An increase in regulatory T cells in peripheral blood correlates with an adverse prognosis for malignant melanoma patients - A study of T cells and natural killer cells.

Malignant melanoma is a highly immunogenic tumour, and the immune profile significantly influences cancer development and response to immunotherapy. The peripheral immune profile may identify high risk patients. The current study showed reduced levels of CD4+ T cells and increased levels of CD8+ T cells in peripheral blood from malignant melanoma patients compared with controls. Percentages of peripheral CD56dimCD16+ NK cells were reduced and CD56brightCD16-KIR3+ NK cells were increased in malignant melanoma patients. Late stage malignant melanoma was correlated with low levels of CD4+ T cells and high levels of CD56brightCD16-KIR3+ NK cells. Finally, high levels of Tregs in peripheral blood were correlated with poor overall survival and disease-free survival. The results indicate that changes in specific immune cell subsets in peripheral blood samples from patients at the time of diagnosis may be potential biomarkers for prognosis and survival. Further studies will enable clarification of independent roles in tumour pathogenesis.

Endometrial HLA-F expression is influenced by genotypes and correlates differently with immune cell infiltration in IVF and recurrent implantation failure patients.

STUDY QUESTION: Is human leukocyte antigen (HLA)-F protein expressed in mid-secretory endometrium, and are its expression levels influenced by HLA-F gene polymorphisms and correlated with the abundance of uterine natural killer (uNK) cells and anti-inflammatory M2 macrophages? SUMMARY ANSWER: HLA-F protein is expressed in mid-secretory endometrium, and levels are correlated with immune cell infiltration, plasma progesterone concentrations and HLA-F single-nucleotide polymorphisms (SNPs), however, women experiencing recurrent implantation failure (RIF) show differences when compared to women attending their first IVF treatment. WHAT IS KNOWN ALREADY: The immunomodulatory HLA class Ib molecules HLA-G and HLA-F are expressed on the extravillous trophoblast cells and interact with receptors on maternal immune cells. Little is known regarding HLA-F expression in endometrial stroma and HLA-F function; furthermore, HLA-F and HLA-G SNP genotypes and haplotypes have been correlated with differences in time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: Primary endometrial stromal cell (ESC) cultures (n = 5) were established from endometrial biopsies from women attending IVF treatment at a fertility clinic. Basic HLA-F and HLA-G protein expression by the ESCs were investigated. A prospective controlled cohort study was performed including 85 women with a history of RIF and 36 control women beginning their first fertility treatment and with no history of RIF. In some analyses, the RIF group was divided into unknown cause, male infertility, female infertility, and both female and male infertility. Endometrial biopsies and blood samples were obtained the day equivalent to embryo transfer in a hormone-substituted cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: HLA protein expression by ESCs was characterized using flow cytometry and western blot. In the cohort study, the specific immune markers HLA-F and HLA-G, CD56 and CD16 (NK cells), CD163 (M2 macrophages), FOXP3 (regulatory T cells) and CD138 (plasma cells) were analysed by immunohistochemistry and a digital image analysis system in endometrial biopsies. Endometrial receptivity was assessed by an endometrial receptivity array test (the ERA® test). Endometrial biopsies were examined according to modified Noyes' criteria. SNPs at the HLA-F gene and HLA-G haplotypes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: HLA-F protein is expressed in the endometrium at the time of implantation. Furthermore, the HLA-F protein levels were different according to the womens HLA-F SNP genotypes and diplotypes, which have previously been correlated with differences in time-to-pregnancy. Endometrial HLA-F was positively correlated with anti-inflammatory CD163+ M2 macrophage infiltration and CD56+ uNK cell abundance for the entire cohort. However, this was not the case for CD56+ in the female infertility RIF subgroup. HLA-F levels in the endometrial stroma were negatively correlated with plasma progesterone concentrations in the RIF subgroup with known female infertility. Conversely, HLA-F and progesterone were positively correlated in the RIF subgroup with infertility of the male partner and no infertility diagnosis of the woman indicating interconnections between progesterone, HLA-F and immune cell infiltration. Glandular sHLA-G expression was also positively correlated with uNK cell abundance in the RIF subgroup with no female infertility but negatively correlated in the RIF subgroup with a female infertility diagnosis. LARGE SCALE DATA: Immunohistochemistry analyses of endometrial biopsies and DNA sequencing of HLA genes. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The control group of women attending their first IVF treatment had an anticipated good prognosis but was not proven fertile. A significant age difference between the RIF group and the IVF group reflects the longer treatment period for women with a history of RIF. The standardization of hormonal endometrial preparation, which allowed consistent timing of endometrial and blood sampling, might be a strength because a more uniform hormonal background may more clearly show an influence on the immune marker profile and HLA class Ib levels in the endometrium by other factors, for example genetic polymorphisms. However, the immune marker profile might be different during a normal cycle. WIDER IMPLICATIONS OF THE FINDINGS: The findings further highlight the importance of HLA-F and HLA-G at the implantation site and in early pregnancy for pregnancy success. Diagnostic measures and modulation of the complex interactions between HLA class Ib molecules, maternal immune cells and hormonal factors may have potential to improve fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Region Zealand Health Sciences Research Foundation and the Zealand University Hospital through the ReproHealth Research Consortium ZUH. The authors declared there are no conflicts of interest.

