Solid phase-based cross-matching for solid organ transplantation: Currently out-of-stock but urgently required for improved allograft outcome.

Transplant recipients who have undergone sensitizing events, such as pregnancy, blood transfusion or previous transplants, frequently develop antibodies directed against the highly polymorphous human leukocyte antigen (HLA)-molecules. These pre-formed, donor-specific antibodies (DSA) present a high risk of causing organ failure or even complete loss of the grafted organ as a consequence of antibody-mediated, hyper-acute or acute allograft rejection. In order to detect DSA, the so-called functional complement-dependent lymphocytotoxicity assay (CDC-XM) was established about 50 years ago. Although effective in improving the outcome of solid organ allo-grafting, for the last ten years this assay has been controversially discussed due to its low sensitivity and especially because of its high susceptibility to various artificial factors, which generally do not yield reliable results. As a consequence, novel immunochemical test systems have been developed using ELISA- or bead-based solid phase assays as replacements for the traditional CDC-based assays. Because these assays are independent of single or vital cells, which are frequently not available, they have provided an additional and alternative diagnostic approach compared with the traditional CDC-based and flow-cytometric analyses. Unfortunately, however, the AMS-ELISA (Antibody Monitoring System), which was the first system to become commercially available, was recently discontinued by the manufacturer after seven years of successful use. Alternative procedures, such as the AbCross-ELISA, had to be either considerably modified, or did not yield reliable results, as in the case of the Luminex-based assay termed DSA. We draw the conclusion that due to the unique features and fields of application reviewed here, the implementation of solid phase cross-matching still represents an urgent requirement for any HLA-laboratory's routine tasks.

Is periodontitis a prognostic factor in order to indicate antibodies against citrullinated peptides in patients with rheumatoid arthritis?

OBJECTIVES: In this cross-sectional study we investigated antibody titres against cyclic citrullinated peptides derived from filaggrin (anti-CCP) and citrullinated α-enolase (anti-CEP-1) among patients with RA as a function of periodontal findings. METHODS: 107 patients with RA (median age 56 years, 75% females) were included. For periodontal diagnoses missing teeth, periodontal epithelial surface area, periodontal inflamed surface area and periodontal diagnosis according to the working group's guidelines of the Center for Disease Control and Prevention were determined. Subgingival bacterial DNA of five periodontopathic bacteria was assessed by PCR with sequence-specific oligonucleotides. Anti-CCP and anti-CEP-1 antibodies in plasma samples were investigated using enzyme-linked immunosorbent assays. Low resolution human leukocyte antigen (HLA) typing was carried out using PCR with sequence-specific primers. RESULTS: PESA was found associated with a low adjusted odds ratio for anti-CCP positivity (OR=1.002, p=0.040). All patients who were infected with Aggregatibacter actinomycetemcomitans were simultaneously anti-CCP positive (p=0.043). HLA-DRB1*13 lowered the adjusted odds ratio for anti-CCP (OR=0.073, p=0.002) and anti-CEP-1 (OR=0.068, p=0.018) positivity whereas HLA-DRB1*07 indicated a lower risk only for demonstrable anti-CCP antibodies (OR=0.079, p=0.004). HLA-DRB1*04 was associated with increased adjusted odds ratio for anti-CEP-1 positivity (OR=4.154, p=0.005) and the simultaneous proof of both investigated autoantibodies (OR=3.725, p=0.011). CONCLUSIONS: Among patients with RA periodontitis may be a minor risk factor for anti-CCP positivity. Our data first provide evidence that an infection with A. actinomycetemcomitans is associated with an increased formation of anti-CCP. HLA phenotype proved to be a significant risk indicator for both investigated antibodies.

Differences in the frequencies of HLA-class I and II alleles between German patients with renal cell carcinoma and healthy controls.

