KIR and HLA haplotype analysis in a family lacking the KIR 2DL1-2DP1 genes.

The killer cell immunoglobulin-like receptor (KIR) gene cluster exhibits extensive allelic and haplotypic diversity that is observed as presence/absence of genes, resulting in expansion and contraction of KIR haplotypes and by allelic variation of individual KIR genes. We report a case of KIR pseudogene 2DP1 and 2DL1 gene absence in members of one family with the children suffering from acute myelogenous leukemia (AML). Killer cell immunoglobulin-like receptor low resolution genotyping was performed by the polymerase chain reaction (PCR)-sequence-specific primers (SSP)/sequence-specific oligonucleotide (SSO) method and haplotype assignment was done by gene content analysis. Both parents and the maternal grandfather, shared the same Cen-B2 KIR haplotype, containing KIR 3DL3, -2DS2, -2DL2 and -3DP1 genes. The second haplotype in the KIR genotype of the mother and grandfather was Tel-A1 with KIR 2DL4 (normal and deleted variant), -3DL1, -22 bp deletion variant of the 2DS4 gene and -3DL2, while the second haplotype in the KIR genotype of the father was Tel-B1 with 2DL4 (normal variant), -3DS1, -2DL5, -2DS5, -2DS1 and 3DL2 genes. Haplotype analysis in all three offsprings revealed that the children inherited the Cen-B2 haplotype with the same gene content but two of the children inherited a deleted variant of the 2DL4 gene, while the third child inherited a normal one. The second haplotype of all three offspring contained KIR 2DL4, -2DL5, -2DS1, -2DS4 (del 22bp variant), -2DS5, -3DL1 and -3DL2 genes, which was the basis of the assumption that there is a hybrid haplotype and that the present 3DL1 gene is a variant of the 3DS1 gene. Due to consanguinity among the ancestors, the results of KIR segregation analysis showed the existence of a very rare KIR genotype in the offspring. The family who is the subject of this case is even more interesting because the father was 10/10 human leukocyte antigen (HLA)-matched to his daughter, all members of the family have the "best" donor KIR-B content and the presence of a rare KIR genotype with KIR 2DP1-2DL1 genes absence.

Distribution of HLA-DRB1 and HLA-DQB1 families of alleles and haplotypes in Vojvodina population.

Major histocompatibility complex encoding human leucocyte antigens (HLA) is a highly polymorphic gene cluster that makes it a valuable tool in the population genetic studies. The aim of our study was to compare HLA class II gene frequencies with other populations from Europe and to determine the relationship between the investigated populations. In this study, one hundred and twenty healthy individuals from Vojvodina, northern Serbia, were studied for 18 of the HLA-DRB1 and HLA-DQB1 loci. The HLA families of alleles were analysed by using sequence-specific primers for polymerase chain reaction (PCR-SSP). The results showed the increased frequency of HLA-DRB1*11(0.333), -DRB1*04(0.300), -DRB1*07(0.250), -DQB1*03(0.730) and -DQB1* 05(0.391), among the tested families of alleles. The two-locus haplotype analysis revealed significant positive linkage disequilibrium for DRB1*11DQB1*03 (Δ = 0.0788, χ(2) = 12.61) and DRB1*04DQB1*03 (Δ = 0.0583, χ(2) = 8.04). A phylogenetic tree constructed on the basis of the DRB1* gene frequencies derived from other populations revealed the clustering among the Vojvodina population together with other populations in Europe (Croats, Austrians and Hungarians). Close relationship of the Vojvodina population with the populations of Hungarians and Austrians can be the result of their historical influence on the region of Vojvodina.

Human leukocyte antigen-b27 and disease susceptibility in vojvodina, serbia.

