Simulation of conformational changes in 2 Zn insulin.

Isolated insulin monomers, the dimer and higher aggregates from the 2 Zn crystal structure are subjected to convergent energy minimization in Cartesian co-ordinates using a force-field that includes the position of all hydrogen atoms. The minimizations are found, for the first time, to produce conformational changes of appreciable magnitude, which agree well with observed structural differences between monomers in the 2 Zn crystal and with the mechanism proposed previously for the coupling between deformations in different parts of the molecule. Our results also suggest that insulin would tend to adopt a molecule 1-like conformation in the absence of crystal packing forces, and that dimer formation is not at the origin of the observed asymmetry in the 2 Zn crystal.

Local ultraviolet B irradiation impairs contact hypersensitivity induction by triggering release of tumor necrosis factor-alpha from mast cells. Involvement of mast cells and Langerhans cells in susceptibility to ultraviolet B.

Our laboratory has previously demonstrated that ultraviolet B radiation impairs contact hypersensitivity induction in ultraviolet B susceptible mice through a tumor necrosis factor-alpha-dependent mechanism, involving calcitonin gene related peptide and cutaneous mast cells. This study was designed to test directly whether mast cells are the source of tumor necrosis factor-alpha, to account for the ultra-violet B-susceptible phenotype. As dermal mast cells seem to release tumor necrosis factor-alpha following exposure to ultraviolet B, we investigated whether tumor necrosis factor-alpha released by mast cells could mediate impairment of contact hypersensitivity in a manner similar to that found with ultraviolet B radiation treatment. First, we loaded Fcepsilon receptors of mast cells of ultraviolet B-susceptible (C3H/HeN), ultraviolet B-resistant (C3H/HeJ), and mast-cell deficient (Sl/Sld) mice by intradermal injections of anti-dinitrophenyl immunoglobulin E antibodies. Twenty-four hours later, dinitrophenyl was injected intravenously, and within 30 min oxazolone was painted on injected skin sites. Contact hypersensitivity induction was impaired in ultraviolet B-susceptible mice, but not in ultraviolet B-resistant or Sl/Sld mice, and treatment with anti-tumor necrosis factor-alpha antibodies was able to reverse this impairment of contact hypersensitivity. Second, we have found that ultraviolet B radiation did not impair contact hypersensitivity induction when haptens were painted on irradiated skin of mast cell deficient mice. As ultraviolet B radiation impairs contact hypersensitivity induction through a tumor necrosis factor-alpha-dependent mechanism, we conclude that ultraviolet B radiation triggers the prompt release of tumor necrosis factor-alpha from dermal mast cells, and that mast cell-derived tumor necrosis factor-alpha interferes with generation of the hapten-specific signal required for contact hypersensitivity induction. In addition, we are providing data that indicate that tumor necrosis factor-alpha levels released from mast cells as well as sensitivity of Langerhans cells to tumor necrosis factor-alpha contribute in defining the phenotypes of resistance versus sensitivity to ultra-violet B radiation.

EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.

The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.

A possible role for specific "anergy" in immunologic hyporeactivity to donor stimulation in human kidney allograft recipients.

The low immunological reactivity toward donor cells usually observed in transplant recipients has been linked to clonal deletion or suppression of alloreactive cells. However, the anergy of donor-specific reactive cells is another possibility not extensively tested until now in humans. In this case, donor-specific reactive cells would be present and eventually be activated without becoming effector cells (i.e., without secreting IL-2 or becoming cytotoxic) after donor-specific stimulation. We studied 8 patients under low-dose immunosuppressive drugs who displayed hyporeactivity toward donor stimulation. IL-2 production, proliferative response, and cytotoxic activity toward donor cell stimulation was decreased (respectively 22, 53, and 19% of response toward third-party stimulation). In order to evidence anergy, we studied two activation markers (cell size increase and expression of IL-2 receptor [CD25]) in allografted recipient T cells after autologous (background), donor (experimental), and third-party cell stimulation (positive control). We showed that the percentage of CD25+ cells and the cell size increase were similar after donor or third-party cell stimulation and clearly above the background as early as days 1-2 after the beginning of the mixed lymphocyte culture. Moreover, CD4+ and CD8+ cells similarly expressed CD25 after donor or third-party stimulation. Thus, donor-specific reactive cells not only were present but could be activated without becoming effector cells. These data suggest that anergy may be an important phenomenon in allograft tolerance.

