A reverse vaccinology approach on transmembrane carbonic anhydrases from Plasmodium species as vaccine candidates for malaria prevention.

BACKGROUND: Malaria is a significant parasitic infection, and human infection is mediated by mosquito (Anopheles) biting and subsequent transmission of protozoa (Plasmodium) to the blood. Carbonic anhydrases (CAs) are known to be highly expressed in the midgut and ectoperitrophic space of Anopheles gambiae. Transmembrane CAs (tmCAs) in Plasmodium may be potential vaccine candidates for the control and prevention of malaria. METHODS: In this study, two groups of transmembrane CAs, including α-CAs and one group of η-CAs were analysed by immunoinformatics and computational biology methods, such as predictions on transmembrane localization of CAs from Plasmodium spp., affinity and stability of different HLA classes, antigenicity of tmCA peptides, epitope and proteasomal cleavage of Plasmodium tmCAs, accessibility of Plasmodium tmCAs MHC-ligands, allergenicity of Plasmodium tmCAs, disulfide-bond of Plasmodium tmCAs, B cell epitopes of Plasmodium tmCAs, and Cell type-specific expression of Plasmodium CAs. RESULTS: Two groups of α-CAs and one group of η-CAs in Plasmodium spp. were identified to contain tmCA sequences, having high affinity towards MHCs, high stability, and strong antigenicity. All putative tmCAs were predicted to contain sequences for proteasomal cleavage in antigen presenting cells (APCs). CONCLUSIONS: The predicted results revealed that tmCAs from Plasmodium spp. can be potential targets for vaccination against malaria.

Diagnostic yield of genetic testing in a heterogeneous cohort of 1376 HCM patients.

BACKGROUND: Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. METHODS: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. RESULTS: A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p < 0.0001), a higher maximum wall thickness (MWT) (p < 0.0001), a positive family history (p < 0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). CONCLUSION: The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.

A genome-wide expression quantitative trait loci analysis of proprotein convertase subtilisin/kexin enzymes identifies a novel regulatory gene variant for FURIN expression and blood pressure.

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN, PCSK5, PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant (p < 5 × 10(-8)) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis-eQTL SNP rs4702 for FURIN is strongly associated with both diastolic (p = 0.012) and systolic (p = 0.035) BP levels, as well as peripheral vascular resistance (p = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease.

The proprotein convertase subtilisin/kexin furinA regulates zebrafish host response against Mycobacterium marinum.

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection.

Donor Haplotype B of NK KIR Receptor Reduces the Relapse Risk in HLA-Identical Sibling Hematopoietic Stem Cell Transplantation of AML Patients.

Successful allogeneic hematopoietic stem cell transplantation (HSCT) depends not only on good HLA match but also on T-cell mediated graft-versus-leukemia (GvL) effect. Natural killer (NK) cells are able to kill malignant cells by receiving activation signal from the killer-cell immunoglobulin-like receptors (KIR) recognizing HLA molecules on a cancer cell. It has been recently reported that the risk of relapse in allogeneic hematopoietic stem cell transplantation (HSCT) is reduced in acute myeloid leukemia (AML) patients whose donors have several activating KIR genes or KIR B-motifs in unrelated donor setting, obviously due to enhanced GvL effect by NK cells. We studied the effect on relapse rate of donor KIR haplotypes in the HLA-identical adult sibling HSCT, done in a single center, in Helsinki University Central Hospital, Helsinki, Finland. Altogether, 134 patients with 6 different diagnoses were identified. Their donors were KIR genotyped using the Luminex and the SSP techniques. The clinical endpoint, that is, occurrence of relapse, was compared with the presence or absence of single KIR genes. Also, time from transplantation to relapse was analyzed. The patients with AML whose donors have KIR2DL2 or KIR2DS2 had statistically significantly longer relapse-free survival (P = 0.015). Our data support previous reports that donors with KIR B-haplotype defining genes have a lower occurrence of relapse in HSCT of AML patients. Determination of donor KIR haplotypes could be a useful addition for a risk assessment of HSCT especially in AML patients.