The human leukocyte antigen (HLA) system is a major part of the human immune system and has an impact on tumor initiation, tumor progression, and immunosurveillance. Renal cell carcinoma tumors are considered to be immunogenic. Therefore, we studied the allele frequencies of four gene loci (HLA-A, -B, -C, and HLA-DR) in a cohort of German renal cell carcinoma (RCC) patients and in healthy controls. HLA-A-C were determined using serological methods, whereas HLA-C12, C14, C16, C18, and HLA-DR were characterized through the use of standard molecular biological methods. The occurrence of the HLA-C*12 allele was significantly increased in German RCC patients compared with healthy controls (P < 0.005; Fisher's exact test), whereas the occurrence of the HLA-DRB1*04 allele was significantly reduced in RCC patients compared with healthy controls (P < 0.05; Fisher's exact test). However, the presence of allele HLA-C*12 was not significantly associated with 10 year overall survival. We suggest that the frequency of HLA alleles can affect development of RCC and could add knowledge as predictive marker for future immunotherapies.

Solid phase-based cross-matching as solution for kidney allograft recipients pretreated with therapeutic antibodies.

In order to select recipients without donor-specific anti-HLA antibodies, the complement-dependent cytotoxicity crossmatch (CDC-CM) was established as the standard procedure about 40 years ago. However, the interpretability of this functional assay strongly depends on the vitality of isolated donors' lymphocytes. Since the application of therapeutic antibodies for the immunosuppressive regimen falsifies the outcome of the CDC-crossmatch as a result of these antibodies' complement-activating capacity in the recipients' sera, we looked for an alternative methodical approach. We here present 27 examples of AB0 blood group-incompatible living kidney allograft recipients who, due to their treatment with the humanized chimeric monoclonal anti-CD20 antibody Rituximab, did not present valid outcomes of CDC-based pretransplant cross-matching. Additionally, four cases of posttransplant cross-matching after living kidney allografting and consequent treatment with the therapeutic anti-CD25 antibody Basiliximab (Simulect) due to acute biopsy-proven rejection episodes are presented and compared regarding CDC- and ELISA-based crossmatch outcomes. In all cases, it became evident that the classical CDC-based crossmatch was completely unfeasible for the detection of donor-specific anti-HLA antibodies, whereas ELISA-based cross-matching not requiring vital cells was not artificially affected. We conclude that ELISA-based cross-matching is a valuable tool to methodically circumvent false positive CDC-based crossmatch results in the presence of therapeutically applied antibodies.

Sensitive solid-phase detection of donor-specific antibodies as an aid highly relevant to improving allograft outcomes.

Transplant recipients who have had sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donor tissue. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. As a first assay to detect DSA, the complement-dependent lymphocytotoxicity assay (CDC) was established more than 40 years ago. However, this assay is characterized by several drawbacks such as a low sensitivity and a high susceptibility to various artificial factors generally not leading to valid and reliable outcomes under several circumstances that are reviewed in this article. Furthermore, only those antibodies that exert complement-fixing activity are detected. As a consequence, novel procedures that act independently of the complement system and that do not represent functional assays were generated in the format of solid phase assays (SPAs) (bead- or ELISA-based). In this article, we review the pros and cons of these sensitive SPA in comparison with the detection of DSA through the use of the traditional methods such as CDC and flow cytometric analyses. Potential drawbacks of the alternative methodological approaches comprising high background reactivity, susceptibility to environmental factors and the possible influence of subjective operators' errors concerning the interpretation of the results are summarized and critically discussed for each method. We provide a forecast on the future role of SPAs reliably excluding highly deleterious DSA, thus leading to an improved graft survival.

A novel ELISA-based crossmatch procedure to detect donor-specific anti-HLA antibodies responsible for corneal allograft rejections.

Previous studies had shown that donor-specific anti-HLA antibodies may highly influence the survival rate of corneal allografts, although the anterior chamber generally represents an immune-privileged compartment of the eye. We postulated that the introduction of a novel crossmatch procedure for the detection of donor-specific anti-HLA antibodies in recipients awaiting a corneal graft would be adequate to investigate their influence on the outcome of the graft survival. The Antibody Monitoring System (AMS) HLA class I & II crossmatch ELISA was adapted for the use of material from the outer scleral rim instead of blood lymphocytes to isolate the donors' HLA molecules. In case of detectable donor-specific anti-HLA class I and/or class II antibodies (DSA) this result was confirmed using an identification ELISA to specify the detectable recipient's anti-HLA antibodies. PCR-based genetic tissue typing of the donors was performed also using their outer scleral rims. 45 recipients of corneal grafts were analyzed for DSA prior to or after grafting, respectively. 75% of the recipients with preformed DSA exhibited immunological complications up to the complete graft loss in four cases during the first two months. In contrast 77% of the recipients without DSA did not show any complications during the follow up period of averagely 18months. Only two cases of graft loss were observed in this group after 17 and 23months, respectively. The results demonstrate the impact of preventing donor-specific anti-HLA antibodies which are for the first time reliably detectable in any laboratory's daily work using the adapted AMS-ELISA.

