Nelson Fernandez

Dendritic Cells: Key to Fetal Tolerance?

Abstract Pregnancy is a unique event in which a fetus, despite being genetically and immunologically different from the mother (a hemi-allograft), develops in the uterus. Successful pregnancy implies avoidance of rejection by the maternal immune system. Fetal and maternal immune cells come into direct contact at the decidua, which is a highly specialized mucous membrane that plays a key role in fetal tolerance. Uterine dendritic cells (DC) within the decidua have been implicated in pregnancy maintenance. DC serve as antigen-presenting cells with the unique ability to induce primary immune responses. Just as lymphocytes comprise different subsets, DC subsets have been identified that differentially control lymphocyte function. DC may also act to induce immunologic tolerance and regulation of T cell-mediated immunity. Current understanding of DC immunobiology within the context of mammalian fetal-maternal tolerance is reviewed and discussed herein.

Anomalous Diffusion of Major Histocompatibility Complex Class I Molecules on HeLa Cells Determined by Single Particle Tracking

Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.

The Th2 Cytokine Environment of the Placenta

It is now accepted that local changes to the balance of Th1/Th2-type cytokines occur during pregnancy within the maternal uterus and fetoplacental unit. These changes in cytokine profiles contribute to implantation of the embryo, development of the placenta, and survival of the fetus to term. Overall within the placenta there is a bias in the ratio of Th1:Th2 cytokines towards the Th2-type cytokines. However, there are specific fluctuations in this balance at implantation and during the initiation of parturition. The predominant cytokines at each stage of gestation function both to limit maternal immune rejection of the semi-allogeneic embryo/fetus, especially at the maternofetal interface; and to facilitate the on-going physiological processes within the maternal reproductive tract. These two, at times conflicting, roles are discussed in this review, with key evidence concerning cytokine expression and function from mouse and humans.

Antigen-presenting cells and materno-fetal tolerance: an emerging role for dendritic cells

During pregnancy, a delicate balance of innate and adaptive immune responses at the maternal–fetal interface promotes survival of the semi‐allogeneic embryo and, at the same time, allows effective immunity to protect the mother from environmental pathogens. As in other tissues, antigen handling and processing in the decidualized endometrium constitutes a primary event in the onset of immune responses and is therefore likely to determine their stimulatory or tolerogenic nature. Maternal antigen‐presenting cells [macrophages and dendritic cells (DCs)] are scattered throughout the decidualized endometrium during all stages of pregnancy and appear to be important players in this feto‐maternal immune adjustment. This review focuses on the characterization of decidual macrophages and DCs, as well as their involvement in cell–cell interactions within the decidual leukocyte network, which are likely to influence uterine and placental homeostasis as well as the local maternal immune responses to the fetus during pregnancy.

In vivo dendritic cell depletion reduces breeding efficiency, affecting implantation and early placental development in mice

Implantation of mammalian embryos into their mother’s uterus ensures optimal nourishment and protection throughout development. Complex molecular interactions characterize the implantation process, and an optimal synchronization of the components of this embryo-maternal dialogue is crucial for a successful reproductive outcome. In the present study, we investigated the role of dendritic cells (DC) during implantation process using a transgenic mouse system (DTRtg) that allows transient depletion of CD11c+ cells in vivo through administration of diphtheria toxin. We observed that DC depletion impairs the implantation process, resulting in a reduced breeding efficiency. Furthermore, the maturity of uterine natural killer cells at dendritic cell knockout (DCKO) implantation sites was affected as well; as demonstrated by decreased perforin expression and reduced numbers of periodic-acid-Schiff (PAS)-positive cells. This was accompanied by disarrangements in decidual vascular development. In the present study, we were also able to identify a novel DC-dependent protein, phosphatidylinositol transfer protein β (PITPβ), involved in implantation and trophoblast development using a proteomic approach. Indeed, DCKO mice exhibited substantial anomalies in placental development, including hypocellularity of the spongiotrophoblast and labyrinthine layers and reduced numbers of trophoblast giant cells. Giant cells also down-regulated their expression of two characteristic markers of trophoblast differentiation, placental lactogen 1 and proliferin. In view of these findings, dendritic cells emerge as possible modulators in the orchestration of events leading to the establishment and maintenance of pregnancy.