HLA Class Ib-receptor interactions during embryo implantation and early pregnancy.

BACKGROUND: Although the immune system intuitively must have an important role in embryo implantation and in the achievement of a pregnancy, the molecular details have for long been controversial. The role of the human leukocyte antigen (HLA) system has been debated. The unique HLA expression profile of the HLA Class Ia molecule HLA-C and the HLA Class Ib molecules HLA-E, HLA-F and HLA-G at the feto-maternal interface is now recognized. However, HLA Class Ib molecules may also have a role in embryo implantation and pregnancy success. OBJECTIVE AND RATIONALE: The aim of this review was to evaluate the literature and recent discoveries on the role of the non-polymorphic HLA Class Ib molecules with a focus on HLA-F and HLA-G molecules at the time of implantation, including the interaction with uterine immune cells through the specific receptors immunoglobulin-like transcript 2 (ILT2), ILT4 and a number of killer cell immunoglobulin-like receptors (KIRs), and the importance of HLA-F and HLA-G genetic variation that influences fertility and time-to-pregnancy. SEARCH METHODS: Drawing on recent advances in basic and clinical studies, we performed a narrative review of the scientific literature to provide a timely update on the role of HLA Class Ib in embryo implantation, fertility and infertility. Pertinent studies were searched in PubMed/Medline using relevant key words. OUTCOMES: Both HLA-F and HLA-G interact with inhibitory or activating ILT2 or ILT4 receptors and KIRs on uterine immune cells, especially uterine natural killer (NK) cells that are highly abundant in the mid-secretory endometrium and in early pregnancy. The binding of HLA-G to ILT2 stimulates the secretion of growth-promoting factors from decidual NK cells. However, functional aspects of a HLA-F-receptor interaction remain to be clarified. Recent studies indicate that HLA-F and HLA-G are expressed in mid-secretory endometrium and HLA-G is expressed in the blastocyst. HLA-F fluctuates during the menstrual cycle with high levels during the implantation window. The level of HLA-F protein expression correlates with the number of CD56-positive NK cells in the mid-secretory endometrium. HLA-F and HLA-G gene polymorphisms, including a single nucleotide polymorphism (SNP) in a progesterone-responsive element, are associated with time-to-pregnancy. Depending on the SNP genotype, the effect of progesterone varies resulting in differences in HLA-F expression and thereby the interaction with receptors on the uterine NK cells. Studies suggest that the expression of HLA-G and HLA-F, both by the embryonic-derived trophoblast cells and by cells in the endometrium and decidua, and the interaction between HLA-G and HLA-F with specific receptors on uterine immune cells, stimulate and facilitate embryo implantation and placentation by secretion of growth factors, cytokines and angiogenic factors. WIDER IMPLICATIONS: A detailed understanding of the molecular mechanisms controlling the expression of HLA-F and HLA-G periconceptionally and in early pregnancy may improve the success of ART and holds promise for further insight into pathophysiological aspects of certain pregnancy complications.

An exploration of immunohistochemistry-based prognostic markers in patients undergoing curative resections for colon cancer.

