Outcomes from a mechanistic biomarker multi-arm and randomised study of liposomal MTP-PE (Mifamurtide) in metastatic and/or recurrent osteosarcoma (EuroSarc-Memos trial).

The phase III clinical study of adjuvant liposomal muramyl tripeptide (MTP-PE) in resected high-grade osteosarcoma (OS) documented positive results that have been translated into regulatory approval, supporting initial promise for innate immune therapies in OS. There remains, however, no new approved treatment such as MTP-PE for either metastatic or recurrent OS. Whilst the addition of different agents, including liposomal MTP-PE, to surgery for metastatic or recurrent high-grade osteosarcoma has tried to improve response rates, a mechanistic hiatus exists in terms of a detailed understanding the therapeutic strategies required in advanced disease. Here we report a Bayesian designed multi-arm, multi-centre, open-label phase II study with randomisation in patients with metastatic and/or recurrent OS, designed to investigate how patients with OS might respond to liposomal MTP-PE, either given alone or in combination with ifosfamide. Despite the trial closing because of poor recruitment within the allocated funding period, with no objective responses in eight patients, we report the design and feasibility outcomes for patients registered into the trial. We demonstrate the feasibility of the Bayesian design, European collaboration, tissue collection with genomic analysis and serum cytokine characterisation. Further mechanistic investigation of liposomal MTP-PE alone and in combination with other agents remains warranted in metastatic OS.

Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions.

Introduction: Trophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells. Methods: After enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT. Results: The trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR. Conclusion: We verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

A Combined microRNA and Chemokine Profile in Urine to Identify Rejection After Kidney Transplantation.

There is an unmet need for noninvasive tools for diagnosis of rejection after kidney transplantation. The aim of this study was to determine the discriminative value of a combined cellular and molecular biomarker platform in urine for the detection of rejection. METHODS: First, microRNA (miR) molecules were screened in transplant biopsies and urine sediments of patients with acute rejection and patients without rejection and stable graft function. Second, the expression of 15 selected miRs was quantified in an independent set of 115 urine sediments of patients with rejection and 55 urine sediments of patients without histological signs of rejection on protocol biopsy. Additionally, CXCL-9 and CXCL-10 protein levels were quantified in the urine supernatant. RESULTS: Levels of miR-155-5p (5.7-fold), miR-126-3p (4.2-fold), miR-21-5p (3.7-fold), miR-25-3p (2.5-fold), and miR-615-3p (0.4-fold) were significantly different between rejection and no-rejection urine sediments. CXCL-9 and CXCL-10 levels were significantly elevated in urine from recipients with rejection. In a multivariable model (sensitivity: 89.1%, specificity: 75.6%, area under the curve: 0.94, P < 0.001), miR-155-5p, miR-615-3p, and CXCL-9 levels were independent predictors of rejection. Stratified 10-fold cross validation of the model resulted in an area under the curve of 0.92. CONCLUSIONS: A combined urinary microRNA and chemokine profile discriminates kidney transplant rejection from stable graft conditions.

Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration.

About 10-15% of couples who want to conceive suffer from subfertility, while in 30% of these cases, a male factor plays a role. Levels of particular microRNAs in seminal plasma, including those involved in spermatogenesis, may serve as an indicative parameter for subfertility. We first optimized a protocol for acquiring microRNAs from seminal plasma. Next, using a test-validation strategy in a male cohort, we aimed to identify microRNAs of which the levels are related to semen motility and concentration. By qPCR, 742 microRNAs were profiled in three normozoospermic samples, three seminal samples with a low semen motility (asthenozoospermia), and three with a low semen concentration (oligozoospermia). MicroRNAs showing significant differences between groups were further validated in a second cohort consisting of 40 samples with normozoospermia (control group), 47 samples with asthenozoospermia, and 19 samples with oligozoospermia (of which 74% also low motility). Highest microRNA yields were obtained with the Biofluids RNA extraction kit, with inclusion of MS2 RNA carrier and proteinase K treatment to the protocol, and when 50 µL of seminal plasma was used as input. Exosome isolation prior to RNA extraction did not lead to enhanced yields. In the test cohort, 236 microRNAs could be detected, of which 54 microRNAs showed a difference between groups. Five microRNAs were analyzed in the validation cohort. MiR-34b-5p levels in the control group were significantly higher compared to the asthenozoospermia group (p < 0.05) and compared to the oligozoospermia group (p < 0.001). We optimized microRNA acquirement from seminal plasma and identified microRNA levels in relation to semen concentration and motility. As recent human and mouse studies show that the miR-34 family is a marker of low semen concentration and is crucial in spermatogenesis, seminal plasma miR-34b-5p may represent a suitable candidate to study further as a marker of male subfertility.

Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells.

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.

Recombinant human monoclonal HLA antibodies of different IgG subclasses recognising the same epitope: Excellent tools to study differential effects of donor-specific antibodies.

In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.

Soluble HLA in the Aqueous Humour of Uveal Melanoma Is Associated with Unfavourable Tumour Characteristics.

A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression and intratumoural inflammation. Aqueous humour from 108 UM patients was analysed for the presence of sHLA, using a Luminex assay specific for HLA Class I. Clinical and genetic parameters were compared between sHLA-positive and negative eyes. A qPCR analysis was performed on tumour tissue using a Fluidigm assay. In 19/108 UM-containing eyes, the sHLA level in the aqueous was above the detection limit. Tumours in sHLA-positive eyes were significantly larger, more frequently involved the ciliary body, and more often showed monosomy 3, gain of chromosome 8q and loss of BAP1 staining. Melanoma-related survival was worse in patients with sHLA-positive aqueous humour. sHLA in the aqueous did not represent the tumour's HLA expression and did not relate to immune cell infiltration in the tumour. We conclude that UM-containing eyes may contain sHLA in the aqueous humour, where it is a prognostically-unfavourable sign and may influence local immune responses.

Soluble HLA-G levels in seminal plasma are associated with HLA-G 3'UTR genotypes and haplotypes.

Soluble HLA-G (sHLA-G) levels in human seminal plasma (SP) can be diverse and may affect the establishment of maternal-fetal tolerance and thereby the outcome of pregnancy. We investigated whether sHLA-G levels in SP are associated with polymorphisms in the 3'-untranslated region (UTR) and UTR haplotypes of the HLA-G gene. Furthermore, we compared the HLA-G genotype distribution and sHLA-G levels between men, whose partner experienced unexplained recurrent miscarriage (RM), and controls. Soluble HLA-G levels (n = 156) and HLA-G genotyping (n = 176) were determined in SP samples. The concentration of sHLA-G was significantly associated with several single-nucleotide polymorphisms (SNPs): the 14 base pair (bp) insertion/deletion (indel), +3010, +3142, +3187, +3196, and + 3509. High levels of sHLA-G were associated with UTR-1 and low levels with UTR-2, UTR-4, and UTR-7 (P < .0001). HLA-G genotype distribution and sHLA-G levels in SP were not significantly different between the RM group (n = 44) and controls (n = 31). In conclusion, seminal sHLA-G levels are associated with both singular SNPs and 3UTR haplotypes. HLA-G genotype and sHLA-G levels in SP are not different between men whose partner experienced RM and controls, indicating that miscarriages are not solely the result of low sHLA-G levels in SP. Instead, it is more likely that these miscarriages are the result of a multifactorial immunologic mechanism, whereby the HLA-G 3'UTR 14 bp ins/ins genotype plays a role in a proportion of the cases. Future studies should look into the functions of sHLA-G in SP and the consequences of low or high levels on the chance to conceive.

Increased infiltration of M2-macrophages, T-cells and PD-L1 expression in high grade leiomyosarcomas supports immunotherapeutic strategies.

Background: Immunotherapy may be a rational strategy in leiomyosarcoma (LMS), a tumor known for its genomic complexity. As a prerequisite for therapeutic applications, we characterized the immune microenvironment in LMS, as well as its prognostic value. Methods: CD163+ macrophages, CD3+ T-cells, PD-L1/PD-L2 and HLA class I expression (HCA2, HC10 and ß2m) were evaluated using immunohistochemistry in primary tumors (n = 75), local relapses (n = 6) and metastases (n = 19) of 87 LMS patients, as well as in benign leiomyomas (n = 7). Correlation with clinicopathological parameters and survival analyses were assessed. Effect of LMS cells on macrophage differentiation was investigated using coculture of CD14+ monocytes with LMS cell lines or their conditioned media (CM). Results: 58% and 52% of the tumors were highly infiltrated with CD163+ macrophages and T-cells, respectively, with HLA class I expression observed in almost all tumors and PD-L1 expression in 30%. PD-L2 expression was also detected in some PD-L1+ tumors. All these immune markers correlated with high tumor grade but only CD163 associated with overall survival (p = 0.003) and disease-specific survival (p = 0.041). In vitro, CD163 was upregulated in the presence of LMS cells producing M-CSF, suggesting that this tumor drives macrophages towards the M2 phenotype. Conclusion: The clinical significance of M2 macrophages, possibly induced by LMS cell-secreted factors, suggests that 2/3 of high-grade LMS patients might benefit from macrophage-targeting agents. Furthermore, PD-L1 expression together with high T-cell infiltrate and HLA class I expression in around 30% of high grade LMS reflects an active immune microenvironment potentially responsive to immune checkpoint inhibitors.