Stat1 and CD74 overexpression is co-dependent and linked to increased invasion and lymph node metastasis in triple-negative breast cancer.

Triple-negative breast cancer is difficult to treat because of the lack of rationale-based therapies. There are no established markers and targets that can be used for stratification of patients and targeted therapy. Here we report the identification of novel molecular features, which appear to augment metastasis of triple negative breast tumors. We found that triple-negative breast tumors can be segregated into 2 phenotypes based on their genome-wide protein abundance profiles. The first is characterized by high expression of Stat1, Mx1, and CD74. Seven out of 9 tumors from this group had invaded at least 2 lymph nodes while only 1 out of 10 tumors in group 2 was lymph node positive. In vitro experiments showed that the interferon-induced increase in Stat1 abundance correlates with increased migration and invasion in cultured cells. When CD74 was overexpressed, it increased cell adhesion on matrigel. This effect was accompanied with a marked increase in the membrane expression of beta-catenin, MUC18, plexins, integrins, and other proteins involved in cell adhesion and cancer metastasis. Taken together, our results show that Stat1/CD74 positive triple-negative tumors are more aggressive and suggest an approach for development of better diagnostics and more targeted therapies for triple negative breast cancer. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Analysis of HLA-G in Maternal Plasma, Follicular Fluid, and Preimplantation Embryos Reveal an Asymmetric Pattern of Expression

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.

Involvement of beta2-microglobulin and integrin alphavbeta3 molecules in the coxsackievirus A9 infectious cycle.

It is becoming apparent that many viruses employ more than one cell surface molecule for their attachment and cell entry. In this study, we have tested the role of integrin alpha(v)beta3 and MHC class I molecules in the coxsackievirus A9 (CAV-9) infectious cycle. Binding experiments utilizing CHO cells transfected and expressing human integrin alpha(v)beta3, revealed that CAV-9 particles were able to bind to cells, but did not initiate a productive cell infection. Antibodies specific for integrin alpha(v)beta3 molecules significantly reduced CAV-9 infection in susceptible cell lines. Moreover, MAbs specific for beta2-microglobulin (beta2-m) and MHC class I molecules completely inhibited CAV-9 infection. To assess the effect of these antibodies on virus binding, we analysed CAV-9 binding by flow cytometry in the presence of alpha2-m- or integrin alpha(v)beta3-specific antibodies. The results showed a reduction in CAV-9 binding in the presence of integrin alpha(v)beta3-specific antibodies while there was no reduction in the presence of beta2-m-specific MAb. Taken together, these data suggest that integrin alphavbeta3 is required for CAV-9 attachment but is not sufficient for cell entry, while beta2-m, although not directly involved in CAV-9 binding, plays a post-attachment role in the CAV-9 infectious process, possibly being involved in virus entry.

Functionalized diphenylchlorins and bacteriochlorins: their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging

A range of 5,15-diphenylporphyrins with symmetric and unsymmetric substitution patterns were subjected to osmium tetroxide-mediated dihydroxylations. The resulting chlorins and bacteriochlorins present an important group of compounds for studying structure-activity relationships of photodynamic sensitizers. The regioselectivity of the dihydroxylation of various 5,15-diphenylporphyrins with unsymmetrical substitution patterns was also examined. Both diphenylchlorin diol (DPC) and diphenylbacteriochlorin tetrol (DPBC) examples were converted into reactive isothiocyanates and conjugated with bovine serum albumin (BSA).

Preimplantation mouse embryos express Mhc class I genes before the first cleavage division

We have used oligonucleotide primers complementary for polymorphic regions of the mouse H-2D gene in a highly sensitive polymerase chain reaction (PCR) assay to detect the transcription of maternal and paternal class I mRNAs in gametes and preimplantation embryos. Using congenic strains of mice differencing only at the major histocompatibility loci, class I (H-2D) mRNA of both the maternal and paternal haplotypes was demonstrated in embryos from the one-cell zygote to the late blastocyst stage of development but could not be detected in vas deferens or in vitro capacitated sperm or in ovulated secondary oocytes. These data clearly show that both paternally and maternally inherited Major histocompatibility complex (Mhc) class I genes are transcribed from the earliest stages of embryonic development, and suggest that developmental regulation of expression of their protein products is principally at the post-transcriptional level.