There are numerous studies showing the role of human leukocyte antigens (HLAs) related with susceptibility or resistance to certain diseases. The aim of this study was to determine the association of HLA-B27 with ankylosing spondylitis (AS), polyarthralgia, lumboishialgia, acute anterior uveitis (AAU), psoriatic arthritis (PA), synovitis coxae and rheumatoid arthritis (RA) in patients from Vojvodina, Serbia. An HLA I class typing was performed by the serological immunomagnetic two-color fluorescence method using peripheral blood T lymphocytes in 97 patients and 224 healthy controls from the population of Vojvodina, Serbia. We calculated HLA-B27 frequencies, relative risk (RR), ethiologic fraction (EF), e.g., population attributive risk, when RR was greater than 1, while, preventive fraction (PF) was calculated when RR was lower than 1. This study revealed the strongest association of AS with HLAB27 antigen: RR = 25.0, while the EF was greater than 0.15, respectively. The χ(2) test showed the significant difference (p <0.05) in HLA-B27 in patients with AS in comparison to controls (χ(2) = 52.5). It was concluded that there is a positive association of HLA-B27 with AS and that HLA-B27 can serve as a marker for predisposition to diseases.

Study of the HLA class II allele polymorphism and phylogenetic analysis in Vojvodina population.

The polymorphism at the HLA DRB1 and DQB1 loci in the population of Vojvodina was studied by PCR-SSP method. A total of 13 DRB1 and 5 DQB1 specificities displaying population-specific frequency distribution pattern were described. The most frequent HLA Class II alleles in Vojvodina population were: HLA-DRB1*11 (af = 0.30), -DRB1*04 (af = 0.28), -DRB1*07(af = 0.21), -DRB1*13 and -DRB1*16 (af = 0.18), -DQB1*03 (af = 0.64), -DQB1*05 (af = 0.39) and -DQB1*02 (af = 0.35). The haplotypes with high frequencies (> or = 0.02) included HLA DRB1*11 DQB1*03 (0.0825), DRB1*04DQB1*03 (0.0725), DRB1*07DQB1*02 (0.0475). The allele DRB1*07 showed the strongest association with DQB1*02 (delta = 0.0261, chi2 = 4.437) and DRB1*13 allele with DQB1*06 (delta = 0.0222, chi2 = 4.247). The allelic frequencies and populations distance dendrogram revealed the closest relationship of Vojvodina population with Hungarians, Croat, and Greeks which can be the result of turbulent migration within this region and admixture with neighbour populations during the history.

Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood.

Chalkbrood (Ascosphaera apis) and stonebrood (Aspergillus flavus) are well known fungal brood diseases of honeybees (Apis mellifera), but they have hardly been systematically studied because the difficulty of rearing larvae in vitro has precluded controlled experimentation. Chalkbrood is a chronic honeybee-specific disease that can persist in colonies for years, reducing both brood and honey production, whereas stonebrood is a rare facultative pathogen that also affects hosts other than honeybees and can likely survive outside insect hosts. Hive infection trials have indicated that accidental drops in comb temperature increase the prevalence of chalkbrood, but it has remained unclear whether virulence is directly temperature-dependent. We used a newly established in vitro rearing technique for honeybee larvae to test whether there are systematic temperature effects on mortality induced by controlled infections, and whether such effects differed between the two fungal pathogens. We found that increasing spore dosage at infection had a more dramatic effect on mortality from stonebrood compared to chalkbrood. In addition, a 24h cooling period after inoculation increased larval mortality from chalkbrood infection, whereas such a cooling period decreased mortality after stonebrood infection. These results raise interesting questions about honeybee defenses against obligate and facultative pathogens and about the extent to which stress factors in the host (dis)favor pathogens with lesser degrees of specialization.

[The HLA antigen system in married couples with recurrent spontaneous abortions]. / Ispitivanje antigena HLA sistema u bracnih parova sa rekurentnim spontanim abortusima.

