Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture.

BACKGROUND: Pancreatic cancer remains a fatal disease. Experimental systems are needed for personalized treatment strategies, drug testing and to further understand tumor biology. Cell cultures can serve as an excellent preclinical platform, but their generation remains challenging. METHODS: Tumor cells from surgically removed pancreatic ductal adenocarcinoma (PDAC) specimens were cultured under novel protocols. Cellular growth and composition were analyzed and culture conditions were continuously optimized. Characterization of cell cultures and primary tumors was performed via hematoxylin and eosin (HE) and immunofluorescence (IF) staining. RESULTS: Protocols for two- and three-dimensional PDAC primary cell cultures could successfully be established. Primary cell culture depended on dissociation techniques, growth factor supplementation and extracellular matrix components containing Matrigel being crucial for the transformation to three-dimensional PDAC organoids. The generated cultures showed to be highly resemblant to established PDAC primary cell cultures. HE and IF staining for cell culture and corresponding primary tumor characterization could successfully be performed. CONCLUSIONS: The work presented herein shows novel and effective methods to successfully establish primary PDAC cell cultures in a distinct time frame. Factors contributing to cell growth and differentiation could be identified with important implications for further primary cell culture protocols. The established protocols might serve as novel tools in personalized tumor therapy.

Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation.

Lipocalin 2 (Lcn2) is rapidly produced by damaged nephron epithelia and is one of the most promising new markers of renal injury, delayed graft function and acute allograft rejection (AR); however, the functional importance of Lcn2 in renal transplantation is largely unknown. To understand the role of Lcn2 in renal AR, kidneys from Balb/c mice were transplanted into C57Bl/6 mice and vice versa and analyzed for morphological and physiological outcomes of AR at posttransplantation days 3, 5, and 7. The allografts showed a steady increase in intensity of interstitial infiltration, tubulitis and periarterial aggregation of lymphocytes associated with a substantial elevation in serum levels of creatinine, urea and Lcn2. Perioperative administration of recombinant Lcn2:siderophore:Fe complex (rLcn2) to recipients resulted in functional and morphological amelioration of the allograft at day 7 almost as efficiently as daily immunosuppression with cyclosporine A (CsA). No significant differences were observed in various donor-recipient combinations (C57Bl/6 wild-type and Lcn2(-/-) , Balb/c donors and recipients). Histochemical analyses of the allografts showed reduced cell death in recipients treated with rLcn2 or CsA. These results demonstrate that Lcn2 plays an important role in reducing the extent of kidney AR and indicate the therapeutic potential of Lcn2 in transplantation.

Treatment with tetrahydrobiopterin overcomes brain death-associated injury in a murine model of pancreas transplantation.

Brain death (BD) has been associated with an immunological priming of donor organs and is thought to exacerbate ischemia reperfusion injury (IRI). Recently, we showed that the essential nitric oxide synthase co-factor tetrahydrobiopterin (BH4) abrogates IRI following experimental pancreas transplantation. We therefore studied the effects of BD in a murine model of syngeneic pancreas transplantation and tested the therapeutic potential of BH4 treatment. Compared with sham-operated controls, donor BD resulted in intragraft inflammation reflected by induced IL-1ß, IL-6, VCAM-1, and P-selectin mRNA expression levels and impaired microcirculation after reperfusion (p < 0.05), whereas pretreatment of the BD donor with BH4 significantly improved microcirculation after reperfusion (p < 0.05). Moreover, BD had a devastating impact on cell viability, whereas BH4-treated grafts showed a significantly higher percentage of viable cells (p < 0.001). Early parenchymal damage in pancreatic grafts was significantly more pronounced in organs from BD donors than from sham or non-BD donors (p < 0.05), but BH4 pretreatment significantly ameliorated necrotic lesions in BD organs (p < 0.05). Pretreatment of the BD donor with BH4 resulted in significant recipient survival (p < 0.05). Our data provide novel insights into the impact of BD on pancreatic isografts, further demonstrating the potential of donor pretreatment strategies including BH4 for preventing BD-associated injury after transplantation.