Hodgkin's lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses.

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.

Novel solid phase-based ELISA assays contribute to an improved detection of anti-HLA antibodies and to an increased reliability of pre- and post-transplant crossmatching.

Antibodies directed against HLA antigens of a given organ donor represent the dominating reason for hyper-acute or acute allograft rejections. In order to select recipients without donor-specific antibodies, a standard crossmatch (CM) procedure, the complement-dependent cytotoxicity assay (CDC), was developed. This functional assay strongly depends on the availability of isolated vital lymphocytes of a given donor. However, the requirements of the donor's material may often not be fulfilled, so that the detection of the antibodies directed against HLA molecules is either impaired or becomes completely impossible. To circumvent the disadvantages of the CDC procedure, enzyme-linked immunosorbent assay (ELISA)-based and other solid phase-based ELISA-related techniques have been designed to reliably detect anti-HLA antibodies in recipients. Due to the obvious advantages of these novel technologies, when compared with the classical CDC assay, there is an urgent need to implement them as complementary methods or even as a substitution for the conventional CDC crossmatch that is currently being applied by all tissue typing laboratories.

Serotonin targets inhibitory synapses to induce modulation of network functions.

The cellular effects of serotonin (5-HT), a neuromodulator with widespread influences in the central nervous system, have been investigated. Despite detailed knowledge about the molecular biology of cellular signalling, it is not possible to anticipate the responses of neuronal networks to a global action of 5-HT. Heterogeneous expression of various subtypes of serotonin receptors (5-HTR) in a variety of neurons differently equipped with cell-specific transmitter receptors and ion channel assemblies can provoke diverse cellular reactions resulting in various forms of network adjustment and, hence, motor behaviour. Using the respiratory network as a model for reciprocal synaptic inhibition, we demonstrate that 5-HT(1A)R modulation primarily affects inhibition through glycinergic synapses. Potentiation of glycinergic inhibition of both excitatory and inhibitory neurons induces a functional reorganization of the network leading to a characteristic change of motor output. The changes in network operation are robust and help to overcome opiate-induced respiratory depression. Hence, 5-HT(1A)R activation stabilizes the rhythmicity of breathing during opiate medication of pain.

The role of C5a in the innate immune response after experimental blunt chest trauma.

The inflammatory response after severe blunt chest trauma often leads to acute lung injury and acute respiratory distress syndrome which are associated with high mortality rates. Whereas the role of innate immunity in acute lung injury has been broadly investigated, the immune response after blunt chest trauma is still poorly understood. Therefore, the role of complement and neutrophils was determined in bilateral lung injury induced by a single blast wave. The following time-points were investigated posttrauma: sham, 1, 6, 12, and 24 h. There was a time-dependent systemic activation of complement as determined by CH-50 and presence of C5a-dependent chemotactic plasma activity. Moreover, factor H, a complement regulatory protein, was increased systemically and locally after injury. Anti-C5a treatment immediately after trauma ameliorated these peaks. After an initial systemic leukopenic phase, a marked leukocytosis occurred. The latter was normalized by C5a blockade. In parallel, white blood cell count in bronchioalveolar lavage fluids was increased as a function of time and was significantly decreased by anti-C5a treatment. Trauma-induced lung injury was also associated with dramatic changes in neutrophil function, namely early enhanced chemotaxis and phagocytosis, followed by prolonged functional defects-all of which were ameliorated by anti-C5a treatment. Furthermore, blockade of C5a ameliorated the buildup of the proinflammatory cytokine TNF-alpha, diminished the increase of cytokine-induced neutrophil chemoattractant 1, and altered the levels of the anti-inflammatory cytokine IL-10. These data suggest that blunt chest trauma leads to systemic activation of complement and robust C5a generation, which causes perturbations in defensive functions of neutrophils. Thus, C5a might represent a potential target for therapeutic immunomodulation to prevent immune dysfunctions post-trauma and thereby, perhaps, the progression to acute respiratory distress syndrome.