Stronger T-Cell Alloreactivity and Diminished Suppressive Capacity of Peripheral Regulatory T Cells in Infertile Women Undergoing In Vitro Fertilization.

PROBLEM: Increasing evidence suggests modulation of the maternal immune response to be essential for successful pregnancy. We studied the immunophenotypic profile and function of peripheral blood T lymphocytes in infertile women undergoing in vitro fertilization (IVF) and fertile control population. METHOD: We collected peripheral blood mononuclear cells (PBMC) from infertile patients with recurrent implantation failure (RIF), infertile patients with successful IVF (IVFs), and normal fertile women. Cells were phenotypically analyzed, and the proliferative response and cytokine production were studied in mixed lymphocyte cultures (MLC), using lymphocytes of the own partner, or a third-party male as stimulators cells. To examine the suppressive capacity of regulatory regulatory T cells (Tregs), we performed MLC studies with a CD45(+) fraction depleted for CD4(+) CD25(bright) T cells. RESULTS: No significant differences in proportions of subsets of circulating T lymphocytes were observed. The proliferative allo-immune response of PBMC of IVF women (RIF and IVFs) was significantly higher, with higher production of T-helper cells (Th1) and Th2 cytokines, compared to the fertile women. This difference in proliferation and cytokine production was associated with a diminished suppressive capacity of Tregs in these women. CONCLUSION: The higher allo-immune response of the IVF women compared to fertile women might be the result of a diminished suppressive capacity of Tregs and emphasizes the important role of Tregs during conception.

Differential immunoregulation in successful oocyte donation pregnancies compared with naturally conceived pregnancies.

In oocyte donation (OD) pregnancies, there is a higher level of antigenic dissimilarity between mother and fetus compared with naturally conceived (NC) pregnancies. We hypothesize that a higher degree and/or a different type of immunoregulation is necessary to maintain an uncomplicated OD pregnancy. Different immunological aspects of successful OD pregnancies (n=28) were compared with those of NC pregnancies (n=51), and non-donor IVF (n=20) pregnancies. Maternal peripheral blood mononuclear cells (mPBMCs) were characterized by flow cytometry; the outcome correlated with the number of mother-child HLA mismatches. The fetus-specific alloreactivity of mPBMCs was measured in a mixed lymphocyte reaction. The percentages of CD4(+)CD25(bright) and CD4(+)CD25(dim) cells were higher in mPBMCs of OD and IVF pregnancies compared with NC pregnancies. The percentage of CD4(+)CD25(dim) cells in mPBMCs of OD pregnancies correlated positively with the number of HLA mismatches. Functional studies showed a lower proliferative response to umbilical cord blood by mPBMCs in OD pregnancies. In conclusion, we found a higher degree of peripheral immunoregulation in OD and IVF pregnancies compared with NC pregnancies. A higher number of HLA mismatches in successful OD pregnancies leads to increased percentages of activated T cells in peripheral blood, but not to a higher alloreactivity to the fetus. These studies show that immunoregulation in OD pregnancy is different from that in NC pregnancies. The antigenic dissimilarity in OD pregnancies may play a role in the pathophysiology of preeclampsia.

Differential effects of activated human renal epithelial cells on T-cell migration.