Decreased levels of serum complement C3 and natural killer cells add to the predictive value of total immunoglobulin G for severe infection in heart transplant recipients.

BACKGROUND: Infection remains a source of mortality in heart recipients. We previously reported that post-transplant immunoglobulin G (IgG) quantification can help identify the risk for infection. We assessed whether other standardized parameters of humoral and cellular immunity could prove useful when identifying patients at risk of infection. METHODS: We prospectively studied 133 heart recipients over a 12-month period. Forty-eight patients had at least one episode of severe infection. An event was defined as an infection requiring intravenous antimicrobial therapy. RESULTS: Cox regression analysis revealed an association between the risk of developing infection and the following: lower IgG2 subclass levels (day 7: relative hazard [RH] 1.71; day 30: RH 1.76), lower IgA levels (day 7: RH 1.61; day 30: RH 1.91), lower complement C3 values (day 7: RH 1.25), lower CD3 absolute counts (day 30: RH 1.10), lower absolute natural killer [NK] cell count (day 7: RH 1.24), and lower IgG concentrations (day 7: RH 1.31; day 30: RH 1.36). Cox regression bivariate analysis revealed that lower day 7 C3 levels, IgG2 concentration, and absolute NK cell count remained significant after adjustment for total IgG levels. CONCLUSIONS: Data suggest that early immune monitoring including C3, IgG2, and NK cell testing in addition to IgG concentrations is useful when attempting to identify the risk of infection in heart transplant recipients.

Intrarenal gene expression of proinflammatory chemokines and cytokines in chronic proteinuric glomerulopathies.

Proteinuria has been recently shown to be an independent risk factor for the progression of chronic nephropathies, but the actual mechanisms by which urinary protein load damages renal tissue in humans remain unsolved. Using real-time RT-PCR method we evaluated intrarenal mRNA expression of various cytokines and chemokines in patients with biopsy-proven IgA nephropathy (IgAN, n=11), membranous nephropathy (MN, n=6) and focal and segmental glomerulosclerosis (FSGS, n=6) who exhibited proteinuria over 0.5 g/day. There was a significant positive correlation between the proteinuria extent and the intrarenal RANTES (regulated upon activation normal T cell expressed and secreted) mRNA expression in patients with IgAN, a similar trend was also observed in patients with MN and FSGS. There were no clear relationships between the proteinuria and intrarenal mRNA expression of tumor necrosis factor alpha, transforming growth factor beta1 and monocyte chemoattractant peptide-1. There were no differences in the pattern of cytokine mRNA expression between different glomerulopathies. In conclusion, our results support the hypothesis that lymphocytes, macrophages and their products provoke tissue injury in response to proteinuria independently of the nature of renal diseases in man.

Cytokines and chemokine gene expression in human kidney transplantation.

Despite advances in immunosuppression in past decades, allograft rejection remains the main reason for kidney graft failure. Recently, despite great improvements in understanding of molecular basis of allograft rejections, renal histology remains the primary method to monitor the onset of graft rejection. The aim of the present study was to ascertain whether cytokine and chemokine expression profiles in kidney allografts contributed to the diagnosis of graft dysfunction. We analyzed mRNA expression in 174 kidney graft biopsies for the following cytokines: TGF-beta1, TNF-alpha, IL-10, and chemokine RANTES. Based on the expression levels obtained by real-time RT-PCR, we correlated data with the results of morphologic examinations. All tested cytokines and chemokines were upregulated (P < .001) during acute rejection compared to nonrejecting controls. Upregulation was also found in chronic allograft nephropathy (CAN) group for TGF-beta1, IL-10 (P < .001), TNF-alpha, and RANTES (P < .01). Upregulated expression of IL-10 (P < .001), TGF-beta1, (P < .01) and RANTES (P < .05) showed borderline changes. Higher expression levels (P < .001) of TGF-beta1 and IL-10 were also found during ATN. IL-10 was upregulated (P < .01) in specimens with recurrent glomerulonephritis. Weakly increased (P < .05) expressions of TGF-beta1 were found during CsA toxicity. Distinctive expression levels between acute rejection and CAN were only found for IL-10 (P < .01). TNF-alpha showed a different expression profile in acute rejection versus ATN (P < .001). These findings suggest that distinct cytokine and chemokine expression profiles in grafts may contribute to the diagnosis for and elucidation of the immunopathologic process during graft dysfunction.