Detection of donor-derived cells by polymerase chain reaction in neonatally tolerant mice. Microchimerism fails to predict tolerance.

Although it has long been appreciated that establishment of chimerism is important in the acquisition and maintenance of allograft tolerance, the importance of this relationship has been reemphasized recently. Using the exquisite sensitivity of the polymerase chain reaction we have studied qualitatively and quantitatively the presence of donor-derived chimeric cells in relation to the ability of neonatally injected mice to display skin graft tolerance or rejection. We have found that virtually all mice that receive neonatal injections of F1 hematopoietic cells acquire donor-derived chimerism that is detectable in blood, spleen, lymph nodes, and thymus. Surprisingly, neither the presence nor the quantity of chimeric cells predicts whether a particular neonatally injected mouse will accept or reject donor-specific skin allografts. Moreover, whether the test skin allograft is accepted (tolerance) or rejected by neonatally injected mice, chimerism typically remains detectable within recipient lymphoid tissues. In functionally tolerant mice, challenge with a test skin allograft actually leads to a remarkable expansion in donor-derived genetic sequences, implying that donor-derived cells have been induced by the graft to undergo proliferation. Since persistence of chimerism without proliferation after test grafting is characteristic of mice that fail to display tolerance, we believe that achievement of a threshold level of donor-derived alloantigen may be necessary to retain the tolerant state. We conclude that chimerism is essential for the induction of neonatally induced tolerance, and its expansion may play an important role in the maintenance of that tolerance, when challenged by an allograft.

Decreased lymphokine-activated killer cells in kidney transplant recipients. Correlation with a diminished number of CD3-/NKH1+ cells.

The activity of lymphokine-activated killer cells, measured either by a clonal or polyclonal technique, was assessed in 30 kidney transplant recipients (TX), in 13 hemodialyzed patients (HD-CRI), and in 18 normal (N) controls. A highly significant decrease of the LAK activity in TX in comparison with HD-CRI or N (P = 0.0001) was observed. Moreover, the percentage of CD3-/NKH1+ cells was decreased in TX in comparison with N (P = 0.01). LAK activity was strongly correlated (r = 0.72; P = 0.0001) with the percentage of CD3-/NKH1+ cells and not with that of double-positive CD3+/NKH1+ cells. Multivariate analysis showed that the sole independent variable that determined the LAK activity was the percentage of CD3-/NKH1+ cells: the pathological status (TX, HD-CRI, and N) variable was statistically not significant. On the other hand, two T cell-specific functions (IL-2 secretion and specific cytotoxic activity) were, on the whole, preserved in TX. Altogether, these results suggest that TX are LAK deficient predominantly because they have a decreased number of CD3-/NKH1+ cells. The normality of T cell functions suggests that the high rate of malignancies seen in TX is related to this LAK deficiency. Moreover, our study indicates that, in vivo, CD3+ cells do not significantly contribute to the LAK precursors.

A versatile ELISA-PCR assay for mRNA quantitation from a few cells.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

A new concept of skin-associated lymphoid tissue (SALT): UVB light impaired cutaneous immunity reveals a prominent role for cutaneous nerves.

