunized mice with vaccinia virus (VV) recombinants expressing the MV haemagglutinin (HA), fusion (F), nucleoprotein (NP) and matrix (M) antigens and challenged them with CDV. A partial protection was observed with the VV recombinants expressing the F, NP and M antigens, but not the HA. In contrast, immunization with a VV recombinant expressing the CDV F protein completely protected mice from CDV.

Animals↗
PubMed1993

Alpha IIb beta 3 integrin dissociation induced by EDTA results in morphological changes of the platelet surface-connected canalicular system with differential location of the two separate subunits.

Treatment of human platelets by EDTA (5 mM at 37 degrees C and pH 7.4 for 30 min) induces ultrastructural morphological changes of the surface-connected canalicular system (SCCS). The first consists in dilations of some portions of the channels, whereas the second is represented by collapse of parts of the canaliculi. The collapsed elements of the EDTA treated SCCS are made up of two parallel limiting membranes and a central striated zone. Some of the EDTA treated platelets form microaggregates, the cohesion of which is apparently due to the appearance of pentalaminar interplatelet structures. EDTA treatment is known to induce an irreversible loss of platelet aggregability which is due to irreversible dissociation of the membrane GPIIb-IIIa complexes. In the present study, we looked for involvement of GPIIb-IIIa in the formation of these pentalaminar structures, and were able to demonstrate that the morphological changes described are in fact directly dependent on the EDTA induced dissociation of GPIIb-IIIa complexes. Indeed, we observed that these changes (a) cannot be induced in type I Glanzmann's thrombasthenia, where GPIIb-IIIa complexes are absent, (b) do not appear when human platelets are preincubated with monoclonal anti-GPIIb-IIIa complex-dependent (CD41a) antibodies, which protect the complex from EDTA induced dissociation, (c) appear only at alkaline pH and at 37 degrees C, which corresponds to the range of pH and temperature where EDTA can dissociate GPIIb-IIIa complexes, (d) are accompanied by the disappearance in fluorescence flow cytometry of the heterodimer complex-dependent epitopes, when using anti-CD41a antibodies and (e) do not appear in rat platelets, where GPIIb-IIIa does not dissociate after EDTA treatment. Furthermore, using gold-labeled mAbs concomitantly with the addition of EDTA, we observed that almost only GPIIb was present in the collapsed regions of the canaliculi. Using double labeling studies with polyclonal anti-GPIIb antibodies coupled to 10 nm gold particles and polyclonal anti-GPIIIa antibodies coupled to 20 nm gold particles, we observed that while both 10 and 20 nm particles were present in the dilated portions of the canaliculi almost only the small particles, coupled to the anti-GPIIb antibodies, labeled the collapsed portions of the SCCS. On Lowicryl thin sections, polyclonal antibodies against GPIIb labeled the central striated zone while both GPIIb and GPIIIa were found in the dilated portions of the SCCS. All these observations lead us to suggest that homopolymers of GPIIb could be responsible for "zipping" of the SCCS.

Blood Platelets↗
PubMed1993

Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines.

Vaccinia virus encodes several proteins, the activity of which is essential for multiplication in different cell types. Both the C7L and K1L open reading frames (ORFs) have been characterized as viral determinants for multiplication in human cells. To confirm and extend these findings we inserted the C7L ORF into the genome of a mutant virus unable to multiply in human cells and showed that this virus recovered its ability to replicate. Deletion of C7L from a wild-type viral genome did not adversely affect virus multiplication in human cells but it did reduce replication in hamster Dede cells. When both C7L and K1L were deleted from the vaccinia virus genome only poor or no viral yields were obtained from various human cell lines. Recombinant viruses were also constructed to facilitate the study of C7L protein synthesis during infection. One virus in which the lacZ ORF was fused downstream and in-frame with the C7L ORF enabled us to characterize the C7L protein as an early gene product. Another recombinant virus was constructed so that the carboxy terminus of the C7L ORF product contained an additional 28 amino acids from the carboxy terminus of K1L. Tagging of C7L in this way allowed us to detect the fusion protein by immunoprecipitation with antibodies against the K1L protein. Furthermore, the hybrid protein retained its biological properties. The recombinant viruses constructed in this work should be useful for studies of the molecular basis of the activity of viral host range proteins.

Animals↗
PubMed1993

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.

Animals↗
PubMed1992

Restricted replication of ectromelia virus in cell culture correlates with mutations in virus-encoded host range gene.

Ectromelia virus (strain Moscow) was shown to replicate poorly or not at all in cell lines derived from the rabbit or hamster. The failure of ectromelia virus to replicate in cell lines derived from the hamster suggested that the virus lacked a functional CHO host range (hr) gene required for multiplication in these cells. A DNA fragment which hybridized to the CHO hr gene was cloned from the ectromelia virus genome and shown by sequence analysis to be deleted of 506 bp within the ectromelia virus CHO hr homologue. Two additional ectromelia viruses (Hampstead and Mill Hill strains) were also shown to lack an intact CHO hr gene. Insertion of the CHO gene from cowpox virus into the ectromelia virus genome extended the host range of ectromelia virus in tissue culture. These results demonstrate that an intact CHO hr gene is not required for maintenance of ectromelia virus in nature and provide a partial explanation for ectromelia virus' narrow host range, as opposed to the broad host range of cowpox virus, which has a functional CHO hr gene.

Animals↗
PubMed1992

Construction of vaccinia virus recombinants expressing several measles virus proteins and analysis of their efficacy in vaccination of mice.