Effect of cytokines and chemokines (TGF-beta, TNF-alpha, IL-6, IL-10, MCP-1, RANTES) gene polymorphisms in kidney recipients on posttransplantation outcome: influence of donor-recipient match.

Posttransplantation alloantigen-dependent and alloantigen-independent processes are both mediated by cytokines and chemokines. Recently cytokines and chemokines, as well as their receptors, have been shown to be highly polymorphic. The cytokine and chemokine gene polymorphisms are associated with variable production, activity, expression, or ligand-receptor affinity. The aim of our study was to analyze the relation between selected cytokine and chemokine gene polymorphisms in kidney donors and recipients as a function of donor-recipient match and posttransplantation outcome. Polymorphisms transforming growth factor-beta (TGF-beta); tumor necrosis factor-alpha (TNF-alpha); interleukin (IL)-6, and IL-10; monocyte chemoattractant protein-1 (MCP-1); and RANTES (regulated upon activation, normal T-cell expressed and secreted) genes were determined using DNA polymerase chain reaction technology in 268 healthy volunteers, 345 kidney transplant recipients (1997 to 1999), and 298 cadaveric donors. Patients were followed up for 4 to 6 years. The distribution of alleles of selected genes was identical in control subjects, cadaveric donors, and recipients. Low TGF-beta production in both the donor and recipient genotypes was associated with risk for early rejection (6 months) and worse graft function at 4 years. The only tendency for worse graft outcome was observed among donor-recipient combinations mismatched for TGF-beta genotype. Genetic determination of TNF-alpha and IL-10 production was associated with delayed graft function and rejection. IL-6 gene polymorphisms had no effect on the incidence of early acute rejections, but was associated with worse 5-year outcomes. Determinations of MCP-1 overproduction and RANTES-109 TT allele were associated with significant deterioration of graft function. Our data support the hypothesis that the strength of the alloimmune response after transplantation is in part genetically determined. Donor-recipient matching of cytokine gene polymorphisms has a marginal effect.

Quantification of donor-derived DNA in serum: a new approach of acute rejection diagnosis in a rat kidney transplantation model.

Clinical and laboratory findings of acute rejection (AR) are often late and misleading. Core needle biopsy, the most reliable diagnostic method, is usually performed late in the course of AR and is associated with several complications. Therefore noninvasive approaches to monitor the immune system for detection of early AR is one of the major aims in transplant medicine. In a fully MHC-mismatched renal allograft model in the rat, we quantified donor-derived DNA (ddDNA) in the recipient serum using real-time RT-PCR as an alternative screening procedure for the early diagnosis of acute rejection. We also investigated the influence of different immunosuppressive protocols on the levels of ddDNA. Our results show that donor-derived DNA is present in the serum of kidney allograft recipients prior to acute rejection. Animals that received a syngeneic graft and animals that received a mismatched allograft but were treated with immunosuppressive drugs did not show significant elevations of ddDNA. When steroid therapy failed to avoid acute rejection, the animals showed a delayed peak of ddDNA. In summary, the detection of ddDNA in recipient serum offers a noninvasive diagnostic approach to uncover ongoing rejection processes in the graft.

Significant reduction of proinflammatory cytokines by treatment of the brain-dead donor.