More than 20 years have passed since the concept that the skin has its own associated immune system was first proposed by Streilein. This proposal was advanced in part on evidence that cutaneous contact hypersensitivity (CH) reactions are closely correlated with Langerhans cells (LC). Recent reports have demonstrated that LC have neural connectivity with cutaneous nerve termini containing calcitonin gene-related peptide (CGRP), suggesting that a link exists between innervation and immune responses in the skin. Here we discuss the neural components which have recently been found to be participants in skin-associated lymphoid tissue (SALT). In part, discovery of a functional link between the nervous system and SALT is based on studies in which cutaneous immunity was impaired by ultraviolet-B radiation (UVR). The deleterious effects of UVR on cutaneous immunity include failed CH induction and promotion of hapten-specific tolerance, effects that are mediated by tumor necrosis factor-alpha and interleukin-10, respectively. The source of these cytokines after UVR appears to be dermal mast cells. Evidence indicates that mast cells are triggered to release these cytokines in response to CGRP, which is released from UVR-damaged cutaneous nerve endings. Moreover, a substance P agonist was able to reverse the deleterious effects of UVR on CH induction, rendering the mice able to develop intense CH. These observations indicate that two cell types not originally included in the SALT concept are critical to the functional integrity of cutaneous immunity: mast cells and cutaneous nerves. We propose that cutaneous nerves dictate whether antigen applied to or arising within skin will lead to sensitivity or tolerance.

Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation.

OBJECTIVE: To delineate the mechanisms by which topical interleukin 1 receptor antagonist (IL-1RA) treatment promotes orthotopic corneal allograft survival. METHODS: Corneal buttons were prepared from eyes of C57BL/6 mice and placed orthotopically in normal or neovascularized (high-risk) eyes of BALB/c mouse recipients. Topical IL-1RA (or vehicle alone) was applied to grafts 3 times daily until the grafted eyes were enucleated. Corneal specimens were evaluated for content of Langerhans cells. A week after enucleation, 1 group of recipients was tested for allospecific delayed-type hypersensitivity elicited by intrapinnae injections of donor splenocytes. In companion experiments, a second group of mice that underwent transplantation, IL-1RA treatment, and enucleation was challenged with orthotopic skin grafts from B10.D2 donor mice (sharing minor H antigens with C57BL/6 mice) to determine whether the second group of mice could reject grafts bearing corneal donor minor H alloantigens in an accelerated fashion. RESULTS: Mice whose orthotopic corneal allografts were treated topically with IL-1RA acquired neither donor-specific delayed-type hypersensitivity (P<.001) nor the capacity to reject orthotopic donor-type skin allografts in an accelerated manner (P<.05), whereas controls treated with vehicle alone developed delayed-type hypersensitivity and rejected B10.D2 grafts in an accelerated manner. Moreover, IL-1RA-treated grafts placed in both high-risk (P = .01) and normal-risk (P = .004) eyes displayed significantly reduced levels of infiltrating Langerhans cells compared with vehicle-treated controls. CONCLUSIONS: Topical IL-1RA promotes corneal allograft survival in large part by preventing activity of recipient Langerhans cells, and thereby preventing these cells from inducing systemic allosensitization. These data suggest that IL-1 plays a key role in promoting allosensitization when corneal allografts are placed orthotopically. CLINICAL RELEVANCE: Suppression of allosensitization by topical IL-1RA may prove a clinically useful method for enhancing corneal transplant survival.

Neural influences on induction of contact hypersensitivity.

Contact hypersensitivity (CH)-induction begins when cutaneous antigen-presenting cells (APC) capture hapten that has been applied epicutaneously, and the process prepares hapten for presentation to T-cells. APCs are functionally plastic, are influenced by the microenvironment in which they reside, and their functional properties have a profound effect on the phenotype of the hapten-specific T-cells that they activate. Ultraviolet B radiation (UVR) distorts the cutaneous microenvironment, thereby altering local APC function, and changing the immune outcome from sensitization to unresponsiveness. Although UVR induces keratinocytes to produce TNF alpha and IL-10 (cytokines that have been implicated in failed CH-induction and tolerance, respectively, after UVR), dermal mast cells turn out to be the source of these immunomodulatory cytokines. Mast cell degranulation is triggered by CGRP released from UVR-exposed cutaneous nerve termini. Even in normal skin, cutaneous nerves influence the immune response to haptens. Substance P released from cutaneous nerves acts as an adjuvant, raising the immunogenicity of epicutaneously applied haptens. Thus, the nerves and the neuropeptides that these processes release contribute to the cutaneous microenvironment. By altering APC function, cutaneous nerves can dictate the quality and the quantity of immune responses to antigens of the skin.