Study of the cytokine polymorphisms in correlation to rejection and graft survival in renal allograft donors and recipients from a homogenous Saudi population.

OBJECTIVES: Allograft outcome can be improved with the discovery of risk factors that influence adverse events and may allow individualization of patients' treatment. Rejection is the main hurdle to successful transplantation and the immune response is the key effecter to rejection development. Hence, the major objective of the present study was to assess the relationship between single nucleotide polymorphisms (SNPs) in 5 cytokine genes, HLA mismatch and graft outcome in a cohort of 100 Saudi kidney transplant recipients and 100 living related donors at a single transplant center. MATERIALS & METHODS: Genotyping of the following positions: TNFA (-308G/A), TGFB1 (codon 10T/C, codon 25C/G), IL-10 (-1082G/A, -819C/T, -592C/A), IL-6 (-174C/G), and IFNG (+874T/A) were performed. RESULTS: The majority of the donors whose recipients presented with either cellular or antibody mediated graft rejection (90% and 100%) respectively were found to be significantly (p=0.0351) associated with intermediate or high IL-10 producing haplotypes, compared to those with stable grafts (58.66%). Haplotypes linked with lower IL-10 production were not detected in the donors or their recipients with antibody mediated graft rejection compared to donors with stable graft (41.33%). The distribution of donor IL-10-1082 haplotypes (GG, GA, AA) showed a statistically significant association of IL-10-1082 GA genotype (p=0.0351) with rejection, when grouped according to patients' rejection status. No other statistically significant deviations were observed in the donors' genotypes. Analyses of cytokine polymorphisms in the recipients revealed no significant association. Multivariate logistic regression analyses showed that only HLA-DRB1 mismatch significantly influenced graft loss (p=0.0135). CONCLUSION: This study demonstrates that the donor IL-10 genotypes and HLA-DRB1 mismatch are key determinants in graft outcome after renal transplantation.

Proprotein convertase subtilisin/kexin type 7 (PCSK7) is essential for the zebrafish development and bioavailability of transforming growth factor ß1a (TGFß1a).

Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFß1a. Consequently, tgfß1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.

Zebavidin--an avidin-like protein from zebrafish.

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P = .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR] = 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P = .078), higher relapse-free survival (P = .029), and higher overall survival (P = .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner.

Genetics of the first seven proprotein convertase enzymes in health and disease.

Members of the substilisin/kexin like proprotein convertase (PCSK) protease family cleave and convert immature pro-proteins into their biologically active forms. By cleaving for example prohormones, cytokines and cell membrane proteins, PCSKs participate in maintaining the homeostasis in a healthy human body. Conversely, erratic enzymatic function is thought to contribute to the pathogenesis of a wide variety of diseases, including obesity and hypercholestrolemia. The first characterized seven PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made up of paired basic amino acid residues. This feature results in a variable degree of biochemical redundancy in vitro, and consequently, shared substrate molecules between the different PCSK enzymes. This redundancy has confounded our understanding of the specific biological functions of PCSKs. The physiological roles of these enzymes have been best illustrated by the phenotypes of genetically engineered mice and patients that carry mutations in the PCSK genes. Recent developments in genome-wide methodology have generated a large amount of novel information on the genetics of the first seven proprotein convertases. In this review we summarize the reported genetic alterations and their associated phenotypes.

S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity.

The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.

SAP30L (Sin3A-associated protein 30-like) is involved in regulation of cardiac development and hematopoiesis in zebrafish embryos.