INTRODUCTION: Experimental studies suggest that brain death in the donor has a significant impact on graft quality; however, there are no data correlating organ-specific cytokine expression and the corresponding serum protein levels in human organ donors. Furthermore, it is unknown whether donor treatment can reduce the up-regulation of proinflammatory cytokines and thereby optimize organ quality. METHODS: We investigated the expression pattern of cytokines comparing serum (n = 53) and tissue expression (n = 25) in brain-dead human donors. The controls were living donors (n = 25). Additionally 41 deceased donors were treated with steroids before organ harvest (250 mg initial, afterward 100 mg/h until laparotomy). Hepatic tissue samples were obtained immediately after donor laparotomy to assess transcription rates of tissue cytokines (IL-6, IL-10, CD3, TGFb, TNFa, BAG, HO-1, Mipla) by RT-PCR. Serum samples were obtained after declaration of brain death and before laparotomy. RESULTS: Transcription of proinflammatory cytokines was significantly increased in brain-dead compared to living donor grafts (P < .005). Donor treatment with steroids led to significantly decreased tissue and serum expression of proinflammatory cytokines (P < .01), which were comparable to living donors. Tissue levels of cytokines (IL-6, IL-10) correlated strongly with serum levels of the corresponding proteins. CONCLUSIONS: Serum protein levels of proinflammatory cytokines proffer a valuable, easy accessible marker to define the immunological status of a graft. Our data suggest a beneficial effect of anti-inflammatory treatment of brain-dead organ donors.

Skin disease is prevented but nephritis is accelerated by multiple pregnancies in autoimmune MRL/LPR mice.

The role of pregnancy in the progression of systemic lupus erythematosus (SLE) is still poorly understood. We analysed the effect of repeated pregnancies in MRL/lpr mice, a murine model of SLE. Seven-week old female mice were used: multiparous mice underwent three consecutive pregnancies (M); age-matched virgin mice served as controls (V). Animals were harvested at 20 weeks of age. Skin lesions were characterized by hair loss and scabs in the dorsum of the neck. Virgin skins showed thickened dermis, fibrosis and mononuclear cell infiltrates, which were practically absent in M. This was accompanied by higher IFN-gamma and lower IL-10 mRNA expression levels in V compared to M skin. Plasma IFN-gamma protein levels were also upregulated in V versus M. However, survival and kidney function were dramatically reduced and accompanied by hypertension after multiple pregnancies. Kidney histology also showed markedly increased renal lesions in M. In contrast to plasma and skin levels, both IL-10 and IFN-gamma mRNA were lower in the kidneys of V versus M mice. Concluding our findings, the pathomechanisms of lupus kidney and skin disease may be regulated differently at the organ level during pregnancy. Both IFN-gamma and IL-10 may be important regulatory cytokines at the local level.

Heightened expression of the cytotoxicity receptor NKG2D correlates with acute and chronic nephropathy after kidney transplantation.

The activating cytotoxicity receptor NKG2D binds to stress-regulated molecules encoded by the major histocompatibility complex class I chain-related (MIC) and UL-16-binding protein (ULBP)/retinoic acid early transcript (RAET) gene family. To assess whether acute allograft rejection leads to an induction of these inducible ligands and their receptor NKG2D, we examined the mRNA profiles in kidney transplant biopsies. Expression levels were correlated with the incidence of acute rejection (aRx) episodes and chronic allograft nephropathy (CAN) proven by histology. Whereas MICA, ULBP1/3 and RAET1-E did not display heightened gene expression, elevated levels of NKG2D mRNA could be associated with aRx (p < 0.001). Immunohistology of kidney biopsies diagnosed with aRx revealed NKG2D+ cells in tubulointerstitial areas positive for CD8+ cells. Most importantly, elevated levels of NKG2D mRNA were associated with restricted long-term graft function assessed by the glomerular filtration rate at 6, 12 and 18 months posttransplantation. Induced NKG2D mRNA expression was still observable in biopsies diagnosed with CAN (p < 0.001), demonstrating a higher sensitivity and specificity compared to CD3, granzyme B and granulysin mRNA measurement. Significant elevated levels of NKG2D mRNA could be further detected in urine sediment prior to aRx, suggesting this receptor as a new candidate marker for the diagnosis of acute and chronic allograft rejection.