MHC class II tolerant T cells undergo apoptosis upon re-exposure to tolerogen in vivo.

Tolerance of MHC class II alloantigens can be achieved by intravenous injection of semiallogeneic hematopoietic cells into neonatal mice. Lymphoid cells of tolerant mice fail to proliferate or secrete interleukins IL-2 or IL-4 when stimulated in vitro with tolerogen. Since the lymphoid organs of B10.T(6R) tolerant mice contain normal levels of I-E reactive (V beta 11+) CD4+ T cells, deletion of alloreactive T cells does not appear to be the mechanism involved in the tolerance induction. To test whether T cells from tolerant animals can become activated under conditions that do not involve alloantigen stimulation, we stimulated these cells with immobilized anti-V beta 11 antibodies. Spleen cells from grafted tolerant and rejector mice proliferated in response to anti-V beta 11+ antibodies, suggesting they were not inert. We then tested whether V beta 11+ T cells from grafted mice can be induced to proliferate following stimulation with alloantigen in vivo. We adoptively transferred T cells from grafted tolerant and rejector mice into irradiated (B10.AQR x B10.T(6R))F1 mice and harvested the lymphoid organs after 65 h. Cells from both grafted tolerant and rejector mice underwent blast transformation, but only cells from rejector mice proliferated when exposed to immobilized anti-V beta 11 antibodies. The failure of V beta 11+ cells from tolerant mice to proliferate after in vivo stimulation may be because they are apoptotic. To test this hypothesis, spleen cells from naive or neonatally tolerized (with (B10.AQR x B10.T(6R))F1 cells) B10.T(6R) mice were adoptively transferred into irradiated (B10.AQR x B10.T(6R))F1 mice and bcl-2 expression was analysed in harvested V beta 11+ cells. Large cells recovered from recipients of naive 6R cells expressed bcl-2 mRNA. By contrast, large cells harvested from recipients of tolerized 6R cells did not express bcl-2 mRNA, suggesting bcl-2 mRNA expression was downregulated in these mice. Moreover, in another experiment, large V beta 11+ cells from grafted tolerant animals recovered after transfer into irradiated (B10.AQR x B10.T(6R))F1 mice did not express the bcl-2 protein as determined by flow cytometry, and contained fragmented DNA as assessed by the TUNEL method. Taken together, these data suggest that MHC class II tolerant T cells undergo apoptosis upon re-exposure to tolerogen in vivo.

Allogenic recognition in 1992.

Many new data have been obtained in the last 4 years concerning the structure and functions of major histocompatibility complex (MHC) molecules. Structural data are summarized and the functions of MHC molecules in the presentation of peptides to T cells are described. The high selectivity of peptides binding to MHC molecules is discussed. The negative and positive selection of T cells in the thymus is described and the notion of repertoire introduced. The consequences of these new data on the understanding of H2 restriction and alloreactivity are described. The four potential types of alloreactivity are defined: i) house keeping gene (HKG) peptides bound to allogenic MHC molecules; ii) allogenic peptides (derived from allogenic MHC molecules) bound to allogenic MHC molecules; iii) empty allogenic MHC molecules; iv) allogenic peptides bound to autologous MHC molecules. In fact, the allogenic response is mostly directed toward HKG peptides bound to the allogenic MHC molecules of the graft cells (type 1). The potential role of type four alloreactivity in rejection is discussed.

