Jacek R. Wilczyński

Does the KIR2DS5 Gene Protect from Some Human Diseases

Background KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation. Methodology/Principal Findings In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (pu200a=u200a0.003, odds ratio [OR]u200a=u200a0.47, confidence interval [CI]u200a=u200a0.28–0.79), endometriosis (pu200a=u200a0.03, ORu200a=u200a0.25, CIu200a=u200a0.07–0.82) and acute rejection of kidney graft (pu200a=u200a0.0056, ORu200a=u200a0.44, CIu200a=u200a0.24–0.80), but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05). In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (pu200a=u200a0.0003, ORu200a=u200a0.35, CIu200a=u200a0.19–0.65), whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (pu200a=u200a0.0017, ORu200a=u200a1.92, CIu200a=u200a1.28–2.89), whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (pu200a=u200a0.005, ORu200a=u200a1.47, CIu200a=u200a1.13–1.92). The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (pu200a=u200a0.017, ORu200a=u200a0.47, CIu200a=u200a0.25–0.89), whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (pu200a=u200a0.0015, ORu200a=u200a2.13, CIu200a=u200a1.34–3.37). Conclusions/Significance Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.

Immunotherapy of Patients with Recurrent Spontaneous Miscarriage and Idiopathic Infertility: Does the Immunization-Dependent Th2 Cytokine Overbalance Really Matter?

Recurrent spontaneous miscarriage (RSM) and idiopathic infertility (IIF) are partially caused by immunologic disturbances. Paternal lymphocyte immunization (PLI) is proposed for restoration of the proper Th1/Th2 balance in these patients, but still there are controversies on PLI mechanism, its efficacy and identification of patients who may benefit from this therapy. The study group consisted of nxa0=xa034 RSM and nxa0=xa042 IIF women with unexplained miscarriage or IIF. PLI was offered as a treatment in both groups. Peripheral blood lymphocyte (PBL) populations (CD3+, CD3−/CD19+, CD3+/CD4+, CD3+/CD8+, CD3−/CD16+CD56+) were studied before immunization, while PBL cytokine secretion (IFN-γ, TNF-α, IL-10, IL-5, IL-4, IL-2), before and after immunization, pre-conceptionally in both groups. The reference PBL ratio and cytokine levels were adopted from previously studied normal fertile women. PBL populations, concentration and ratio of Th1/Th2 cytokines did not differ between RSM and IIF patients. Compared to the results observed in normal fertile women the levels of IFN-γ, TNF-α and IL-2 were higher, while IL-10 lower in both RSM and IIF patients (pxa0<xa00.01). After immunization a decrease of IFN-γ (RSM and IIF groups) and IL-4 and IL-10 (RSM group) were observed, as well as an increase in TNF-α/IL-4 ratio (RSM group) (pxa0<xa00.05). No differences in Th1/Th2 concentration and ratio between patients with successful and unsuccessful pregnancy were observed. No significant correlations between success and particular cytokine concentration were observed. Concentrations of Th1/Th2 cytokines and PBL populations did not differ between RSM and IIF women. Th1 shift in both RSM and IIF patients was observed in comparison to fertile women. Treatment with PLI-induced pre-conceptionally cytokine changes which neither indicated Th2 shift nor correlated with subsequent pregnancy success.

How do Tumors Actively Escape from Host Immunosurveillance

The immunological background for the process of tumor growth is still obscure. However, our understanding of what happens could have important consequences, namely in the context of cancer immunotherapy. A tumor is able to grow in the host environment either because it is recognizable as normal tissue and tolerated by host immune cells, or because it can “escape” from host immunosurveillance. According to the second option the mechanisms of tumor recognition and consequent destruction are actively disturbed by such processes as: change of tumor immunogenicity, production of tumor-derived regulatory molecules, and interaction of cancer cells with tumor-infiltrating immune cells. The results of studies devoted to the problem of immunoregulation in the tumor environment seem to support the “escape” hypothesis.

The Effect of Cadmium on Steroid Hormones and Their Receptors in Women with Uterine Myomas