Measles virus genes encoding the haemagglutinin (HA), fusion protein (F) or nucleoprotein (NP) have been inserted into the vaccinia virus genome either alone or in various combinations. In each case the measles virus genes were expressed from the 7.5K promoter and were incorporated into the thymidine kinase (tk) or K1L loci of the Copenhagen strain of vaccinia virus. Cells infected by the recombinants synthesized measles virus proteins indistinguishable from those induced in measles virus-infected cells. However, in some instances the level of expression in cells infected by recombinants expressing more than one measles virus gene was reduced when compared to those encoding a single gene. The sera from mice immunized with recombinants containing either HA, HA.F, HA.NP or HA.F.NP had similar levels of measles virus neutralizing antibodies which remained constant throughout a 7 month period. Analysis of these sera by immunoprecipitation of radiolabelled measles virus confirmed the presence of specific antibody to each of the antigens where appropriate. The introduction of the measles virus genes into the K1L and the tk sites despite attenuating the virus for mice by 10-fold and 1000-fold respectively did not affect the vaccination efficiency, i.e. ability to induce measles virus antibody and protect mice. Vaccination of BALB/c (H2d) mice with HA and F, but not NP, recombinants completely protected the animals against a lethal measles virus challenge. In contrast, although the HA recombinant protected CBA (H2k) mice, the F recombinant did so poorly. However, by immunizing CBA mice with a recombinant expressing both F and NP, protection was increased to more than 75%. Our findings demonstrate the ability of three measles virus antigens expressed from the vaccinia virus genome alone or in combination to contribute to protective immunity against measles virus infection of mice. They also suggest that the association of measles virus antigens in a single recombinant DNA vaccine could be beneficial to overcome host-related restriction of the immune response to particular antigens.

Animals↗
PubMed1992

Alpha IIB beta 3 integrin dissociation induced by EDTA results in formation of Birbeck granule-like structures in human blood platelets.

EDTA (5 mM at 37 degrees C and pH 7.4 for 30 min) induces morphologic changes in the platelet surface-connected canalicular system (SCCS), in particular the collapse of some portions of the canaliculi. These collapsed elements are made up of two parallel limiting membranes and a central irregular striated zone, a pentalaminar organization that clearly resembles that of Langerhans cell Birbeck granules (BG). Such BG-like structures are also seen between adjacent platelets in EDTA-treated platelet microaggregates. EDTA is known to induce an irreversible dissociation of the platelet membrane glycoprotein(GP)IIb-IIIa, the alpha IIb beta 3 platelet-specific intergrin, a calcium-dependent heterodimer that serves as an inducible receptor for fibrinogen and is essential for platelet aggregation. Hence, we looked for involvement of the GPIIb-IIIa in the formation of these BG-like modifications. We observed that these changes i) cannot be induced in type I Glanzmann's thrombasthenia, where the GPIIb-IIIa complexes are absent; ii) did not appear when human platelets were pre-incubated with MoAb anti-GPIIb-IIIa complex, which protected GPIIb-IIIa from EDTA-induced dissociation; iii) appeared only at alkaline pH and 37 degrees C, which corresponds to the range of pH and temperature where EDTA can dissociate the GPIIb-IIIa complexes; iv) are accompanied by the dissappearance on fluorescence flow cytometry analysis of the heterodimer specific epitopes; and v) do not appear in rat platelets at pH 7.4 where GPIIb-IIIa does not dissociate after EDTA treatment. Thus, the appearance of BG-like structures in the platelet SCCS is directly dependent on the EDTA-induced dissociation of the GPIIb-IIIa complexes. Furthermore, using gold-labeled MoAb concomitantly with the addition of EDTA, we observed that only GPIIb is present in the collapsed portions of the canaliculi. On Lowicryl thin sections essentially polyclonal antibodies to GPIIb labeled the central striated zone. All these observations lead us to suggest that homopolymers of GPIIb could be responsible for "zipping" of the SCCS and raise the question of the participation of a Langerhans cell integrin in the formation of Langerhans cell BG.

Blood Platelets↗
PubMed1991

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.

Animals↗
PubMed1991

A specific inhibitor of cysteine proteases impairs a Vif-dependent modification of human immunodeficiency virus type 1 Env protein.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) regulates viral infectivity. Virions produced in cell culture after transfection by a Vif-negative molecular clone show a dramatic decrease in infectivity for susceptible CD4+ cell lines, although the Vif protein does not appear to be a constituent of the viral particle. The exact mechanism by which Vif affects HIV-1 infectivity is so far unknown. We report the existence of structural homologies between Vif and a family of cysteine proteases and present evidence which suggests that one of the targets of Vif is the Env protein and more precisely the cytoplasmic domain of gp41. Vif was found to modify both the processing and conformation of the Env protein. Ethyl(25, 35)- 3[(5)-3-methyl-1-(3-methylbutylcarbamoyl)]oxirane-2-carboxylate, a specific inhibitor of cysteine proteases, inhibits the effect of Vif, as does the mutation of Cys-114 to Leu in Vif. Furthermore, Cys-114 of Vif produced in Escherichia coli, interacts directly with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane. These observations suggest that a cysteine protease activity is associated with Vif and that this activity plays a role in Env maturation.

Amino Acid Sequence↗
PubMed1990

Fowlpox virus recombinant encoding the measles virus fusion protein: protection of mice against fatal measles encephalitis.

A recombinant Fowlpox virus engineered to encode the measles virus fusion protein was shown to protect mice against a challenge measles infection. A vaccine dose of about 10(6) p.f.u. was needed to protect nearly 100% of the animals. Mice failed to develop a significant level of antibodies directed against measles virus suggesting that other components of the immune system may be involved.

Animals↗