BACKGROUND: The immune system recognizes and destroys cancer cells. However, cancer cells develop mechanisms to avoid detection by expressing cell surface proteins. Specific tumour cell surface proteins (e.g. HLA-G, PD-L1, CDX2) either alone or in combination with the relative presence of immune cells (CD3 and CD8 positive T-cells) in the tumour tissue may describe the cancer cells' ability to escape eradication by the immune system. The aim was to investigate the prognostic value of immunohistochemical markers in patients with colon cancer. METHODS: We conducted a retrospective study including patients diagnosed with pT3 and pT4 colon cancers. Immunohistochemical staining with HLA-G, PD-L1, CDX2, CD3, and CD8 was performed on tissue samples with representation of the invasive margin. PD-L1 expression in tumour cells and immune cells was reported conjointly. The expression of CD3 and CD8 was reported as a merged score based on the expression of both markers in the invasive margin and the tumour centre. Subsequently, a combined marker score was established based on all of the markers. Each marker added one point to the score when unfavourable immunohistochemical features was present, and the score was categorized as low, intermediate or high depending on the number of unfavourable stains. Hazard ratios for recurrence, disease-free survival and mortality were calculated. RESULTS: We included 188 patients undergoing colon cancer resections in 2011-2012. The median follow-up was 41.7 months, during which 41 (21.8%) patients had recurrence and 74 (39.4%) died. In multivariable regression analysis positive HLA-G expression (HR = 3.37, 95%CI [1.64-6.93]) was associated with higher recurrence rates, while a preserved CDX2 expression (HR = 0.23, 95%CI [0.06-0.85]) was associated with a lower risk of recurrence. An intermediate or high combined marker score was associated with increased recurrence rates (HR = 20.53, 95%CI [2.68-157.32] and HR = 7.56, 95%CI [1.06-54.16], respectively). Neither high expression of PD-L1 nor high CD3-CD8 score was significantly associated with recurrence rates. Patients with a high CD3-CD8 score had a significantly longer DFS and OS. CONCLUSIONS: In tumour cells, expression of HLA-G and loss of CDX2 expression were associated with cancer recurrence. In addition, a combination of certain tumour tissue biomarkers was associated with colorectal cancer recurrence.

Peripheral blood immune markers in breast cancer: Differences in regulatory T cell abundance are related to clinical parameters.

BACKGROUND: Cancer development is among other factors driven by tumor immune escape and tumor-mediated changes in the immune response. Investigating systemic immune changes may provide important knowledge for the improvement of patient prognosis and treatment opportunities. METHODS: The systemic immune profile of patients with ER-positive breast cancer (n = 22) and healthy controls (n = 30) was investigated based on complete blood counts, flow cytometric analysis of T cell subsets including regulatory T cells (Tregs), and immune assays investigating soluble (s)HLA-G and the cytokine profile in plasma. We further examined the correlation between the immune markers and clinical parameters including tumor size, tumor grade and lymph node involvement. RESULTS: Results indicated that breast cancer patients possessed a higher amount of neutrophils and monocytes and fewer lymphocytes and eosinophils compared with healthy controls. Breast cancer patients had significantly more CD25+CD127low Tregs than controls, and both lymphocyte and Treg numbers were negatively correlated with tumor size. Furthermore, Treg numbers were elevated in grade I tumors compared with grade II tumors and with healthy controls. No difference in sHLA-G levels was observed between patients and controls. Higher levels of IL-6 and TNF-α were observed in breast cancer patients. Cytokine and sHLA-G levels were not associated with clinical parameters. CONCLUSION: The results of this exploratory study contribute to the elucidation of the systemic immune response in breast cancer indicating a potential use of peripheral immune cell counts and Tregs to distinguish patients from healthy controls and as potential diagnostic and prognostic biomarkers to be investigated in future studies.

Tumour-infiltrating CD4-, CD8- and FOXP3-positive immune cells as predictive markers of mortality in BRCA1- and BRCA2-associated breast cancer.

