Serge Montplaisir

Persistence of host Langerhans cells following allogeneic bone marrow transplantation: possible relationship with acute graft-versus-host disease

Langerhans cells (LC) are bone marrow‐derived dendritic antigen‐presenting cells found in the epidermis. In an effort to determine the origin (host versus donor) of LC at different intervals following bone marrow transplantation, we performed skin biopsies in 16 recipients of sex‐mismatched marrow. LC were identified using monoclonal antibody OKT6 in an indirect immunoperoxidase assay and their donor or host origin determined according to the presence or absence of Y body. The presence of Y‐positive (donor) LC could be demonstrated in all (6/6) skin biopsies of female recipients of male marrow tested between days 39 and 730 post‐transplant. Persistence of host LC in male recipients of female marrow was documented in all (6/6) recipients studied on day 39 and in two out of seven patients tested on day 120 post‐transplant. From day 365 onward, no residual host LC could be detected, suggesting that by this time all epidermal LC are donor‐derived. Our study demonstrates that host LC usually persist for 39 and up to 120 d following bone marrow transplantation. The relevance of this observation to the possible role of LC and other host dendritic antigen‐presenting cells in the graft‐versus‐host reaction is discussed.

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.

A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b

Monoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (10(3)) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 X 10(3)-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype-specific surface-exposed OMP of H. influenzae type b.

The human thymic dendritic cell phenotype and its modification in culture.

In order to extend our study of human thymic dendritic cells (DC) we have purified DC by density gradient separation followed by treatment with CD1 and CD2 mAb and antibody-coated immunobeads. The resulting population contains 60 to 75% brightly HLA-DR+ cells. Morphological and functional studies demonstrate that these cells share the common characteristics of dendritic cells. Extensive phenotypic analysis of the purified DC has been made using a panel of mAb. Cytofluorometric assays with mAb reactive with common leucocyte antigen confirm that the brightly HLA-DR+ cells are of mesenchymal origin. Thymic DC express HLA-DQ and HLA-class I antigens. They are also positive for the expression of CD45RA molecules and some express the ICAM-1 and the LFA-1 molecules. DC do not stain with a wide variety of anti-T, -B, and -monocyte or -M phi mAb and lack Fc gamma RIII, CR2, and CR3. Freshly isolated DC failed to stain with OKT6 mAb; however, they progressively acquire the CD1 molecule after a few days culture. The acquisition of CD1 molecule is selective since CD4, CD2, and HLA-ABC molecules are not upregulated under the same conditions. From phenotypic results, it was therefore possible to sort brightly HLA-DR+ or -DQ+ cells and so obtain greater than 90 to 95% purified human thymic DC. Such homogeneous DC populations are obviously of great interest for the study of thymic DC functions.

Liposome-encapsulated antibiotics: preparation, drug release and antimicrobial activity against Pseudomonas aeruginosa

Ticarcillin- and tobramycin-resistant strains of Pseudomonas aeruginosa were shown to have a markedly increased sensitivity to antibiotics enclosed in liposomes. This was demonstrated by growth inhibition of two resistant P. aeruginosa strains in the presence of the liposome-enclosed ticarcillin and tobramycin at 2 per cent and 20 per cent of their respective minimum inhibitory concentration. The liposome-enclosed antibiotic was as effective against the beta-lactamase-producing strain as against the non-beta-lactamase producing strain. Entrapment efficiency of the two antibiotics with the dehydration-rehydration vesicle (DRV) method was largely superior to other methodologies used. Kinetic studies with DRV demonstrated that tobramycin and ticarcillin-loaded liposomes still contained 83 per cent and 67 per cent of drug respectively after 24 h at 37 degrees C.

Histochemical and immunochemical study of the fate of Candida albicans inside human neutrophil phagolysosomes.

To further define the urtrastructural events associated with the killing of Candida albicans by human neutrophils, four methods were used: (1) the periodate‐thiocarbohydrazidesilver proteinate (PA‐TCH‐SP) staining of vicinal‐glycol‐containing complex carbohydrates; (2) the localization of thermostable immunodeterminants of the yeast cell wall, mannans or mannoproteins, using monospecific antibodies and a protein A‐gold complex (monAb‐gold); (3) the localization of mannose residues with concanavalin A labeled with gold particles (Con A‐gold); (4) the localization of chitin oligomers using wheat germ agglutinin and ovomucoid labeled with gold particles (WGA‐gold). The mannan‐rich cell wall layers were progressively lost as shown by altered PA‐TCH‐SP reactivity and a diffuse pattern of staining with Con A‐gold and monAb‐gold. The de novo appearance of conspicuous amounts of glycogen‐like particles near the plasmalemma and in the cell wall was interpreted as evidence of a reparative process of the yeast cell wall. Chitin was seemingly unaltered and readily demonstrated by the WGA‐gold in the wall remnants of ghost cells.