Brain death activates donor organs and is associated with a worse I/R injury after liver transplantation.

The majority of transplants are derived from donors who suffered from brain injury. There is evidence that brain death causes inflammatory changes in the donor. To define the impact of brain death, we evaluated the gene expression of cytokines in human brain dead and ideal living donors and compared these data to organ function following transplantation. Hepatic tissues from brain dead (n = 32) and living donors (n = 26) were collected at the time of donor laparotomy. Additional biopsies were performed before organ preservation, at the time of transplantation and one hour after reperfusion. Cytokines were assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and cytometric bead array. Additionally, immunohistological analysis of tissue specimens was performed. Inflammatory cytokines including IL-6, IL-10, TNF-alpha, TGF-beta and MIP-1alpha were significantly higher in brain dead donors immediately after laparotomy compared to living donors. Cellular infiltrates significantly increased in parallel to the soluble cytokines IL-6 and IL-10. Enhanced immune activation in brain dead donors was reflected by a deteriorated I/R injury proven by elevated alanin-amino-transferase (ALT), aspartat-amino-transferase (AST) and bilirubin levels, increased rates of acute rejection and primary nonfunction. Based on our clinical data, we demonstrate that brain death and the events that precede it are associated with a significant upregulation of inflammatory cytokines and lead to a worse ischemia/reperfusion injury after transplantation.

TGF-beta1 mRNA upregulation influences chronic renal allograft dysfunction.

Acute rejection (AR) is a dominant risk factor for developing chronic allograft nephropathy (CAN) after kidney transplantation. CAN is characterized by progressive interstitial fibrosis. It has been associated with increased transforming growth factor (TGF)-beta1 expression, however, kinetic studies are absent. We investigated whether intragraft TGF-beta1 expression in various causes of early graft dysfunction may influence late renal allograft dysfunction. A total of 174 human renal biopsies were quantified for TGF-beta1 mRNA expression using real-time reverse transcriptase-polymerase chain reaction. Expression levels were correlated with the Banff histopathological grades, TGF-beta1 immunohistology, and clinical follow-up. TGF-beta1 was most markedly upregulated in AR, CAN, and acute tubular necrosis - delayed graft function compared to non-rejecting controls (P < 0.001). TGF-beta1 expression was heightened in borderline changes (P < 0.01), recurrence of glomerulonephritis, and cyclosporine toxicity (P < 0.05). There was no correlation between intragraft TGF-beta1 expression during AR and short-term outcome of a rejection episode. TGF-beta1 gene overexpression during CAN has been shown to be associated with the increased risk for renal allograft dysfunction 18 months after biopsy (odds ratios 9.9 vs 3.2, respectively). Intragraft TGF-beta1 mRNA expression is significantly upregulated in both AR and CAN. Thus, our results support the hypothesis that TGF-beta1 might play a key role in chronic allograft dysfunction.

Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function.

The early identification of renal transplant recipients at enhanced risk of developing acute and subclinical rejection would allow individualized adjustment of immunosuppression before functional graft injury occurs and would exclude these patients from drug-weaning studies. Protein and reverse transcriptase-polymerase chain reaction-based analyses of candidate markers in urine open the opportunity to closely monitor kidney-transplanted patients non-invasively. The chemokine interferon-inducible protein 10 (IP-10; CXCL10) might be an interesting candidate to uncover ongoing immune processes within the graft. Urine samples from kidney-transplanted recipients were retrospectively analyzed for IP-10 mRNA and protein expression. IP-10 levels were correlated with the incidence of acute rejection episodes proven by histology and long-term graft function assessed by the glomerular filtration rate 6 months post transplantation. IP-10 expression in urine identified patients with ongoing acute rejection episodes several days before a biopsy was indicated by rising serum creatinine levels. Most importantly, elevated levels of urinary IP-10 protein within the first four postoperative weeks were predictive of graft function at 6 months even in the absence of acute rejection. These data reveal a correlation between elevated IP-10 expression in urine at early time points post-transplantation and intragraft immune activation that leads to acute rejection and compromised long-term graft function.