BACKGROUND: The prognostic value of tumour-infiltrating lymphocytes (TILs) in breast cancer is well-established. However, the investigation of specific T-cell subsets exclusively in BRCA-associated breast cancer is sparse. METHODS: Tumour tissues from 414 BRCA-mutated breast cancer patients were analysed by immunohistochemistry and digital image analysis for expression of CD4, CD8 and FOXP3 immune markers. Distribution of CD4-, CD8- and FOXP3-positive cells and clinicopathological characteristics were assessed according to groups of low or high expression. The prognostic value was evaluated as continuous variables in univariate and multivariate analyses of overall survival and disease-free survival. RESULTS: Both CD4 and CD8 expression are associated with histological diagnosis, tumour grade and oestrogen and progesterone receptor expression status. CD4 expression is associated with BRCA gene status. A high percentage of tumour-infiltrating CD4-, CD8- or FOXP3-positive cells is significantly associated with lower mortality in BRCA1- and BRCA2-associated breast cancer and CD8-positive cells are associated with disease-free survival. No heterogeneity according to BRCA gene status was found for the prognostic value of the immune markers. CONCLUSIONS: The results support a prognostic role of specific T-cell subsets in BRCA-associated breast cancer and the promising potential of targeting the immune system in the treatment of these patients.

Are different markers of endometrial receptivity telling us different things about endometrial function?

PROBLEM: To what extent do endocrine, immunological, gene expression and histological markers of endometrial receptivity correlate? METHOD OF STUDY: Between November 2017 and September 2019, 121 women referred to a University Hospitals Fertility Clinic consented to inclusion in this cohort study. The women underwent timed endometrial biopsy followed by blood samples in a hormone-substituted cycle. Of these, 37 women had just started IVF treatment, and the remaining 84 had experienced recurrent implantation failure following IVF/ICSI. The hormone-substituted cycle consisted of initiation with oral oestradiol followed by addition of vaginal progesterone treatment for five full days. Endometrial biopsies were subject to histological examination, immune cell markers by immunohistochemistry (CD56+ , CD16+ , CD163+ , FoxP3) and gene expression microarray analyses with the endometrial receptivity array (ERA® ) test (Igenomix). Plasma progesterone and oestradiol were measured on the day of biopsy. RESULTS: CD56+ uterine natural killer (uNK) cell counts correlate with transcriptional markers of endometrial receptivity assessed by the ERA test. Endometrial maturation, receptivity and immunological markers were not correlated with mid-luteal blood plasma progesterone level. Mid-luteal serum oestradiol level correlated with markers of endometrial maturation and receptivity. The tests were carried out during a standard hormone substitution cycle, and the findings may not apply in the natural cycle. CONCLUSION: CD56+ uNK cell counts and endometrial receptivity assessed by the ERA test appear to be linked. Mid-luteal progesterone levels were not correlated to the tested markers of endometrial receptivity. In contrast, mid-luteal oestradiol level was inversely related to markers of endometrial receptivity and maturation.

A role for both HLA-F and HLA-G in reproduction and during pregnancy?

The human major histocompatibility complex includes a group of non-classical HLA class I genes, HLA-E, -F and -G. While nearly all focus since the discovery of these class Ib molecules have been on basic biochemistry and molecular biology of HLA-G and HLA-E, as well as their expression patterns, functions in immune modulation and during pregnancy, and also possible implications in a range of diseases, in infertility and pregnancy complications, HLA-F has nearly been ignored. However, recent discoveries show that HLA-F can be expressed as both open conformers binding to a number of KIRs on primarily NK cells, as well as peptide-bound HLA-F binding to ILT2 and ILT4. Furthermore, a number of reports indicate a possible involvement of HLA-F in viral infections, in cancer immunology, and in fertility and reproduction, which may initiate more interest in this rather unknown HLA class I molecule. In this short review, we focus on recent discoveries that indicate a functional role for HLA-F in reproduction and during pregnancy, and the role of HLA-F in relation to HLA-G.

Soluble HLA-G and TGF-ß in couples attending assisted reproduction - A possible role of TGF-ß isoforms in semen?

Soluble isoforms of the non-classical Human Leukocyte Antigen (HLA)-G as well as Transforming Growth Factor (TGF)-ß is expressed in seminal plasma possibly influencing the pregnancy potential. We wanted to examine the association of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGFß3 with pregnancy success in a cohort of 127 couples and 4 single women attending fertility treatment with the use of assisted reproduction technologies (ART). Soluble HLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in seminal plasma did not fluctuate significantly over time. We did not find any impact of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 on time-to-pregnancy measured as number of treatment cycles. There was a significant association between concentrations of seminal plasma sHLA-G and HLA-G variations in the 3'untranslated region (3'UTR) of the HLA-G gene, supporting and extending previous findings. Furthermore, by comparing seminal plasma concentrations of sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in male subjects with reduced semen quality, male subjects with normal semen quality, and sperm donors, we found that TGF-ß2 was significantly lower, and TGF-ß3 was significantly higher, in seminal plasma from sperm donors. These findings suggest that TGF-ß isoforms may influence semen quality and fertility.