Aqueous humor induces transforming growth factor-beta (TGF-beta)-producing regulatory T-cells.

PURPOSE: The intraocular microenvironment is an immune-privileged site where immunogenic inflammation is suppressed. Suppression of immunogenic inflammation has been associated with immunosuppressive factors found in aqueous humor produced by ocular tissues. To further understand the mechanisms suppressing immunogenic inflammation in the eye, we have examined the production of lymphokines by primed T-cells activated in the presence of aqueous humor. METHODS: Enriched in vivo primed T-cells were T-cell receptor-stimulated in the presence of fresh aqueous humor. The culture supernatant was assayed for IFN-gamma and IL-4 by sandwich ELISA. TGF-beta production by T-cells stimulated in the presence of aqueous humor was assayed by a TGF-beta bioassay of the culture supernatant and by quantitative RT-PCR for TGF-beta 1 mRNA expression. Aqueous humor-treated T-cells were assayed for their capacity to suppress IFN-gamma production by stimulated, primed T-cells. RESULTS: Aqueous humor-enhanced proliferation but irreversibly suppressed production of both IFN-gamma and IL-4 by in vitro-activated, in vivo-primed T-cells. Aqueous humor induced in vivo primed T-cells to produce TGF-beta in vitro, and these TGF-beta-producing T-cells suppressed IFN-gamma production by other T-cells activated in co-cultures. CONCLUSIONS: Aqueous humor alters the functional program of TCR-ligand-activated, primed T-cells, converting the cells to TGF-beta-producing regulatory cells.

FcγRI is required for TGFß2-treated macrophage-induced tolerance.

Macrophages treated with TGFß2 (TGFß2-Mϕ) and antigen are highly tolerogenic in vivo, and induce antigen-specific and long-lasting tolerance in both naïve and primed mice via induction of suppressor/regulatory T cells. In this study, we examined the molecular pathways, including the requirements for Smad-dependent signaling, that are involved in the induction and function of tolerogenic TGFß2-Mϕ. Treatment of murine macrophages with TGFß2 induced translocation of Smad2/3 to the nucleus, and impairment of Smad3-, but not Smad2-, dependent signaling inhibited the tolerogenic function of a TGFß2-treated murine macrophage cell line. Gene expression in murine macrophages treated with TGFß2 was evaluated by microarray analysis. The FcγRI gene was one of a number of immune-related genes differentially expressed in TGFß2-Mϕ, and appeared to be critical for tolerance in this system, since TGFß2-Mϕ from FcγRI deficient mice were unable to induce tolerance. The role that FcγRI plays in TGFß2-Mϕ-mediated tolerance is currently unclear. The results of this study provide important information about the factors that are critical for the induction of TGFß2-Mϕ-mediated tolerance, and a better understanding of these mechanisms could lead to the development of more effective tolerance-inducing strategies for the treatment of autoimmune/inflammatory diseases.

[Current concepts on the alloreactive response]. / Conceptions actuelles de la réponse alloréactive.

Many new data have been obtained in the last 4 years about the structure and the functions of Major Histocompatibility Complex (MHC) molecules. Structural data are summarized and the functions of MHC molecules in the presentation of peptides to T cells is described. The high selectivity of peptides binding to MHC molecules is explained. The negative and positive selection of T cells in the thymus is described and the notion of repertoire introduced. The consequences of these new data on the understanding of H2 restriction and alloreactivity are explained. The 4 potential types of alloreactivity are defined: 1) House keeping gene (HKG) peptides bound to allogenic MHC molecules, 2) allogenic peptides (derived from allogenic MHC molecules) bound to allogenic MHC molecules, 3) empty allogenic MHC molecules, 4) allogenic peptides bound to autologous MHC molecules. In fact, the allogenic response is mostly directed toward HKG peptides bound to the allogenic MHC molecules of the graft cells (type 1). The potential role of type 4 alloreactivity in rejection is discussed.