Structure, expression and function of HLA-G in renal cell carcinoma.

Tumors have developed different strategies to escape from immune cell recognition which include the downregulation or loss of the classical HLA class I antigens as well as aberrant expression of non-classical HLA antigens like HLA-G. Abnormalities in MHC class surface expression have also been described in renal cell carcinoma (RCC) and represent mechanisms to avoid elimination by immune effector cells. We here review the structure/polymorphism, mRNA and protein expression profile of HLA-G in RCC and corresponding normal kidney epithelium, its mode of regulation and its functional consequences on immune responses. A heterogeneous constitutive HLA-G mRNA and/or protein expression was found in both RCC lesions and RCC cell lines, whereas normal kidney epithelium totally lack HLA-G mRNA and protein expression. In comparison to other tumor entities, the frequency of HLA-G expression is relatively high in RCC. Since HLA-G expression is lost during cultivation of RCC cells, the tumor microenvironment and/or endothelium appear to be involved in the regulation of HLA-G expression in this disease. HLA-G expression could be transcriptionally upregulated in RCC by interferons, IL-10 and gangliosides. Silencing of HLA-G expression in RCC is often associated with methylation of the HLA-G promoter which could be reverted by the treatment with demethylating agents. Functional studies using natural killer cells, lymphokine activated killer cells as well as antigen-specific CD8+ cytotoxic T lymphocytes demonstrated that HLA-G expression prevents lysis of RCC cells by these different immune effector cells. In contrast, HLA-G-negative normal kidney cells as well as HLA-G-negative RCC cells were not recognized by NK and T cells. Thus, HLA-G represents one important immune escape mechanism of human RCC which has an impact on the design of T and NK cell-based immunotherapies in this disease.

High variation of individual soluble serum CD30 levels of pre-transplantation patients: sCD30 a feasible marker for prediction of kidney allograft rejection?

BACKGROUND: Previous studies have suggested that the pre-transplant levels of the soluble CD30 molecule (sCD30) represent a non-invasive tool which can be used as a biomarker for the prediction of kidney allograft rejections. METHODS: In order to evaluate the feasibility of sCD30 for pre-transplantation monitoring the sera of potential kidney recipients (n = 652) were collected four times in a 3 months interval. Serum from healthy blood donors (n = 203) served as controls. The sCD30 concentrations of all samples were determined using a commercially available ELISA. This strategy allowed the detection of possible variations of individual sCD30 levels over time. RESULTS: Heterogeneous sCD30 concentrations were found in the samples obtained from individual putative kidney transplant recipients when quarterly measured over 1 year. Total 95% of serum samples obtained from healthy controls exhibited sCD30 values <30 U/ml, whereas most recipients displayed higher serum levels (>30 U/ml). Total 524 patients (80.4%) constantly exhibited serum concentrations of <100 U/ml during the period investigated, whereas 109 patients (16.7%) showed variations by exceeding the proposed 'cut off' of 100 U/ml for one to three times. The frequency of samples exhibiting sCD30 values >100 U/ml was significantly lower than that previously reported. CONCLUSIONS: The high degree of variation does not allow the stratification of patients into high and low immunological risk groups based on a single sCD30 value > 100 U/ml. Due to the heterogeneity of sCD30 levels during time course and the high values of SD, its implementation as a pre-transplant marker cannot be justified to generate special provisions for the organ allocation to patients with single sCD30 values > 100 U/ml.

C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cells.