The Tolerogenic Function of Regulatory T Cells in Pregnancy and Cancer.

Regulatory T cells, a subpopulation of suppressive T cells, are potent mediators of self-tolerance and essential for the suppression of triggered immune responses. The immune modulating capacity of these cells play a major role in both transplantation, autoimmune disease, allergy, cancer and pregnancy. During pregnancy, low numbers of regulatory T cells are associated with pregnancy failure and pregnancy complications such as pre-eclampsia. On the other hand, in cancer, low numbers of immunosuppressive T cells are correlated with better prognosis. Hence, maternal immune tolerance toward the fetus during pregnancy and the escape from host immunosurveillance by cancer seem to be based on similar immunological mechanisms being highly dependent on the balance between immune activation and suppression. As regulatory T cells hold a crucial role in several biological processes, they may also be promising subjects for therapeutic use. Especially in the field of cancer, cell therapy and checkpoint inhibitors have demonstrated that immune-based therapies have a very promising potential in treatment of human malignancies. However, these therapies are often accompanied by adverse autoimmune side effects. Therefore, expanding the knowledge to recognize the complexities of immune regulation pathways shared across different immunological scenarios is extremely important in order to improve and develop new strategies for immune-based therapy. The intent of this review is to highlight the functional characteristics of regulatory T cells in the context of mechanisms of immune regulation in pregnancy and cancer, and how manipulation of these mechanisms potentially may improve therapeutic options.

Expression of Circadian Clock Genes in Human Colorectal Cancer Tissues Using Droplet Digital PCR.

Increasing evidence indicates that disruption of circadian rhythms may be directly linked to cancer. Here we report that the expression levels of the core clock genes Per1 and Per3 measured by droplet digital polymerase chain reaction are significantly decreased in tumour tissue from 16 patients undergoing colorectal cancer surgery compared to paired normal mucosa. No differences were observed in the expression of Per2, Bmal1, and Clock. In conclusion, abnormal expression levels of the clock genes Per1 and Per3 in CRC tissue may be related to tumourigenesis and may provide future diagnostic and prognostic information.

Implications of uterine NK cells and regulatory T cells in the endometrium of infertile women.

A range of studies have shown that the complex process of implantation and an establishment of a pregnancy also involves immune factors. Disturbances in these underlying immune mechanisms might lead to implantation and pregnancy failure and may be involved in the pathogenesis of unexplained infertility. Several studies have reported that imbalances in uterine NK (uNK) cell abundance are associated with infertility; however, controversies exist. An increased amount of CD56+ uNK cells along with a decrease in CD16+ uNK cells have been associated with normal fertility in some studies. Very few studies of FoxP3+ regulatory T cells (Tregs) in the pre-implantation endometrium have been performed. Results are sparse and controversial, studies reporting both increased and decreased numbers of Tregs, respectively, in women suffering from infertility. In conclusion, studies imply that uNK cells, Tregs and HLA-G carry pivotal roles regarding the establishment of a healthy pregnancy, and that abnormal immune mechanisms involving these parameters may be associated with infertility. However, more research in early phases of the reproductive cycle, such as investigating the conditions in the endometrium before implantation, is needed to further clarify the underlying mechanisms.

The choriocarcinoma cell line JEG-3 upregulates regulatory T cell phenotypes and modulates pro-inflammatory cytokines through HLA-G.