The Sin3A-associated proteins SAP30 and SAP30L share 70% sequence identity and are part of the multiprotein Sin3A corepressor complex. They participate in gene repression events by linking members of the complex and stabilizing interactions among the protein members as well as between proteins and DNA. While most organisms have both SAP30 and SAP30L, the zebrafish is exceptional because it only has SAP30L. Here we demonstrate that SAP30L is expressed ubiquitously in embryonic and adult zebrafish tissues. Knockdown of SAP30L using morpholino-mediated technology resulted in a morphant phenotype manifesting as cardiac insufficiency and defective hemoglobinization of red blood cells. A microarray analysis of gene expression in SAP30L morphant embryos revealed changes in the expression of genes involved in regulation of transcription, TGF-beta signaling, Wnt-family transcription factors, and nuclear genes encoding mitochondrial proteins. The expression of the heart-specific nkx2.5 gene was markedly down-regulated in SAP30L morphants, and the cardiac phenotype could be partially rescued by nkx2.5 mRNA. In addition, changes were detected in the expression of genes known to be important in hemoglobin synthesis and erythropoiesis. Our results demonstrate that SAP30L regulates several transcriptional pathways in zebrafish embryos and is involved in the development of cardiac and hematopoietic systems.

In vitro functional and immunomodulatory properties of the Lactobacillus helveticus MIMLh5-Streptococcus salivarius ST3 association that are relevant to the development of a pharyngeal probiotic product.

The use of proper bacterial strains as probiotics for the pharyngeal mucosa is a potential prophylactic strategy for upper respiratory tract infections. In this context, we characterized in vitro the functional and immunomodulatory properties of the strains Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 that were selected during previous investigations as promising pharyngeal probiotics. In this study, we demonstrated in vitro that strains MIMLh5 and ST3, alone and in combination, can efficiently adhere to pharyngeal epithelial cells, antagonize Streptococcus pyogenes, and modulate host innate immunity by inducing potentially protective effects. In particular, we found that the strains MIMLh5 and ST3 activate U937 human macrophages by significantly inducing the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Nonetheless, the induction of the anti-inflammatory interleukin-10 (IL-10) by MIMLh5 or ST3 was never lower than that of TNF-α, suggesting that these bacteria can potentially exert a regulatory rather than a proinflammatory effect. We also found that the strains MIMLh5 and ST3 induce cyclooxygenase 2 (COX-2) expression and demonstrated that toll-like receptor 2 (TLR-2) participates in the recognition of the strains MIMLh5 and ST3 by U937 cells. Finally, we observed that these microorganisms grow efficiently when cocultured in milk, suggesting that the preparation of a milk-based fermented product containing both MIMLh5 and ST3 can be a practical solution for the administration of these bacteria. In conclusion, we propose the combined use of L. helveticus MIMLh5 and S. salivarius ST3 for the preparation of novel products that display probiotic properties for the pharyngeal mucosa.

Identification of proprotein convertase substrates using genome-wide expression correlation analysis.

BACKGROUND: Subtilisin/kexin-like proprotein convertase (PCSK) enzymes have important regulatory function in a wide variety of biological processes. PCSKs proteolytically process at a target sequence that contains basic amino acids arginine and lysine, which results in functional maturation of the target protein. In vitro assays have showed significant biochemical redundancy between the seven family members, but the phenotypes of PCSK deficient mice and patients carrying an inactive PCSK allele argue for a specific biological function. Modeling the structures of individual PCSK enzymes has offered little insights into the specificity determinants. However, previous studies have shown that there can be a coordinated expression between a PCSK and its target molecule. Here, we have surveyed the putative PCSK target proteins using genome-wide expression correlation analysis and cleavage site prediction algorithms. RESULTS: We first performed a gene expression correlation analysis over the whole genome for all PCSK enzymes. PCSKs were found to cluster differently based on the strength of correlations. The screen for putative PCSK target proteins showed a significant enrichment (p-values from 1.2e-4 to < 1.0e-10) of putative targets among the most positively correlating genes for most PCSKs. Interestingly, there was no enrichment in putative targets among the genes that correlated positively with the biologically redundant PCSK7, whereas PCSK5 showed an inverse correlation. PCSKs also showed a highly variable degree of shared target genes that were identified by expression correlation and cleavage site prediction. Multiple alignments were used to evaluate the putative targets to pinpoint the important residues for the substrate recognition. Finally, we validated our approach and identified biochemically PAPPA1 and ADAMTS6 as novel targets for FURIN proteolytic activity. CONCLUSIONS: Most PCSK enzymes display strong positive expression correlation with predicted target proteins in our genome-wide analysis. We also show that expression correlation screen combined with a cleavage site-prediction analysis can be used to identify novel bona fide target molecules for PCSKs. Exploring the positively correlating genes can thus offer additional insights into the biology of proprotein convertases.