Induction of peripheral T cell tolerance and allo/xenoimmunity.

It is theoretically impossible or at least difficult to tolerize animals in xenogeneic situation, particularly in non-concordant species. Both humoral defense and several cellular components (T and non T) are offensive weapons which can be directed at myriad of self-antigens in a normal situation, at allo-antigens in abnormal situations, at xeno-antigens in exceptional situations. In either cases, the fine epitopic definition of allo/xeno antigens seen by the TCR is far from being totally known. From recent studies showing the precise requirement of peptides assembly composition in the MHC class I and II groove it should be theoretically possible to tolerize any group of peptides if they are presented, some other, because not presented, may remain ignored, thus apparently tolerized. It is also know, that transplantation tolerance is not a law of "either nothing or all" but a multiple process depending of both the affinity of the TCR and the nature/compositions of the target. In view of the complex array of factors influencing the pathway of T cell activation, three forms of T cell non responsiveness may be suggested in the context of xenogeneic recognition: physical deletion of potentially reactive T cells occurring predominantly in the thymus, non reactivity of T cells resulting from their failure to be influenced by antigens, and anergy possibly due to inappropriate signals from non professional antigen presenting cells. Future investigations must elucidate the requirements for inducing these events in a purpose of xenogeneic organ transplantation.

Alterations in cytokine production following intraocular injection of soluble protein antigen: impairment in IFN-gamma and induction of TGF-beta and IL-4 production.

Immune deviation induced by intraocular injection of soluble protein Ag, referred to as anterior chamber-associated immune deviation (ACAID), is characterized by impairment of delayed hypersensitivity (DH). Two populations of splenic regulatory cells that impair the induction and expression phases of DH are involved in the ACAID response and may mediate their effects through cytokines. The purpose of the present study was to evaluate the role that cytokines play in ACAID. IFN-gamma production in draining lymph nodes induced by conventional immunization with protein Ag and adjuvant was suppressed after intraocular injection of protein Ag administered either before or after sensitization; IL-12 production in these mice was not decreased, suggesting that suppression of IL-12 may not be the mechanism involved in the impairment in IFN-gamma production. Surprisingly, although significant amounts of IL-4 (but not IL-10) were produced by spleen and lymph node cells from several different strains of mice, experiments in IL-4 knockout mice showed that impairment of neither DH nor IFN-gamma production required IL-4. Interestingly, significant levels of TGF-beta were detected in cultures of spleen cells from mice with ACAID. As determined by quantitative RT-PCR, TGF-beta was produced primarily by the splenic CD4 and non-T cells and was of the TGF-beta1 type. These results suggest that the Th1 response is impaired in ACAID by a mechanism(s) that does not require Th2-type cytokines, but may involve TGF-beta at several different (including the effector) phases during the response.

The attenuated V60 strain of channel catfish virus possesses a deletion in ORF50 coding for a potentially secreted glycoprotein.

A wild-type strain of channel catfish virus was compared at the genomic level with the attenuated strain V60. In addition to several minor differences, restriction mapping revealed one major deletion (approximately 1200 bp) in ORF50 of the V60 strain. Cloning and sequencing of part of this ORF confirmed the presence of a 1164-bp deletion. It should result in a protein of 282 amino acids instead of 670. The predicted truncated protein lacks most of a threonine-rich, highly repetitive region in its central part. Since the protein encoded by ORF50 possesses a hydrophobic N-terminal leader sequence and no membrane anchor sequence, we suggest that it could be a secreted glycoprotein. This protein might be N-glycosylated (35 potential sites) and, given the repetitive arrangement of its residues (mainly threonines), also heavily O-glycosylated like the mucin-type glycoproteins. The deletion observed in ORF50 of the V60 strain implies the loss of 24 potential N-glycosylation sites and should considerably reduce the extent of O-glycosylation.