The 155-kDa complement regulator factor H (FH) is the predominant soluble regulatory protein of the complement system. It acts as a cofactor for the factor I-mediated conversion of the component C3b to iC3b, competes with factor B for a binding site on C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. The primary site of synthesis is the liver, i.e. FH-specific mRNA and protein were identified in both hepatocytes (HC) and Kupffer cells (KC). Previous studies in rat primary HC and KC had shown that the proinflammatory cytokine IFN-gamma influences the balance between activation and inhibition of the complement system through up-regulation of the inhibitory FH. In this study we show that C5a, as a product of complement activation, stimulates the expression of FH-specific mRNA and protein in KC and thus induces a negative feedback. Quantitative-competitive RT-PCR showed an approximate threefold C5a-induced up-regulation of FH. ELISA analyses revealed a corresponding increase in FH protein in the supernatants of KC. The up-regulation of FH was completely inhibited by the C5a-blocking monoclonal antibody 6-9F. Furthermore, an involvement of LPS and IFN-gamma was excluded, which strongly indicates a direct effect of C5a on the expression of FH in KC.

Differences in the involvement of prostanoids from Kupffer cells in the mediation of anaphylatoxin C5a-, zymosan-, and lipopolysaccharide-dependent hepatic glucose output and flow reduction.

Various inflammatory stimuli such as anaphylatoxin C5a, zymosan, and lipopolysaccharides (LPSs) have been reported both to enhance glucose output in the perfused rat liver and to induce prostanoid (ie, prostaglandin and thromboxane) release from Kupffer cells, the resident liver macrophages. Because prostanoids can enhance glucose output from hepatocytes, it was the aim of this study to compare the possible roles of prostanoids released after C5a, zymosan, and LPS in the mediation of hepatic glucose output. In perfused livers both C5a and zymosan immediately enhanced glucose output, reduced flow, and induced prostanoid overflow into the hepatic vein, but with different quantities and kinetics. Only the C5a-induced but not the zymosan-induced effects were abrogated by inhibitors of prostanoid signaling as the prostanoid synthesis inhibitor indomethacin and the thromboxane receptor antagonist daltroban. In contrast to C5a and zymosan, LPS had no effect on glucose output, flow rate, or prostanoid overflow. In isolated Kupffer cells, C5a and zymosan induced maximal release of prostaglandins D(2) and E(2) and of thromboxane A(2) within a period of 0 to 2 minutes and 5 to 15 minutes, respectively. In pulse-chase experiments, maximal prostanoid release was already observed after 2 minutes of continuous stimulation with C5a, but only after 10 to 15 minutes of continuous stimulation with zymosan. LPS-dependent prostanoid release was not seen before 1 hour. Thus, even though C5a, zymosan, and LPS induced prostanoid release from Kupffer cells, only C5a quickly regulated hepatic glucose metabolism in a prostanoid-dependent manner (due to the kinetics and quantities of prostanoids released).

Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: mediation by interleukin-6 from Kupffer cells.

BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.

Constitutive expression and regulation of rat complement factor H in primary cultures of hepatocytes, Kupffer cells, and two hepatoma cell lines.

The 155-kd soluble complement regulator factor H (FH), which consists of 20 short consensus repeats, increases the affinity of complement factor I (FI) for C3b by about 15 times. In addition to its cofactor activity, it prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. The primary site of synthesis of FH, as well as of FI, is the liver, but the cell types responsible for the hepatic synthesis of both factors have not yet been clearly identified. In contrast to FI-mRNA, which was detectable only in hepatocytes (HC), FH-specific mRNA was identified in both HC and Kupffer cells (KC). As calculated for equal amounts of mRNA isolated from both cell types, FH-specific mRNA was found to be nearly 10-fold higher in KC than in HC, leading to the conclusion that KC are an abundant source of FH. Of the investigated proinflammatory cytokines IL-6, TNF-alpha, IL-1beta, and IFN-gamma, only IFN-gamma up-regulated FH-specific mRNA up to 6-fold in both primary HC and KC. This was also demonstrable on the protein level. However, FH-specific mRNA was not inducible in the rat hepatoma cell line H4IIE, which did not express FH-specific mRNA and could not be up-regulated in FAO cells that constitutively expressed FH-specific mRNA. This demonstrates that transformed cell lines do not reflect FH regulation in isolated primary HC. In addition to IFN-gamma, the endotoxin lipopolysaccharide (LPS) up-regulated FH-specific mRNA nearly 10-fold in KC after stimulation at concentrations of 10 or 1 ng/ml. In contrast, concentrations of up to 2 microg LPS/ml did not show any effect on HC. Our data suggest that LPS does not regulate the expression of FH in HC.