INTRODUCTION: Investigations of factors responsible for the disturbance of fetomaternal tolerance during pathological pregnancies and causes of recurrent spontaneous abortions are pointing that HLA antigens play a key role in their development. Rejection of semiallogeneic allograft in this pregnancy disorder is a consequence of interaction between genetic and immunologic phenomena. MATERIAL AND METHODS: This article reports results of an investigation of human lymphocyte antigens in 130 couples with recurrent spontaneous abortions and in control group of 57 healthy couples. In both investigated groups the first class of human lymphocyte antigens was detected using microlymphocytotoxicity test by Terasaki. The phenotypic frequencies in the group with recurrent spontaneous abortions were compared with the corresponding results in the control group. We investigated the relative risk for development of this disorder and statistically significant difference of human lympocyte antigen frequencies between both investigated groups. RESULTS: The results of investigation showed that in the group of couples with recurrent spontaneous abortions the relative risk is hihger than 1 for the following human lymphocyte antigens: A2 = (1.53), A9 = (1.149), A10 = (1.07), A29 = (1.70), B18 = (2.24), B40 = (3.31). A significant difference in human lymphocyte antigen frequencies was established for HLA A2, HLA B18 and HLA B40, in comparison with the results of control group. Couples with recurrent spontaneous abortions had a statistically significantly higher rate of identical human lymphocyte antigens among spouses in comparison with the control group, at the level of significance p < 0.05 = 6.32. CONCLUSION: The results of this investigation showed that human lymphocyte antigens play an etiopathogenic role in development of recurrent spontaneous abortions and that some of these antigens are increasing susceptibility to pregnancy disorders.

[Thrombocyte and leukocyte antigens]. / Trombocitni i leukocitni antigeni.

INTRODUCTION: By analogy, existence of erythrocyte antigens, classified in certain blood group systems, presence of specific alloantigens on platelets and leukocytes has been confirmed. CLINICAL SIGNIFICANCE OF PLATELET AND GRANULOCYTE ANTIGENS: Platelet and leukocyte antigens have a complex clinical significance, especially in hematology and blood transfusion. Immune response occurring after allosensibilization with platelet and granulocyte antigens is involved in pathogenesis of several clinical syndromes including: neonatal alloimmune thrombocytopenia, post-transfusion purpura, refractoriness to platelet transfusion, neonatal alloimmune neutropenia, transfusion related acute lung injury and chronic benign neutropenia in children. METHODS OF DETECTION: Application of various serological and molecular techniques enables phenotypization of platelet and granulocyte antigens and genomic analysis of DNA coding regions, providing determination of specific platelet and granulocyte alloantigen polymorphism. It confirms antigenic diversity of formed blood products.

[Association between HLA antigens and leukemia in the population of Vojvodina]. / Ispitivanje asocijacije HLA antigena i leukemija u populaciji Vojvodine.

INTRODUCTION: One of the most fascinating areas of research within the field of histocompatibility at present time concerns an observation that a major human histocompatibility system, HLA, is deeply involved in development of a great number of diseases. MATERIALS AND METHODS: HLA class I antigens were investigated in 225 cases with various kinds of leukemia: 112 patients with acute myeloblastic leukemia (AML), 31 with acute lymphoblastic leukemia (ALL), 44 with chronic myelocytic leukemia (CML) and 38 with chronic lymphocytic leukemia (CLL). Applying method of microlymphocytotoxicity, 13, 19 and 8 antisera were used for A, B and C loci respectively. Control group comprised 300 unrelated persons, whose phenotypic frequencies were used to calculate the relative risk (RR), while for values of RR greater than 1, we calculated etiologic fraction (EF), and for negative RR values we calculated preventive fraction (PF). RESULTS: Results of investigation showed that RR in AML was: for A2 = 1.144, for A3 = 1.038, for A29 = 1.814, for A34 = 2.69, for B7 = 1.06, for B14 = 1.74, for B17 = 1.65 and for B21 = 2.49 and B35 = 1.77 with value of chi 2 test of 4.62 and 4.63; that RR in ALL was: for A1 = 1.61, for A2 = 1.1, for A10 = 1.23, for A11 = 1.57, for A30 = 1.4, for A32 = 2.2, for B7 = 2.81 with value of chi 2 test 4.39; that RR in CML was: for A2 = 1.21, for A32 = 1.89, for B7 = 1.52, for B12 = 1.2 and for B15 = 3.28 with value of chi 2 test of 5.89; and that RR in CLL was: for A1 = 1.35 with value of chi 2 test of 3.973, RR for A2 = 1.02, for A28 = 1.97, for A32 = 1.25, for B5 = 1.44, for B8 = 1.27, for B13 = 1.91 without statistically significant differences of frequencies except for A1. Investigation of differences between haplotype frequencies among controls and patients showed statistically significant difference of A10 B40 haplotype in CML with RR value 7.24, while there were no statistically significant differences between controls and other leukemias. DISCUSSION: Our results of investigation showed statistically significant differences between HLA frequencies in the control group and investigated diseases, and that the relative risk is under 1, and values of chi 2 test under borderline values for B21 and B35 in AML, for B7 in ALL, for B15 in CML and for A1 in CLL. CONCLUSION: Results of this investigation point to an association between HLA system and leukemias. This association is of great importance because it provides a new tool for investigation of genetics and etiology of abovementioned diseases.