Cadmium (Cd) is one of the environmental metalloestrogens, and its role in uterine tissues has not yet been fully elucidated. The aim of the study was to investigate estrogenic properties of Cd in uterine tissues by analyzing the expression of estrogen receptor (ER) and progesterone receptor (PR) as well as estrogen and progesterone levels in serum and Cd concentrations in blood and tissues. The samples of tissues (leiomyoma and surrounding myometrium) collected intrasurgically and blood samples drawn from 53 women (age 39 to 52xa0years) with uterine myomas were thoroughly analyzed. In the study group, blood Cd concentration ranged from 0.33 to 3.5xa0μg/L. Cd concentration in leiomyoma tissues was twice as high as that in surrounding myometrium (0.48 and 0.75xa0μgxa0Cd/g wet tissue, respectively), albeit the difference was not statistically significant. Cd concentrations in blood significantly correlated with Cd concentrations in tissues (leiomyoma and surrounding myometrium). The measurement of ER expression showed no difference between leiomyoma tissues and surrounding myometrium. The statistical analysis showed a significant correlation between ER expression and Cd concentration in both tissues under study. An additional statistical analysis (path analysis) demonstrated the correlation of uterine tissue levels of Cd and ER expression. However, there was no association between ER expression in both tissues and E2 level in serum. Our results suggest a metalloestrogenic effect of Cd by way of ER stimulation in the uterus.

HLA-C C1C2 heterozygosity may protect women bearing the killer immunoglobulin-like receptor AA genotype from spontaneous abortion.

Spontaneous abortion is the most common complication of human pregnancy. Natural killer (NK) cells expressing killer immunoglobulin-like receptors (KIRs), which may recognize HLA-C (i.e. its C1 or C2 groups) on trophoblast cells, constitute a large leukocyte population in the endometrium. This study investigated whether genetic polymorphisms in the KIR and HLA-C genes are risk factors for spontaneous abortion. One hundred and twenty-five couples with at least two spontaneous abortions, including eighty-five couples with idiopathic recurrent abortion (RSA; three or more abortions), and 117 control couples (with two or more healthy-born children) were tested. The frequencies of the individual KIR genes in the patients were similar to those in the controls. In the group of KIR AA women with HLA-C C2C2 partners, the HLA-C C1C2 heterozygotes were present in the controls but not in the patients (p=0.015 for all patients and p=0.0048 for RSA, but both comparisons lost significance after Bonferroni correction), whereas both homozygotes, C1C1 and C2C2, were absent in the control women but present among the aborting ones. Therefore, our results suggest that among KIR AA women who have HLA-C C2C2 partners, HLA-C heterozygous females show a trend towards an increased chance of successful pregnancy.

Possible Role of HLA-G, LILRB1 and KIR2DL4 Gene Polymorphisms in Spontaneous Miscarriage

The KIR2DL4 receptor and its ligand HLA-G are considered important for fetal-maternal immune tolerance and successful pregnancy. The absence of a particular variant of KIR2DL4 might be a bad prognostic factor for pregnancy outcome. However, it could be compensated by the presence of the respective LILRB1 allele. Therefore, we investigated the KIR2DL4, LILRB1 and HLA-G polymorphisms in 277 couples with spontaneous abortion and 219 control couples by HRM, PCR-SSP and RFLP methods. We found a protective effect of women’s heterozygosity in −716 HLA-G (pxa0=xa00.0206) and LILRB1 (pxa0=xa00.0131) against spontaneous abortion. Surprisingly, we observed more 9A/10A genotypes of KIR2DL4 gene carriers in the group of male partners from the miscarriage group in comparison to the men from the control group (pxa0=xa00.0288). Furthermore, there was no association of women’s KIR2DL4 polymorphism with susceptibility to spontaneous abortion. Multivariate analysis indicated that women’s −716 HLA-G and LILRB1 and men’s KIR2DL4 9A/10A are important in terms of the protection or susceptibility to miscarriage, respectively (pxa0=xa00.00968). In conclusion, a woman’s heterozygosity in HLA-G and LILRB1 might be an advantage for a success of reproduction, but the partner’s heterozygosity in 9A/10A KIR2DL4 alleles might not.

Genetic polymorphism of KIR2DL4 in the Polish population

The KIR2DL4 gene is characterized by alleles with either 9 or 10 consecutive adenines in exon 7, which encodes the transmembrane domain. The 9A variant produces either a protein with a truncated cytoplasmic tail or one lacking the transmembrane region. This causes a lack of KIR2DL4 expression. In contrast, 10A alleles encode receptors that may be expressed at the cell surface. We tested 438 healthy individuals for polymorphism of the KIR2DL4 gene. KIR2DL4 9A/10A alleles were distinguished by the high resolution melting (HRM) method, and restriction fragment length polymorphism (RFLP) was used for genotyping of three other single nucleotide polymorphisms (SNPs) spanning the near vicinity of the poly-adenine fragment. We found a weak difference between males and females in 9769 C/A genotypes and alleles. In addition, we observed complete linkage disequilibrium (LD) between 9A insertion/deletion in the 9620 position and the 9571T/C position of the gene (r(2) u2009=u20091) both in females and males and almost complete LD with the 9797G/A position (r(2) u2009=u20090.963 for females and r(2) u2009=u20090.892 for males). Most importantly, we detected, in a group of fertile women, a high frequency (30.2%) of homozygosity for the defective 9A variant, which suggests that KIR2DL4 as a functional cell surface receptor is not absolutely necessary for reproduction. On the other hand, lower representation of 10A/10A homozygotes and high frequency of 10A/9A heterozygotes indicates a need for both cell membrane-anchored and soluble KIR2DL4 molecules. Finally, cost-reducing RFLP instead of HRM is proposed for typing 9A and 10A variants.