Proprotein convertases in human atherosclerotic plaques: the overexpression of FURIN and its substrate cytokines BAFF and APRIL.

BACKGROUND: Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave proproteins into mature end products. Previously, MBTPS1 and PCSK9 have been shown to regulate cholesterol metabolism and LDL receptor recycling, whereas FURIN and PCSK5 have been suggested to inactivate lipases and regulate inflammation in atherosclerosis. Here, we systematically analyzed the expression of PCSKs and their targets in advanced atherosclerotic plaques. METHODS AND RESULTS: Microarray and quantitative real-time PCR experiments showed that FURIN (42.86 median fold, p = 2.1e-8), but no other PCSK, is universally overexpressed in the plaques of different vascular regions. The mRNA expression screen of PCSK target proteins in plaques identified many known factors, but it also identified the significant upregulation of the previously overlooked furin-processed B cell activating cytokines APRIL (TNFSF13, 2.52 median fold, p = 3.0e-5) and BAFF (TNFSF13B, 2.97 median fold, p = 7.6e-6). The dysregulation of FURIN did not associate with its htSNPs or the previously reported regulatory SNP (-229, rs4932178) in the promoter. Immunohistochemistry experiments showed the upregulation of FURIN in the plaque lymphocytes and macrophages where it was co-expressed with BAFF/TNFSF13B and APRIL/TNFSF13. CONCLUSIONS: Our data unequivocally show that FURIN is the primary PCSK that is dysregulated in the immune cells of advanced human atherosclerotic plaques, which implies a role for this enzyme in plaque pathology. Therefore, drugs that inhibit FURIN in arteries may modulate the course of this disease.

Introduction of real patients into problem-based learning in preclinical first-year anatomy curriculum.

BACKGROUND: Early patient contacts are considered important in medical education. AIMS: We studied the influence of a real patient trigger on study motivation and learning in problem-based study groups of first-year medical and dentistry students. METHODS: 156 eligible students were allocated into 17 groups. Six randomly selected groups received both the real patient and paper trigger, and 11 groups received only the paper trigger. The immediate and later effects of the trigger were assessed with qualitative and quantitative questionnaires and exam scores. The tutors answered questionnaires concerning learning outcomes. RESULTS: The students reported that the real patient trigger significantly improved their study motivation, understanding of the learning objectives and confidence in future patient encounters. The real patient trigger was considered significantly more interesting than the paper case. No statistically significant difference was observed in the exam scores. The tutors observed that groups with poor previous performance gained better results in study sessions. CONCLUSIONS: Real patient triggers motivate students to learn basic medical sciences. Ways to present real patients to students should be considered in medical curricula from early on.

Killer cell immunoglobulin-like receptor gene diversity in the Saudi population.

Killer cell immunoglobulin-like receptors (KIRs) influence the outcome of haematopoetic stem cell transplantation by modulating the cytotoxic ability of natural killer (NK) cells and a subset of T cells. KIRs are also highly polymorphic and could therefore be good population genetic markers, much like their human leukocyte antigen (HLA) ligands. This study represents the first report on distribution of 16 KIR genes in 162 unrelated healthy Saudi individuals. All the 16 KIR genes were observed in the studied population and the four framework genes (KIR2DL4, 3DL2, 3DL3 and 3DP1) were present in all individuals. Forty- one distinct KIR profiles were expressed in our population, 11 of which had not been previously described in other populations including the Middle Eastern population. AA1, the most common genotypic profile was observed at a frequency of 26.5%. The group A haplotype was more frequent (53%) in the Saudi population compared to the group B haplotype (47%). The pattern of the inhibitory KIR/HLA ligands were also analyzed and 52.3% of the Saudi population was found to express two pairs of the inhibitory KIR/HLA-C. The KIR gene frequencies suggests that the Saudi population shares common general features with the Middle Eastern and other populations, but still has its own unique frequencies of several KIR loci.