[Distribution of HLA class II antigens and allele linkage disequilibrium in the population of Vojvodina]. / Distribucija HLA antigena II klase i neravnoteza vezivanja alela u populaciji Vojvodine.

INTRODUCTION: Polymorphism of human leucocyte antigens (HLA) is important in transplantation medicine, anthropological studies and paternity testing. MATERIAL AND METHODS: We investigated polymorphisms of HLA class II in population of Vojvodina by means of serologic typing using microlymphocitotoxicity test. We calculated the HLA-DR and HLA-DQ gene frequencies in 174 subjects. Haplotype frequencies, coefficient of linkage disequilibrium (delta values) and their statistical significant levels, were analyzed on the basis of these data. RESULTS: The most frequent HLA-DR and HLA-DQ alleles were: DR5(11) (gene frequency 0.138), DR4 (gene frequency 0.083), DR1 (gene frequency 0.077), DQ1 (gene frequency 0.388), DQ3 (gene frequency 0.197), and DQ2 (gene frequency 0.09). The highest positive values of coefficient of linkage disequilibrium (delta) were calculated for the following haplotypes: DR1DQ1 (delta value 0.07308708), DR2DQ1 (delta value 0.059846528), DR3DQ2 (delta value 0.06193263), DR2(15)DQ1 (delta value 0.039186022), DR5(11)DQ3(7) (delta value 0.04969439), DR7DQ2 (delta value 0.057985517). Significant differences between observed vs. expected haplotype frequencies were also considered for these haplotypes. CONCLUSION: This study indicates distinctiveness and specificity of the population of Vojvodina and highlights the importance of determining HLA frequencies in endogamic groups of Serbia.

[Acquired B antigen in a pregnant woman belonging to the A1 blood group: case report]. / Steceni B antigen u trudnice A1 krvne grupe: prikaz slucaja.

INTRODUCTION: This article presents a case of acquired B antigen in a 24 year old pregnant woman, with A1 Rh D positive blood group verified by expression ABO tube test, secretor status, and investigation of group specific substance in saliva, by agglutinating inhibition test and examination of blood groups in patient's family (both parents and sister). CASE REPORT: Since the patient was a clinically healthy woman, without anamnestic data and clinical signs of any kind of disease (such as diseases of the gastrointestinal system, especially colonic disorders), and with regular course of pregnancy, this acquired irregular agglutination of red blood cells, suggested a possible infection with Gram-negative bacteria. That is why she underwent additional investigations of blood and urine. RESULTS AND CONCLUSION: Hemocultures were negative, but urine tests revealed a urinary infection with uropathogenic strains of Escherichia coli O86. Eight months later the patient was retested, and findings of acquired B antigen and latent urinary infection with E. coli still persisted, proving the cause of irregular agglutination.

The mechanism of increased renal susceptibility to toxic substances in the elderly. Part I. The role of increased vasoconstriction.

The mechanisms of increased susceptibility to nephrotoxins in aging are complex and incompletely understood. It is very important to try to increase our knowledge of them because adults become increasingly vulnerable to nephrotoxic substances, as they grow older. In addition, the percentage of elderly people will increase markedly in the near future, at least in the developed countries. Drugs such as diuretics, laxatives, NSAIDs, aminoglycosides and other nephrotoxic antibiotics, and converting enzyme inhibitors are used a lot by aging people and can produce severe renal problems. Beside drugs, the clinical use of radiocontrast agents also rises in older patients. It seems that the main mechanism of the increased renal susceptibility to toxic substances in the elderly is a disbalance between vasoconstrictor and vasodilator factors (in favor of vasoconstrictor ones). Increased propensity to vasoconstriction (to Ang II, ET and PAF), as well as increased levels of oxidatively modified biomolecules in the elderly, may enhance susceptibility of old kidney to toxic substances. In addition, all mechanisms that influence both mesangial and fibroblast cell proliferation and over-production of extracellular matrix might also be involved in the processes that make the old kidney prone to drug-induced chronic toxic injury.