Leiomyomatosis peritonealis disseminata of unusual course with malignant transformation: case report

Leiomyomatosis peritonealis disseminata is a very rare, benign entity of unknown pathogenesis, characterized by the presence of multiple subperitoneal or peritoneal smooth muscle nodules throughout the peritoneal surface. Mostly the course is asymptomatic and it is found incidentally during laparotomy, laparoscopy or cesarean section. Non‐specific symptoms such as abdominal pain, vaginal bleeding, abdominal mass or gastrointestinal signs are described. Rare cases of malignant transformation have been reported. We present a case of disseminated peritoneal leiomyomatosis with an unusual course and transformation to endometrial sarcoma in a 26‐year‐old previously healthy woman, where the appearance of peritoneal nodules was preceded by multiple incidents of fast fibroid growth and delivery of myomatous growth into the cervical canal.

TP53 and MDM2 polymorphisms and the risk of endometrial cancer in postmenopausal women.

The aim of the study was to determine an association of TP53 codon 72 (Arg72Pro, G>C transversion, rs1042522) and MDM2 SNP309 (T>G change, rs2279744) polymorphisms in endometrial cancer (EC) of postmenopausal women, regarding grading and staging of EC. In the study, endometrial samples from 202 postmenopausal female patients (the study group, nxa0=xa0152, was women with EC; the control group, nxa0=xa050, cancer-free patients) were taken for the evaluation of two gene polymorphisms: TP53 codon 72 and MDM2 SNP309, respectively. Genotypic analyses were performed using the PCR–RFLP technique. There were significant differences in the frequency of TP53 and MDM2 genotypes in EC patients—increased EC occurrence was observed with the presence of MDM2 G/G and TP53 Arg/Arg genotypes, while allele Pro of TP53 decreased cancer risk. Analysis of combined MDM2/TP53 polymorphisms revealed that T/T-Pro/Arg genotype decreased EC risk, whereas G/G-Arg/Arg genotype increased it. Association of these genetic polymorphisms with histological grading showed increased MDM2 G/G homozygote and TP53 Arg/Arg homozygote frequencies in grading 2 as well as allele G overrepresentation in G1 and G3 EC patients. Finally, with clinical FIGO staging under evaluation, an increase in MDM2 G/G and TP53 Arg/Arg homozygote frequencies in staging I and TP53 Arg/Arg homozygote frequencies in staging II were observed. Co-occurrence of some MDM2 SNP309 and TP53 codon 72 polymorphisms seems to influence EC risk, involving grading and staging of this neoplasm at the same time.

Heterogeneity of the Mac-1 expression on peripheral blood neutrophils in patients with different types of epithelial ovarian cancer.

The expression level of Mac-1 on the surface of neutrophils is an important indicator of neutrophil activation. Under pathological conditions, Mac-1 is believed a key adhesion molecule that facilitates cancer progression and mediates the adhesion of tumour cells to the endothelium of blood vessels. Our previous findings indicated that circulating peripheral blood neutrophils in patients with advanced epithelial ovarian cancer (EOC) expressed enhanced levels of Mac-1, which was functionally associated with an increased adhesive function of neutrophils. The objective of the current study was to analyse whether the value of individual components of the differential white cell count, including the neutrophil and lymphocyte ratios, which are markers of blood neutrophil activation, might be associated with certain types of ovarian cancer. We showed the increase in Mac-1 expression along with a parallel decrease of L-selectin and PSGL-1 on peripheral blood neutrophils of patients with EOC of early and advanced FIGO stages, which indicates an activated state of neutrophils in comparison to neutrophils of individuals without cancer. Despite a significant difference between Mac-1 expression in patients with and without cancer, a dramatic increase in Mac-1 expression was observed in the blood of patients with undifferentiated carcinomas compared with patients with other histological types of EOC. Moreover, the expression level of Mac-1 correlated with the number of neutrophils in patients with serous, endometrioid and undifferentiated EOC. The results of an ROC analysis demonstrated that the patients with the undifferentiated type of EOC form a distinct group with regard to Mac-1 expression on blood neutrophils. The results suggested a diverse biological cadre of immune cells in patients with undifferentiated ovarian carcinomas compared with patients with other histological types of EOC.