BACKGROUND: Renal tubular epithelial cells (TECs) are one of the main targets of inflammatory insults during interstitial nephritis and kidney transplant rejection. While Th1 cells are know to be essential in the pathogenesis of rejection, the role of Th17 is still under debate. We hypothesize that TECs modulate the outcome of rejection process by production of distinct chemokines and cytokines that determine the attraction of different T-cell subsets. Therefore, we studied differential effects of activated human renal epithelial cells on T-cell migration. METHODS: Human primary TECs were stimulated by IFN-γ and TNF-α in vitro. Chemokines and cytokines produced by activated TECs were measured using Luminex or ELISA. Chemotaxis assay was performed using activated peripheral blood mononuclear cells composed of CD4+CXCR3+ and CD4+CCR6+ T cells migrating towards stimulated and unstimulated TECs. RESULTS: While activated TECs secreted abundant amounts of the pro-inflammatory cytokines IL-6 and IL-8, the T helper cell differentiation cytokines IL-1ß, IL-12p70, IL-23 or TGF-ß1 were not produced. The production of Th1 chemokines CXCL9, CXCL10 and CCL5 were significantly upregulated after TEC stimulation. In contrast, Th17 chemokine CCL20 could not be detected. Finally, activated TECs attracted significantly higher numbers of CD4+CXCR3+ T cells as compared to unstimulated TECs. No migration of CD4+CCR6+ T cells could be observed. CONCLUSION: Activated primary renal tubular epithelial cells do not attract Th17 cells nor produce cytokines promoting Th17 cell differentiation in our experimental system mimicking the proinflammatory microenvironment of rejection.

Heart failure determines the myocardial inflammatory response to injury.

AIMS: Systemic complications after cardiac surgery are common in heart failure patients. However, the pathophysiological mechanisms, such as a different local inflammatory response of failing hearts, remain in question. This study examines whether failing hearts respond differently to cardioplegic arrest and reperfusion compared with non-failing hearts (controls). METHODS AND RESULTS: The inflammatory response was evaluated in samples collected simultaneously from the radial artery and coronary sinus, and myocardial tissue in 62 patients undergoing cardiac surgery. No myocardial release of inflammatory mediators was observed upon reperfusion in controls (n = 19). In contrast, in patients with heart failure, reperfusion was characterized by a myocardial release of several cytokines. Myocardial interleukin-6 was 115% increased in non-ischaemic heart failure (n = 18, P = 0.002) as compared with a 117% increase in patients with ischaemic heart failure (n = 25, P = 0.01). Furthermore, a myocardial release of monocyte chemoattractant protein-1 was observed in both patient groups: a 109% (P = 0.001) and 114% (P = 0.01) increase in patients with non-ischaemic heart failure and ischaemic heart failure, respectively. Post-operative myocardial damage, expression of inflammatory mediators, and p65-nuclear factor-κB activity were similar in all patient groups. Inflammatory cell content was increased in early ischaemic myocardial tissue in both heart failure groups compared with controls. CONCLUSION: Heart failure patients show a clear myocardial inflammatory response upon reperfusion, probably explained by degranulation of infiltrated inflammatory cells. Results in controls indicate that they better withstand cardioplegic arrest and reperfusion without an explicit myocardial inflammatory response.

The functional polymorphism Ala258Ser in the innate receptor gene ficolin-2 in the donor predicts improved renal transplant outcome.

BACKGROUND: Innate immunity plays a role in controlling adaptive immune responses. METHODS: We investigated the clinical relevance of single nucleotide polymorphisms in 22 genes encoding innate, secreted, and signaling pattern recognition receptors in a total of 520 donor-recipient pairs of postmortem, human leukocyte antigen-DR-compatible kidney transplantations. Associations with rejection incidence were tested in an a priori randomized training set and validation set. RESULTS: Polymorphisms in TLR-3 (rs3775296) in the recipients and in ficolin-2 (rs7851696; Ala258Ser) and C1qR1 (rs7492) in the donors showed the strongest association with severe rejection. In multivariate analysis, presence of the ficolin-2 Ala258Ser variant in the donor predicted lower incidence of severe rejection (odds ratio=0.3; 95% confidence interval, 0.1-0.9; P=0.024) and of graft loss (hazard ratio=0.5; 95% confidence interval, 0.2-1.0; P=0.046) independently of clinical risk factors. Ficolin-2 messenger RNA expression was detected in pretransplantation biopsies from 69 donor grafts. Serum and tissue ficolin-2 levels were unaffected by genotype. Ficolin-2 protein, which bound to dying cells, was detected in donor kidneys in a passenger leukocyte-like pattern. Indeed, monocytes, monocyte-derived macrophages, and peripheral blood mononuclear cells expressed ficolin-2. Donor grafts with the ficolin-2 Ala258Ser variant contained significantly elevated expression of interleukin 6, having ascribed cytoprotective effects. It has been described that Ala258Ser leads to increased binding capacity of ficolin-2 to N-acetylglucosamine. CONCLUSIONS: Presence of the ficolin-2 Ala258Ser polymorphism in the donor independently predicts improved graft outcome. Based on mechanistic data, we propose that this functional polymorphism leads to more efficient handling of injured cells by phagocytozing cells, resulting in decreased intragraft exposure to danger signals and dampened alloimmune responses.

