Distribution of HLA-G extended haplotypes and one HLA-E polymorphism in a large-scale study of mother-child dyads with and without severe preeclampsia and eclampsia.

The etiological pathways and pathogenesis of preeclampsia have rendered difficult to disentangle. Accumulating evidence points toward a maladapted maternal immune system, which may involve aberrant placental expression of immunomodulatory human leukocyte antigen (HLA) class Ib molecules during pregnancy. Several studies have shown aberrant or reduced expression of HLA-G in the placenta and in maternal blood in cases of preeclampsia compared with controls. Unlike classical HLA class Ia loci, the nonclassical HLA-G has limited polymorphic variants. Most nucleotide variations are clustered in the 5'-upstream regulatory region (5'URR) and 3'-untranslated regulatory region (3'UTR) of HLA-G and reflect a stringent expressional control. Based on genotyping and full gene sequencing of HLA-G in a large number of cases and controls (n > 900), the present study, which to our knowledge is the largest and most comprehensive performed, investigated the association between the HLA-G 14-bp ins/del (rs66554220) and HLA-E polymorphisms in mother and newborn dyads from pregnancies complicated by severe preeclampsia/eclampsia and from uncomplicated pregnancies. Furthermore, results from extended HLA-G haplotyping in the newborns are presented in order to assess whether a combined contribution of nucleotide variations spanning the 5'URR, coding region, and 3'UTR of HLA-G describes the genetic association with severe preeclampsia more closely. In contrast to earlier findings, the HLA-G 14-bp ins/del polymorphism was not associated with severe preeclampsia. Furthermore, the polymorphism (rs1264457) defining the two nonsynonymous HLA-E alleles, HLA-E*01:01:xx:xx and HLA-E*01:03:xx:xx, were not associated with severe preeclampsia. Finally, no specific HLA-G haplotypes were significantly associated with increased risk of developing severe preeclampsia/eclampsia.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications.

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with ß2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use.

A fast and easy real-time PCR genotyping method for the HLA-G 14-bp insertion/deletion polymorphism in the 3' untranslated region.

Human leukocyte antigen-G (HLA-G) is a non-classical HLA class Ib molecule shown to exhibit immunomodulatory function in a wide range of immune-based disorders. A number of functional HLA-G gene polymorphisms have been identified, including a 14-bp insertion/deletion polymorphism in exon 8 of the 3' untranslated region of the HLA-G gene, which has been associated with HLA-G mRNA stability. Moreover, studies show that homozygosity for the 14-bp insertion/deletion polymorphism is associated with lower HLA-G mRNA and protein levels and unique alternative splicing patterns. Here, we introduce a quick and reliable method to screen for the HLA-G 14-bp insertion/deletion polymorphism using an optimized real-time polymerase chain reaction protocol. The genotyping assay has been validated by comparison with conventional methods. As results can be obtained within a few hours, the assay will have a potential for clinical use.

[The characteristics of mediastinal neuroblastoma and perspectives on surgical excision]. / Osobine neuroblastoma medijastinuma i perspektive hirurskog lecenja.