An understanding of the interactions between immune cells and trophoblast cells, as well as choriocarcinoma cells, are of extreme importance in reproductive immunology and cancer immunology. In this study, we found that the human HLA-G-positive choriocarcinoma cell line JEG-3 upregulates CD4+CD25hiCD127lo T cells, increases the expression of HLA-G+CD4+ and CD8+ T cells, and decreases the expression of ILT2+ on CD4+ T cells in resting PBMCs after six days of co-culture. Expression of HLA-G on JEG-3 cells did not affect regulatory T cell phenotypes, but promoted modulation of pro-inflammatory cytokines IFN-γ, TNF-α and IL-17A. When JEG-3 cells were stimulated with rhIFN-γ prior to co-culture, CD4+HLA-G+ T cells were significantly increased, and IFN-γ and TNF-α elevated. Taken together, the results indicate that JEG-3 cells upregulate regulatory T cell phenotypes and modulate the level of pro-inflammatory cytokines, which might be important mechanisms in the tumor microenvironment and at the feto-maternal interface during pregnancy.

HLA class Ia and Ib molecules and FOXP3+ TILs in relation to the prognosis of malignant melanoma patients.

HLA class Ia (HLA-ABC) and HLA class Ib (HLA-E, -F and -G) molecules and FOXP3+ tumor-infiltrating lymphocytes (TILs) are often reported as relevant factors of tumor immune regulation. We investigated their expression as prognostic factors in 200 patients with primary cutaneous melanoma (PCM). In our cohort, patients with tumors showing upregulation of HLA-ABC molecules had significantly thicker tumors (32% vs 7%, P<0.001), frequent ulceration (20% vs 6%, P=0.007) and frequent nodular melanomas (20% vs 4%, P=0.001). Additionally, high expression of HLA-G in the tumor was a sign of bad prognosis for the patients, being associated with thick tumors (30% vs 12%, P=0.017), ulceration (24% vs 5%, P<0.001) and positive sentinel node (13% vs 6%, P=0.015). HLA-E, HLA-F and FOXP3+ TILs were not indicative of the prognosis in PCM. High HLA-ABC and HLA-G were associated with tumor aggressiveness and could be relevant predictive markers for effective immunotherapy of melanoma tumors.

The Pathophysiological Impact of HLA Class Ia and HLA-G Expression and Regulatory T Cells in Malignant Melanoma: A Review.

Malignant melanoma, a very common type of cancer, is a rapidly growing cancer of the skin with an increase in incidence among the Caucasian population. The disease is seen through all age groups and is very common in the younger age groups. Several studies have examined the risk factors and pathophysiological mechanisms of malignant melanoma, which have enlightened our understanding of the development of the disease, but we have still to fully understand the complex immunological interactions. The examination of the interaction between the human leucocyte antigen (HLA) system and prognostic outcome has shown interesting results, and a correlation between the down- or upregulation of these antigens and prognosis has been seen through many different types of cancer. In malignant melanoma, HLA class Ia has been seen to influence the effects of pharmaceutical drug treatment as well as the overall prognosis, and the HLA class Ib and regulatory T cells have been correlated with tumor progression. Although there is still no standardized immunological treatment worldwide, the interaction between the human leucocyte antigen (HLA) system and tumor progression seems to be a promising focus in the way of optimizing the treatment of malignant melanoma.

Urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion increases in normal pregnancy but not in preeclampsia.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) serum values have been shown to increase in preeclampsia. The goal of the present study was to evaluate changes in urinary NGAL concentrations during uncomplicated pregnancy and in cases of preeclampsia and hypertension. METHODS: Fifty-one pregnant women who developed preeclampsia and 28 diagnosed with essential or gestational hypertension were investigated for urinary NGAL concentrations during pregnancy. As controls, 100 healthy pregnant women with uncomplicated singleton pregnancies were randomly selected. Urinary NGAL as well as urinary creatinine and albumin were measured by a standardized clinical chemistry platform (ARCHITECT®; Abbott Diagnostics, Abbott Park, IL, USA). RESULTS: Urinary NGAL concentrations increased during pregnancy in healthy pregnant women, whereas this increase was not detected in preeclampsia. In order to correct for diuresis, spot urine concentrations were also determined as NGAL/creatinine ratio. NGAL/creatinine ratio in pregnancy week 36-38 was significantly lower in preeclampsia than in healthy pregnant women or pregnant women with hypertension. NGAL urinary concentrations did not correlate with albumin concentration in urine. CONCLUSIONS: Urinary NGAL is not a valuable early biomarker for preeclampsia.

The expression and functional activity of membrane-bound human leukocyte antigen-G1 are influenced by the 3'-untranslated region.