Changes in cytokine production and composition of peripheral blood leukocytes during pregnancy are not associated with a difference in the proliferative immune response to the fetus.

We analyzed peripheral blood from women at term pregnancy for leukocyte composition, in vitro proliferative responses and cytokine production after nonspecific and fetus-specific stimulation. Maternal peripheral blood mononuclear cells (PBMCs) were collected and stimulated with umbilical cord blood (UCB) of the mother's own child, third-party UCB, nonspecific stimulus phytohemagglutinin, and anti-CD3 antibody, with PBMCs of nonpregnant women (cPBMC) as controls. Nine combinations of patient, child, third party child, and controls were selected on basis of sharing one human leukocyte antigen (HLA)-DR antigen. The response of mPBMC upon specific stimulation with fetal antigens was similar to that of cPBMC. No differences were found when comparing the mother's response upon stimulation to her own child with stimulation to that with a control child. Nonspecific stimulation with phytohemagglutinin and anti-CD3 antibody did not reveal a difference in proliferation rate between mPBMC and cPBMC. However, mPBMC contained a higher percentage of CD14(+) cells (p = 0.001) and activated T cells (CD25(dim), p < 0.0001), but a lower percentage CD16(-)CD56(bright) natural killer (NK) cells (p = 0.001) and CD16(+)CD56(+) NK cells (p = 0.003). mPBMC produced more interleukin (IL)-6, IL-10, and IL-17 compared with cPBMC (p < 0.05). We found differences in lymphocyte composition and cytokine production between mPBMC and cPBMC. These differences did not result in quantitative changes in proliferative responses during pregnancy compared with responses in nonpregnant controls.

Baseline serum adipokine levels predict radiographic progression in early rheumatoid arthritis.

OBJECTIVE: Adipose tissue can secrete soluble mediators (adipokines) with potent immune regulatory functions. Some adipokines have been previously associated with radiographic damage in patients with rheumatoid arthritis (RA). In the present study, we investigated the capacity of baseline adipokine levels to predict radiographic progression over a period of 4 years and studied their contribution relative to that of other known risk factors, such as anti-cyclic citrullinated peptide (anti-CCP) antibodies. METHODS: Serum concentrations of leptin, visfatin, resistin, adiponectin, adipsin, tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) were determined in serum samples obtained at baseline from 253 patients with RA from the Early Arthritis Cohort. The association between levels of these adipokines and radiographic progression was determined using a multivariate normal regression model correcting for age, sex, treatment strategy, body mass index (BMI), and the presence of anti-CCP antibodies. RESULTS: Levels of IL-6, TNFα, visfatin, and adiponectin were positively associated with radiographic progression over 4 years. This association was independent of BMI. However, only adiponectin levels remained significantly associated with radiographic progression when the model was corrected for the presence of anti-CCP antibodies, whereas a trend was observed for IL-6. The association of both TNFα and visfatin with radiographic damage disappeared after correction for the presence of anti-CCP antibodies, which is consistent with the fact that the levels of both cytokines correlated significantly with anti-CCP antibody levels in these patients. CONCLUSION: Our results indicate that adipokines are predictors of radiographic progression in RA, possibly through distinct underlying biologic mechanisms.

Differential distribution and phenotype of decidual macrophages in preeclamptic versus control pregnancies.

Maternal immune tolerance of the semiallogeneic fetus is a complex phenomenon. Macrophages are an abundant cell population in the human decidua, and changes in distribution or phenotype may be involved in the development of preeclampsia. The aim of this study was to assess the distribution and phenotype of macrophages in preterm preeclamptic, preterm control, and term control placentas. Placentas of preterm preeclamptic (n = 6), preterm control (n = 5), and term control pregnancies (n = 6) were sequentially immunohistochemically stained for CD14, CD163, DC SIGN, and IL-10. The distributions of CD14(+), CD163(+), DC SIGN(+), IL-10(+), CD163(+)/CD14(+), DC SIGN(+)/CD14(+), and Flt-1/CD14(+) cells were determined by double staining and by digital image analysis of sequential photomicrographs. CD14 and CD163 expression increased significantly in preterm preeclamptic decidua basalis compared with preterm control pregnancies (P = 0.0006 and P = 0.034, respectively). IL-10 expression was significantly lower in the decidua parietalis of preterm preeclamptic pregnancies compared with preterm control pregnancies (P = 0.03). The CD163/CD14 ratio was significantly lower in the decidua basalis (P = 0.0293) and the DC SIGN/CD14 ratio was significantly higher in the decidua basalis (P < 0.0001) and parietalis (P < 0.0001) of preterm preeclamptic pregnancies compared with preterm control pregnancies. CD14(+) macrophages did express Flt-1. Alterations in distribution and phenotype of macrophages in the decidua of preterm preeclamptic pregnancies compared with control pregnancies may contribute to the pathogenesis of preeclampsia.