Human thymic dendritic cells

Human thymic dendritic cells (DC) represent a member of the bone marrow–derived dendritic cell family. They have a dendritic shape and are found in small numbers mainly at the corticomedullary border and in medullary regions of the thymus. Human thymic DC were isolated by density gradient separation, followed by treatment with CD2, CD7, CD1, and CD11b mAb and immunobeads magnetic separation. The resulting population contains 60–75% brightly HLA‐DR+ cells which present the morphological characteristics of DC observed in situ. Extensive phenotypic analysis confirmed that they are of mesenchymal origin and that some express CD11a and CD54 molecules. Freshly isolated DC do not stain with a wide variety of anti‐T‐B and ‐monocyte or ‐macrophage mAb. However, they acquire the CD1 molecule after a few days in culture. By using a cell sorter we obtained 90–95% of purified human thymic DC. Functional studies have shown that human thymic DC are potent activators in mixed lymphocyte reactions, act as accessory cells in mitogenic thymocyte proliferation, increase the thymocyte proliferative response to a toxin signal, and produce IL‐1. They also formed spontaneous physical associations with thymocytes, which raises questions about the implication of DC in differentiation and/or maturation processes of thymocytes. Microsc. Res. Tech. 38:267–275, 1997.

IL-1 production by human thymic dendritic cells : studies on the interrelation with DC accessory function

Thymic dendritic cells (DC) have been proposed to play a critical role in the generation of immunocompetent T lymphocytes. Since IL-1 is widely considered to be an important second signal in T cell stimulation, we have studied the ability of isolated human thymic DC to produce IL-1. Using the EL4/CTLL conversion assay standardized with recombinant IL-1 beta (rIL-1 beta), we demonstrate that upon LPS-stimulation thymic DC produce small amounts of IL-1 as compared to peripheral blood monocytes (PBM). In contrast with PBM, DC IL-1 production is not influenced by indomethacin. IL-1 activity was detected in the supernatants of DC cultures from all thymuses tested, although quantitative variability was noted among individual thymic donors. The specificity of the active factor was confirmed by neutralization assays with anti-IL-1 beta mAb. On the other hand, we demonstrate that rIL-1 beta cannot substitute for nor amplify the accessory function of thymic DC and that anti-IL-1 beta mAb fails to block the DC accessory function. Thus we conclude that IL-1 beta might not be a major factor for the efficient DC accessory function toward mature thymocytes recently demonstrated in our laboratory. Of interest, IL-1 beta was also detected in the supernatants of DC-thymocyte cocultures in the absence of mitogenic factor, suggesting that thymocyte contacts can constitute a sufficient signal to induce DC to produce IL-1. These observations indicate that human thymic DC represent an intrathymic source of IL-1 whose role in thymocyte proliferation or maturation remains to be understood.

An improved method for purifying human thymic dendritic cells

Thymic dendritic cells (DC) play a prominent role in the immune response as they constitute a key element involved in the maturation of thymocytes in the thymus. Human thymic DC, like DC from other lymphoid organs, represent a minor cell population (< 2%) of the thymus. Since these cells cannot replicate in vitro, the development of efficient purification methods is an essential prerequisite for extensive functional studies. DC express high levels of HLA-DR, a cell surface marker of the MHC class II antigen which is not exclusive to DC. Since no specific human thymic DC marker has been identified so far, DC purification methods are mainly based on depletion of particular subgroups of cells. We report here an improved method for purifying human thymic dendritic cells. In contrast to prior work, CD2+ thymocytes were first depleted by rosetting with neuraminidase treated sheep red blood cells. The nonrosetted cells were separated in a Percoll gradient, and the low-density cells were subsequently depleted of nondendritic cells by using thymocyte and macrophage specific monoclonal antibodies and either magnetic bead depletion or cytofluorometry. Cell populations (18-55 x 10(6) cells) obtained following magnetic bead purification were at least 80% HLA-DR+/CD2- and exhibited ultrastructural morphological features and functional activities such as those described previously for thymic DC. This improved method was compared with different purification approaches that use various combinations of cell density-based separation techniques and cell surface specific markers antibody reactivity. The magnetic beads depletion approach provided higher yields.

Influence of muramyl dipeptide on renal candidiasis in genetically distinct mice

Susceptible (DBA/2) and resistant (C57BL/6) mice were inoculated intravenously with Candida albicans to evaluate the effect of a four‐day prophylaxis with muramyl dipeptide (MDP) on the renal burden of organisms during the first week after infection. In sham‐treated DBA/2 mice injected with 8 times 104 candida cells, renal CFU (LOG10, ± SEM) on days 1, 4 and 7 after infection were found to average 5.050 ± 0.109, 4.882 ± 0.133 and 5.482 ± 0.245. In sham‐treated C57BL/6 mice injected with 2 times 105 candida cells, renal CFU on days 1, 4 and 7 reached only 3.610 ± 0.118, 3.404 ± 0.107 and 4.176 ± 0.580. MDP‐treated DBA/2 mice achieved significant reduction in CFU of C. albicans on day 1 (1.3 log units) and day 4 (0.6 log unit), while MDP‐treated C57BL/6 mice had significant reduction in CFU of C. albicans only on day 1 (0.6 log unit) after infection. Sham‐treated mice of both strains had a 28.6 to 30% increase in kidney weights on day 4 only, a transient change not seen in MDP‐treated mice. Histopathological examination on days 8, 15 and 21 after infection revealed a higher incidence of renal papillary necrosis in DBA/2 mice than C57BL/6 mice (approximately 70%vs 10%). The incidence of granulomas and of chronic interstitial inflammation was much higher in MDP‐treated mice. We conclude that the genetic makeup of the host influences the potential effectiveness of MDP as a biological response modifier.