Human Leukocyte Antigen (HLA)-G is an immunosuppressive molecule acting on both the innate and adaptive immune system. A 14 bp insertion/deletion polymorphism (rs66554220) in the 3'-untranslated region (3'UTR) of the HLA-G gene has been associated with a number of diseases, pregnancy complications, and graft rejection after organ transplantation. We have investigated the effect of HLA-G polymorphism in the 3'UTR on the processing and stability of the membrane-bound HLA-G1 (mHLA-G1) isoform, as well as its functional significance. Different HLA-G1 cDNA sequences were transduced into the human K562 cell line. Flow cytometry, immunohistochemistry, and ELISA were used to examine HLA-G1 protein expression. A quantitative RT-PCR assay was used to quantify transduced HLA-G1 DNA and mRNA transcript levels. Stability of mRNA and functional significance of HLA-G were investigated via Actinomycin D and NK cytotoxicity assays, respectively. Human leukocyte antigen-G mRNA from the 14 bp insertion K562-G1 cells showed a higher degree of stability than the other constructs, and increased mHLA-G1 expression relative to transductants lacking the 14 bp sequence. In line with this, transductants carrying the 14 bp insertion were the most efficient in inhibiting NK cytotoxicity but showed a lower soluble HLA-G1 per mHLA-G1 ratio than the HLA-G1 K562 cells lacking the 14 bp insertion. Our data suggest 3'UTR polymorphism may play an important role in HLA-G regulation with implications on a range of diseases.

Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

Porcine skin collagen implants for anterior vaginal wall prolapse: a randomised prospective controlled study.

INTRODUCTION AND HYPOTHESIS: The effect of a Pelvicol graft compared with a conventional anterior vaginal repair was evaluated in this randomised controlled study. METHODS: Only patients with a stage II or higher (Ba >or= -1) defect were included. RESULTS: Thirty-one patients were allocated to a conventional anterior repair; 30 to Pelvicol graft. At 12 months follow-up, four patients among controls (15%) and two in the graft group (7%) had objective recurrence. Among controls, the difference at 3 months follow-up in Ba was 6.0 cm when compared with the position of Ba prior to surgery. In the graft group, the difference was 7.0 cm (P < 0.05). This difference was still present at 12 months follow-up (6.0 vs. 7.0 cm; P < 0.05). CONCLUSIONS: The implantation of a Pelvicol graft does not improve the POP-Q stage.

CARD15 single nucleotide polymorphisms 8, 12 and 13 are not increased in ethnic Danes with sarcoidosis.

BACKGROUND: Mutations of the caspase-activating recruitment domain 15 (CARD15) gene on chromosome 16 are associated with chronic inflammatory granulomatous bowel disease (Crohn's disease). Sarcoidosis is a systemic granulomatous disease with unknown etiology, which shares histological features with Crohn's disease. OBJECTIVES: To evaluate whether ethnic Danes with sarcoidosis have an increased frequency of CARD15 mutations compared to healthy control subjects. METHODS: Genotyping for CARD15 mutations R702W, G908R, and L1007fsinsC, also designated single nucleotide polymorphism (SNP) SNP8, SNP12 and SNP13, respectively, were performed by capillary electrophoresis single-strand confirmation polymorphism in 53 patients with histologically verified sarcoidosis and in 103 healthy controls. RESULTS: The frequencies of CARD15 mutations in sarcoidosis patients were: SNP8, 4/106 chromosomes (3.8%); SNP12, 2/106 chromosomes (1.9%); SNP13, 2/106 chromosomes (1.9%); SNP8+SNP12+SNP13, 8/106 chromosomes (7.6%). All 8 patients were heterozygous. The frequencies in controls were: SNP8, 9/206 chromosomes (4.4%); SNP12, 2/206 chromosomes (1.0%); SNP13, 4/206 chromosomes (1.9%); SNP8+SNP12+SNP13, 15/206 chromosomes (7.3%). All controls were heterozygous. The differences were not statistically significant (p>0.05). Furthermore, the course of disease was not significantly different in the 8 patients with CARD15 mutations and the 45 patients without mutations. CONCLUSION: The frequency of CARD15 mutations is not increased in ethnic Danish patients with sarcoidosis, and heterozygosity for such mutations apparently has no influence on the course of disease.