Donor-recipient mismatch for common gene deletion polymorphisms in graft-versus-host disease.

Transplantation and pregnancy, in which two diploid genomes reside in one body, can each lead to diseases in which immune cells from one individual target antigens encoded in the other's genome. One such disease, graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT, or bone marrow transplant), is common even after transplants between HLA-identical siblings, indicating that cryptic histocompatibility loci exist outside the HLA locus. The immune system of an individual whose genome is homozygous for a gene deletion could recognize epitopes encoded by that gene as alloantigens. Analyzing common gene deletions in three HSCT cohorts (1,345 HLA-identical sibling donor-recipient pairs), we found that risk of acute GVHD was greater (odds ratio (OR) = 2.5; 95% confidence interval (CI) 1.4-4.6) when donor and recipient were mismatched for homozygous deletion of UGT2B17, a gene expressed in GVHD-affected tissues and giving rise to multiple histocompatibility antigens. Human genome structural variation merits investigation as a potential mechanism in diseases of alloimmunity.

Association of IL-10 and IL-10Rbeta gene polymorphisms with graft-versus-host disease after haematopoietic stem cell transplantation from an HLA-identical sibling donor.

BACKGROUND: Extensive allelic matching in the human leukocyte antigen (HLA) genes is regarded as a prerequisite for good clinical success of allogeneic haematopoietic stem cell transplantation (HSCT). Also other genetic factors can be assumed to play a role in preventing and controlling the complications associated with allogeneic HSCT, in particular graft-versus-host disease (GvHD). Interleukin-10 (IL-10) and its receptor (IL-10R), key regulators of the immune response, are among these candidates. We studied the association of IL-10 and IL-10Rbeta gene polymorphisms with the occurrence of GvHD in 309 HLA-identical sibling donor and recipient pairs. RESULTS: The difference in genotypic IL-10 production between patient and donor in combination with patient IL-10Rbeta A/A genotype predisposed strongly to acute GvHD (OR = 7.15, p = 0.000023). On the other hand, a combination of same genotypic IL-10 production with patient IL-10Rbeta A/A genotype protected from chronic GvHD (OR = 0.407, p = 0.0097). CONCLUSION: Our results suggest that IL-10 and IL-10Rbeta genes have a synergistic effect on the risk of GvHD.

Genetic similarity of chromosome 6 between patients receiving hematopoietic stem cell transplantation and HLA matched sibling donors.

BACKGROUND: Matching for HLA genes located on chromosome 6 is required in hematopoietic stem cell transplantation to reduce the incidence of graft-versus-host disease. However, a considerable proportion of patients still suffer from it, obviously due to genetic differences outside the HLA gene region. DESIGN AND METHODS: We studied the similarity of almost 4,000 single nucleotide polymorphisms on chromosome 6 between patients receiving hematopoietic stem cell transplantation and their HLA-matched sibling donors. RESULTS: We observed that as a result of routine HLA matching the siblings in fact shared surprisingly long chromosomal fragments with similar single nucleotide polymorphism genotypes--from 11.65 Mb to 134.66 Mb. The number of genes mapped on these shared fragments varied from 402 to 1,302. Considering the whole chromosome 6, the HLA-matched siblings were apparently identical for 65.2-97.8% of the single nucleotide polymorphisms. CONCLUSIONS: Potentially, genes similar in some transplantation pairs while different in others might have a significant role in determining the outcome after hematopoietic stem cell transplantation.