[Inhibitory activity of blood group antigens M and N in inhibition of virus hemagglutination reactions of influenza viruses]. / Inhibitorna aktivnost M i N krvnogrupnih antigena u reakciji inhibicije virusne hemaglutinacije sa virusom influence.

INTRODUCTION: M and N blood group antigens demonstrate inhibitory activity in inhibition reaction of viral hemagglutination with some influenza virus strains, with help of N-acetylneuraminic acid (sialic acid) which occurs in glycophorins on the red blood cells surface, and represent specific hemagglutunation receptors and substrate for action of influenza virus neuraminidase. MATERIAL AND METHODS: Reactivity of human O red blood cells with MM and NN phenotypes is established in inhibition reaction of viral hemagglutination by influenza virus A2 Singapore, with intention to fortify possibility of using human red blood cells in viral hemagglutination, to determine their reactivity in titration, retitration of hemagglutinins and inhibition reaction of hemagglutination. The aim of investigation was to describe destinations between different red blood cells in view of speed of reaction and receptor capacity. Material included 69 samples of sera from persons infected with influenza virus, among them 32 samples were positive with titres 1/80 and more. RESULTS: Reactivity of erythrocytes with MM and NN phenotypes in titration of hemagglutinins of influenza virus A2 Singapore in which base is viral hemagglutination is identical, because there are no statistically significant differences of average geomaterical levels of antibody titers. Enzymatically derived red cells by papain, which do not contain M and N blood group antigens, not cause viral hemagglutination phenomenon, because they sediment in all dilutions. Reactivity of red blood cells with MM and NN phenotypes in retitration of haemagglutinins and inhibition reaction of hemagglutination is identical, because there are no statistically significant differences in results with two kinds red blood cells. DISCUSSION: Results of investigation revealed that the reactivity of O human red blood cells different in MN phenotype is identical in regard to speed of reaction and receptor capacity in titration, retitration and inhibition reaction of viral hemagglutinatination and also showed that they demonstrate viral hemagglutination phenomenon in contrast with papainised red blood cells which do not contain M and N blood group antigens, which indirectly means that M and N blood group antigens contain receptors for influenza virus. CONCLUSIONS: Human red cells with MM and NN phenotypes cause viral hemagglutination phenomenon with influenza virus A2 Singapore, and could be used in routine virusological diagnostic procedures. O blood group red cells (MM and NN) in reaction of viral hemagglutination result identically in view of speed of reaction and receptor capacity, and have the same impact on result of this reaction. Enzymatically derived red cells by papain do not cause viral hemagglutination phenomenon, because they do not contain receptors for viral hemagglutinin on red cell membrane surface, which are hydrolazed by papain. Receptors for influenza virus on red cell membrane surface are a component part of M and N blood group antigens which are destroyed by papain.

The MNSs blood group system in the population of South Backa.

Blood groups represent qualitative characteristics of humans which are controlled by one or a few genetic loci and due to this it is easy to determine various kinds of alleles and their frequencies in certain samples. Analyzing variability of characteristics such as blood groups, it is possible to get an insight in genetic structure of a population and it represents a suitable manner for detecting similarities and differences between populations. The intention of this study was to determine the genetic structure of the population of South Backa in a classic manner by phenotypic characteristics in MNSs blood group system, to assess frequencies of alleles, phenotypes, genotypes, allelic frequencies and genetic distances between the population of South Backa and populations of various geographic regions. Immunohematologic investigation of 1.000 persons included blood group testing in the MNSs system, which was a basis for applying population-genetic analysis based on Hardy-Weinberg equilibrium in populations. Results of investigation revealed that in population of South Backa, s allele has the highest frequence (85.59%); the most frequent genotypes are ss (48.5%), MN (43.4%), Ss (37.4%); the most frequent phenotypes are MNss (19.3%), MNSs (18.4%) and NNss (15.3%). Genetic distances between populations are greatest among geographically most distant populations as Eskimos, Australian Aborigines, populations of Papua-Gvinea, Fiji and low in populations of Turkey, Italy, Albania and Cyprus.