Thoracic (mediastinal) neuroblastomas (NB) have been reported to differ from abdominal (suprarenal and retroperitoneal) NB and to be associated with better prognosis. The comparison between them is rarely published. In this retrospective study, the characteristics of thoracic NB (17 cases) are investigated and compared with abdominal NB (51 cases). Regarding the diagnosis, thoracic NB presented in lower clinical stages I and II in 35.3% of cases, compared to 11.7% of abdominal NB in stages I and II (p<0.001). The disease was initially diagnosed at less than one year of age in 7/17 (41.2%) of thoracic NB and in 12/51 (23.5%) in abdominal cases (p<0.001). The median age at the time of initial diagnosis was 15.3 months for thoracic NB and 27.6 months for abdominal neuroblastoma (p<0.05). The cases with an elevated vanillylmandelic acid (VMA) and homovanyillic acid (HVA) excretion were 9/17 (52.9%) in the mediastinal NB, and 43/51 (84.3%) in the abdominal NB, respectively (p<0.05). The quantitative values of tumour markers were significantly lower in thoracic NB (0.85 vs. 2.14, p<0.001). Regarding surgery, complete tumour resection was achieved in 15/17 thoracic NB (88.2%) compared to 36/51 (70.6%) radicality in abdominal NB. Surgical complications developed in 5/17 thoracic procedures (29.4%) without a lethal outcome. The mean tumour mass of thoracic NB was 56.5 g vs. 106.3 g of abdominal neuroblastoma (p<0.001). The incidence of ganglioneuroblastoma in mediastinal tumours was 3/17 (17.6%) compared to 8/51 (15.7%) in abdominal NB (non significant). A favorable histology based on Shimada classification was found in 37% of the mediastinal neuroblastoma cases and in 22% in the abdominal NB cases (p<0.05). Regarding the biological properties, genetic malformations associated with NB were identified in 2 thoracic cases (1p deletion and polyploidy). Genetic changes were identified in 12 cases of abdominal NB (1p deletion in 4 cases, DNA ploidy in 6 cases, N-myc amplification in 1 case). One additional abdominal NB had 1p deletion, DNA ploidy and N-myc amplification. This study supports results of other investigations that thoracic NB differs significantly in many aspects from abdominal NB. Important differences in favorable histology and biological properties of thoracic NB have changed the concept of surgical treatment, although unnecessary attempts of surgical radicality still lead to serious complications. Complete excision remains the mainstay of therapy of localised thoracic NB, while in most abdominal tumours the aim of an initial operation should be sampling of tumour tissue for histology and molecular biological examination, with complete excision of the mass as the second priority.

[Factors affecting the efficiency (sensitivity, specificity) of diagnostic procedures for the detection of Chlamydia trachomatis infections of the male and female genital tract]. / O faktorima od uticaja na efikasnost (osetljivost, specificnost) dijagnostickih postupaka za utvrdjivanje infekcije genitalnog trakta zena i muskaraca Chlamydijom trachomatis.

This paper presents and analyzes some factors which may exert influence on the outcome of the diagnostic procedure in establishing male and female genital tract infections with Chlamydia Trachomatis. On the basis of this study it has been suggested that diagnostic procedures used nowadays should be used selectively, either individually or as a group within defined optimal conditions.

Haemagglutination-inhibiting (HI) antibodies against four prototype strains of influenza A virus in different age groups.

Sera of 197 apparently well persons were tested for residual haemagglutination-inhibiting antibodies against live Hong Kong/68, A/FM/47 and A/PR/34 strains. Sera of 62 well persons, regularly exposed to contacts with swine, were tested against an inactivated A/New Jersey/76 antigen. Those born some time before and during a certain influenza era showed a significantly greater proportion of homologous residual titres against the subtype prevailing in that influenza era, than those born after the termination of the same era. In each of the seven age groups tested both the percentage of positives and the geometric mean titres were usually highest against the Hong Kong strain (representing the most recent era); the next highest were those against the FM1 strain and the lowest were those against the PR8 strain (representing the most distant of these three influenza eras). The serological involvement of donors exposed to regular contacts with swine was relatively stronger against the New Jersey antigen than the response of other serum donors shown against the other three, more recent, prototypes of influenza virus A. The oldest age groups showed significantly lower antibody response against the PR8, FM1 and Hong Kong strains (but not against the New Jersey antigen) than the next one or two of the younger age groups.

Detection of "alpha-type" inhibitors in the presence of low levels of specific influenza antibodies.

In native, heated or otherwise treated egg white and in sera of men and guinea pigs, haemagglutination inhibition titres were determined against three inhibitor-sensitive (IS) strains and one inhibitor-resistant (IR) variant on influenza A virus. A few human sera with no detectable antibody revealed high inhibition titres even against the IR variant. Human sera after treatment with trypsin and periodate revealed mostly a reduction or no change, and exceptionally an increase of their inhibition titre. The extent of these changes varied with different influenza virus strains and showed a positive correlation with the inhibitor content and no correlation with the antibody levels of the sera. The so-called "nonspecific" thermostable inhibitors possess a certain degree of specificity for different influenza virus strains.