Improved long-term graft survival after HO-1 induction in brain-dead donors.

Brain death (BD) of the donor, a risk factor uniquely relevant for organs derived from cadaver donors, influences organ quality by induction of various inflammatory events. Consequently ischemia/reperfusion injury is deteriorated and acute and chronic rejections accelerated. Donor treatment might be an approach to improve the quality of the graft. The induction of heme oxygenase 1 (HO-1) has been shown to exert beneficial effects in living-donor transplantation models. Therefore, we examined the impact of donor treatment with the selective inducer of HO-1, cobalt protoporphyrin (CoPP), on organ quality and transplant outcome in a standardized BD model in a F344-->LEW kidney transplant rat model. Immediately after BD induction, donor animals were administered a single dose of CoPP (5 mg/kg) and in control groups, HO-1 activity was blocked with zinc protoporphyrin (ZnPP, 20 mg/kg). Recipients of organs from brain-dead donors treated with CoPP survived significantly better than those from untreated brain-dead donors (p < 0.05) and intra-graft analysis showed improved histology (p < 0.05). Blockade of HO-1 with ZnPP decreased the survival rates (p < 0.05) comparable to untreated brain-dead donors. Our results demonstrate that HO-1 induction by one single treatment of CoPP in brain-dead donors leads to enhanced allograft survival.

The noncoding regions of HLA-DRB uncover interlineage recombinations as a mechanism of HLA diversification.

The mechanisms generating new alleles at the MHC loci are still unknown in detail, and several proposals have been made to explain the extent of polymorphism. The patchwork pattern of polymorphism in the 2nd exon of HLA-DRB1 recommends this locus as a model for the study of the potential of interallelic gene conversion. In general, the inference of gene conversion-like events based exclusively on exon sequence comparisons may be misleading because the identity of the putative donor allele remains unknown. In this study, we describe five alleles of the HLA-DRB1 gene, which intron regions give evidence for interlineage recombination events either strictly located at the 2nd exon or involving the adjacent introns. Furthermore, we show that the noncoding regions provide important clues to the mechanisms of the generation of new alleles, and our results indicate that interlineage recombinations may be hidden and are perhaps more frequent than currently expected.

The nature of polymorphism of the HLA class I non-coding regions and their contribution to the diversification of HLA.

The sequence database of HLA class I genes is mainly derived from mRNA analysis. Little is known about the non-coding sequences of the different class I alleles. In this study we have determined the sequence of the 1st through 3rd introns of the majority of HLA-A and -B alleles. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic with a variability of 14.6% in the 1st intron decreasing to 6.2% in the 3rd intron. Against all expectations, this variability is not characterised by random point mutations but by a highly systematic diversity reflecting the ancestral relationship of the HLA alleles. The variability is arrested on the level of the serological diversity. The striking conservation within each ancestral lineage suggests that point mutations have been negatively selected. This finding could be explained by the evolutionary pressure on base order, promoting the potential to extrude single-strand stem-loops from supercoiled duplex DNA, which is believed to be important for combination. Moreover, the GC content was found to be as high as 78% in the 1st and 2nd introns and 55% in the 3rd intron. These CpG islands are directly involved in the exchange of short stretches of DNA in unequal crossing-over events. Additionally, conversion between different class I sequences is facilitated by regions of strong homology, stabilizing the pairing of variable regions. All these observations indicate the potential of a substantial contribution of introns to the recombinational activity of class I genes. The exclusive clustering of CpG islands in the 1st and 2nd introns restricts the gene conversion events to the regions of the 2nd and 3rd exons and therefore protects the conservation of the 5 flanking region and the 3 part of the gene. Since there are less diversification forces acting on introns they may be more conserved in a trans-species manner than exons. Therefore, they could provide the answer for the controversy regarding intra- or trans-species evolution.

Sequencing of HLA class II genes based on the conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes.