Inflammatory cytokines in eyes with uveal melanoma and relation with macrophage infiltration.

PURPOSE: The presence of an inflammatory phenotype, characterized by an increased expression of HLA antigens and an immunologic infiltrate, carries a bad prognosis in uveal melanoma. This study was conducted to determine whether the aqueous humor (AqH) from eyes with uveal melanoma contains inflammatory cytokines and whether their presence is associated with inflammation. METHODS: Immediately after enucleation, AqH was obtained from 37 eyes containing uveal melanoma. Samples were stored at -80°C until use. Fifteen different cytokines were measured with a multiplex bead array. Intratumoral macrophages were analyzed by immunohistochemistry and immunofluorescence staining. The presence of specific cytokines was compared with histopathologic, genetic, and clinical tumor characteristics, as well as patient survival. RESULTS: Several cytokines showed significantly higher expression in the AqH of uveal melanoma-containing eyes than in the AqH of eyes undergoing cataract surgery. MCP-3 was associated with the presence of CD68(+) macrophages. Correlations were found between some cytokine levels and a few known prognostic factors of uveal melanoma, but cytokine levels were not of predictive value for survival. CONCLUSIONS: Uveal melanoma-containing eyes often carry increased levels of inflammation-related cytokines in their AqH. However, the presence of most specific cytokines was not related to the presence of macrophages, clinical or histopathologic parameters, or prognosis.

Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells.

Protective mechanisms are likely to be present at the fetomaternal interface because fetus-specific alloreactive T cells present in the decidua do not harm the fetus. We tested the immunosuppressive capacity of maternal and fetal multipotent stromal cells (MSC). Single cell suspensions were made from second-trimester amnion, amniotic fluid, and decidua. Culture-expanded cells were identified as MSC based on phenotype and multilineage potential. Coculture of MSC in a primary mixed lymphocyte culture of unrelated responder-stimulator combinations resulted in a dose-dependent inhibition of proliferation. Fetal MSC demonstrated a significantly higher inhibition compared with maternal MSC. This stronger inhibition by fetal MSC was even more prominent in a secondary mixed lymphocyte reaction (MLR) with primed alloreactive T cells. Analysis of cytokine production revealed that fetal MSC produced significantly more interleukin (IL)-10 and vascular endothelial growth factor than maternal MSC. Cell-cell contact is needed for part of the inhibitory effects of MSC. In addition, soluble factors play a role because blocking experiments with anti-IL-10 revealed that the inhibition of the MLR response by fetal MSC is mainly mediated by IL-10. For maternal MSC, other soluble factors seem to be involved. Fetal MSC derived from the fetomaternal interface have a stronger inhibitory effect on naive and antigen-experienced T cells compared with maternal MSC, which is probably related to their higher IL-10 production.

The reactivity of Bw4+ HLA-B and HLA-A alleles with KIR3DL1: implications for patient and donor suitability for haploidentical stem cell transplantations.

Natural killer (NK)-cell alloreactivity can be exploited in haploidentical hematopoietic stem cell transplantation (HSCT). NK cells from donors whose HLA type includes Bw4, a public epitope present on a subset of HLA-B alleles, can be alloreactive toward recipients whose cells lack Bw4. Serologically detectable epitopes related to Bw4 also exist on a subset of HLA-A alleles, but the interaction of these alleles with KIR3DL1 is controversial. We therefore undertook a systematic analysis of the ability of most common HLA-B alleles and HLA-A alleles with Bw4 serologic reactivity to protect target cells from lysis by KIR3DL1-dependent NK cells. All Bw4(-) HLA-B alleles failed to protect target cells from lysis. All Bw4(+) HLA-B alleles with the exception of HLA-B*1301 and -B*1302 protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis, whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors with HLA-A*2402 but not in donors with HLA-B*1301 or -B*1302. These findings clarify the HLA types that donors and recipients need in haploidentical HSCT and other NK allotherapies in order to benefit from NK alloreactivity.