Linkage disequilibrium in MNSs blood group system in population of south Backa.

Linkage disequilibrium is a characteristic of MNSs blood group system which performs more frequent association of linked alleles than it is expected in comparison with their allelic frequencies. Measure of linkage disequilibrium is the difference between expected and actual genotypic frequencies which is stated in coefficient of linkage disequilibrium. The purpose of this study was to fortify existence of linkage disequilibrium phenomenon inside MNSs blood group system in population of South Backa in comparison with calculated allelic frequencies. Blood group typing in MNSs system included 1,000 healthy nonrelated persons where allelic frequencies, expected and actual genotype frequencies had been calculated, as well as the differences between them. Results of investigation showed that allele has the highest allelic frequency (0.672), S allele has the lowest frequency (0.33), the highest expected frequencies are calculated for Ms/Ns (Ms/NS) (0.218) and Ms/Ns (0.2235) genotypes, while the actual frequencies are highest for MS/NS (Ms/NS) (0.184), Ms/Ns (0.193) and Ns/Ns (0.153) genotypes. Statistically significant difference between expected and actual genotypic frequencies occurs for Ms/MS and Ns/Ns genotypes, which means that S and M and N and s alleles are more frequently associated and prove the existence of linkage disequilibrium phenomenon in population of South Backa.

L-arginine reduces tubular cell injury in acute post-ischaemic renal failure.

BACKGROUND: The pathophysiology of renal ischaemia, resulting in tubular cell injury and leading to acute renal failure (ARF), remains unclear. An ever-increasing number of investigations focus on a possible role of nitric oxide (NO) in regulating circulation during ARF. In this context, we investigated the influence of chronic stimulation or inhibition of NO synthesis, or both, on haemodynamic parameters, histology and plasma renin activity (PRA) after ischaemia-reperfusion injury of rat kidneys. METHODS: Experiments were performed on adult, male Wistar rats. Before induction of ARF, a group of animals was treated with a NO synthesis inhibitor (L-NAME) and another group was treated with a precursor of NO synthesis (L-arginine). The animals received those substances for 4 weeks. Control groups received the same amount of tap water for 4 or 8 weeks and were divided into groups with ARF (4 weeks--ARF group and 8 weeks ARF group) and a sham-operated group. Another group of rats was treated first with L-NAME and then with L-arginine in their drinking water, for 4 weeks for each of these two substances. All parameters were evaluated 24 h after the induction of ischaemic ARF or the sham operation. RESULTS: Our results show that such long-term stimulation of NO release by L-arginine improved renal haemodynamics in the ischaemic form of ARF. Renal blood flow (RBF) increased by 96% in the L-arginine-treated rats with ARF compared with the group with ARF alone. Inhibition of NO synthesis worsens renal haemodynamics after ARF. However, this aggravation can be reversed by L-arginine. The rate of water reabsorption was reduced in all groups with ARF, but this reduction was least in the group treated with L-arginine. The rate of Na+ reabsorption was reduced in all groups 24 h after renal ischaemia, but a significant decrease was observed after the inhibition of NO synthesis. Histological examination of the kidney specimens showed that morphological changes were least in the rats treated with L-arginine, when compared with all other groups with ARF. Nevertheless, the lesions were most prominent in the L-NAME+ARF group. In this group, the areas of corticomedullar necrosis were more widespread in comparison with other groups, especially the L-arginine group where only swelling of the proximal tubular cells was observed. Treatment with L-NAME was not accompanied by any significant alteration in the plasma concentration of angiotensin I (ANG I), while in the group treated with L-arginine ANG I had a tendency to decrease. CONCLUSIONS: Acute post-ischaemic renal failure may be alleviated by administering the NO substrate (L-arginine). NO acts cytoprotectively on tubular epithelial cells in ischaemia--reperfusion injury of rat kidney. Evidence of this comes from both histopathological findings and increased tubular water and sodium reabsorption. However, inhibition of NO synthesis (provoked by L-NAME) worsens renal haemodynamics and aggravates morphological changes after ARF. These aggravations can, however, be reversed by L-arginine.