In this paper, we present a novel sequencing based typing strategy for the HLA-DRB1, 3, 4 and 5 loci. The new approach is based on a group-specific amplification from intron 1 to intron 2 according to the serologically-defined antigens. For this purpose, we have determined the 3' 500 bp-fragment of intron 1 and the 5' 340 bp-fragment of intron 2 of all serological antigens and their most frequent subtypes. We discovered a remarkably conserved diversity characterized by lineage-specific sequence motifs. This lineage-specificity of non-coding motifs in the 1st and 2nd intron offered the possibility to establish a clear serology-related amplification strategy. The method allows the complete analysis of the 2nd exon and the definition of the cis/trans linkage of sequence motifs by intron-mediated polymerase chain reaction (PCR)-based separation of the haplotypes in nearly all serologically heterozygous samples. In particular, the non-coding variabilities between the DR52-associated DRB1 groups made their independent amplification possible. Thus, compared to the standard procedures using exon-based amplification primers, the groups DR3, DR12, some DR13 alleles (1301, 1302) and the DR14 group could be amplified by specific primer mixes. The DR8 could be amplified with an individual primer mix not co-amplifying the DR12. The DR11 and DR13 did not show any individual motif in intron 1 or intron 2. In order to achieve a separate amplification, they had to be amplified by multispecific primer mixes (DR3/11/13/14; DR3/11/13 or DR11/13/14) excluding the other haplotype. Thus, exclusively the alleles in rare DR11,13 heterozygosities without a DRB1*1301 or 1302 could not be amplified separately. Fourteen primer mixes are used to amplify the specificities DR1-14, and 6 primer mixes for the specificities DR51-53. The sequence homology of the 3' end of intron 1 facilitated the application of only three different sequencing primers for all DRB alleles.

The nature of polymorphism of the HLA-DRB intron sequences is lineage specific.

The sequence database of HLA-DRB genes is mainly derived from mRNA analysis or has focused exclusively on the polymorphism of the 2nd exon. Little is known about the non-coding sequences of the different DRB alleles which represent about 94% of the genes. In this study we have determined the sequence of the 3' 500 bp intron 1 fragment adjacent to exon 2 in all serologically defined HLA-DRB genes and their most frequent allelic subtypes. The intron sequences turned out to be highly polymorphic. Similar to the class I introns, this variability was not characterized by random point mutations but by a highly systematic diversity reflecting the lineage-specific relationship of the HLA-DR alleles. With a few exceptions in DRBI*15, 13 and 08 as well as DRB4 and 5, the variability mirrors the serological diversity. As well as delivering insight into the genetic relationship between the different DRB alleles, these sequences will provide an extremely valuable basis for developing advanced DRB sequencing strategies for clinical purposes.

Sequencing of HLA class I genes based on the conserved diversity of the noncoding regions: sequencing-based typing of the HLA-A gene.

We present a sequencing-based typing strategy for the HLA-A locus that is generally applicable to all HLA class I genes. Sequencing-based typing is the method of choice for matching in unrelated bone marrow transplantation on the allelic level. We determined the noncoding sequences of all serological antigens and most of their subtypes and discovered a remarkably conserved diversity characterized by polymorphic sequence motifs. In this study we took advantage of this diversity we uncovered in the 5' flanking region, 5' untranslated region and in the introns 1, 2 and 3, which was related to serological families. We established 12 primer mixes for setting up a PCR-based template preparation. Our strategy is based on the separate amplification of haplotypes and therefore defines the cis/trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples heterozygous for the broad antigens as well as the complete analysis of the polymorphic exons 2 and 3. All templates included the 2nd intron which was used as a priming site for the gene-specific 5' and 3' universal sequencing primers regardless of the amplified haplotypes. The independent sequencing of the haplotypes allows the application of the dye terminator cycle sequencing technique, which is less time-consuming and less-laborious than dye primer chemistry. The lack of heterozygous positions essentially facilitates on the one hand the data analysis and on the other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. The results will remain unambiguous regardless of a growing HLA sequence data bank since this sequencing technique defines the cis/trans linkage of sequence motifs in more than 95% of the cases.