Gerald Schlaf

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-HT1AR 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-HT1AR activation stabilizes the rhythmicity of breathing during opiate medication of pain.

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.

Determination of synapsin I and synaptophysin in body fluids by two-site enzyme-linked immunosorbent assays

Two-site enzyme-linked immunosorbent assays (ELISA) have been established for the specific and sensitive determination of two membrane proteins of the small synaptic vesicles (SSV), namely: peripheral synapsin I and integral synaptophysin. The ELISA used highly specific capture monoclonal antibodies (mAB) and polyclonal antibodies (pAB) as detectors. For synapsin I, the mAB were newly generated, whereas for synaptophysin, the commercially available mAB SY38 was applied. In order to calibrate the ELISA and to raise pAB, both proteins were purified in the mg-range. Synapsin I was purified by conventional means from human and porcine brain and synaptophysin was purified by immunoaffinity chromatography from porcine brain. Using the ELISA, neither synapsin I nor synaptophysin could be determined in serum or cerebrospinal fluid (CSF) from healthy donors or patients suffering various neurological disorders or pheochromocytomas. For this reason, the degradation of both proteins in serum and CSF was investigated. With the exception of synaptophysin measured in serum, both proteins exhibited fast rates of degradation. Despite the negative results in human body fluids, the two ELISA are appropriate for the quantification of these membrane proteins in neuronal or neuroendocrine cell extracts or preparations of SSV.

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-α, IL-1β, and IFN-γ, only IFN-γ 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-γ, 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 μg LPS/ml did not show any effect on HC. Our data suggest that LPS does not regulate the expression of FH in HC.

Artificially Positive Crossmatches Not Leading to the Refusal of Kidney Donations due to the Usage of Adequate Diagnostic Tools

Allografting patients with human leukocyte antigens (HLA) which are recognized by preformed antibodies constitutes the main cause for hyper-acute or acute rejections. In order to select recipients without these donor-specific antibodies, the complement-dependent cytotoxicity crossmatch (CDC-CM) assay was developed as a standard procedure about forty years ago. The negative outcome of pretransplant crossmatching represents the most important requirement for a successful kidney graft survival. The artificially positive outcomes of CDC-based crossmatches due to the underlying disease Systemic Lupus Erythematosus (SLE), however, may lead to the unjustified refusal of adequate kidney grafts. Two prospective female recipients destined for a living as well as for a cadaver kidney donation, respectively, exhibited positive CDC-based crossmatch outcomes although for both patients no historical immunizing events were known. Furthermore, solid phase-based screening or antibody differentiation analyses never led to positive results. Immediate reruns of the CDC-based crossmatch assays using the alternative antibody monitoring system (AMS-)crossmatch ELISA resulted in unequivocally negative outcomes. Consequently both transplantations were performed without any immunological complications for the hitherto follow-up time of 25 and 28 months, respectively. We here show two case reports demonstrating an alternative methodical approach to circumvent CDC-based artefacts and point to the urgent need to substitute the CDC-based crossmatch procedure at least for special groups of patients.

A novel HLA-B*57 (B*5714) allele in a healthy male Caucasian individual.

A novel human leucocyte antigen (HLA)-B57 (HLA-B*5714) allele has been identified in a male Caucasian individual from Middle Europe using single allele-specific sequencing strategy. This allele is identical to the HLA-B*570101 allele except for two point mutations in exon 3 at codon 138 (ACG-->ACC) with no amino acid change [persisting threonine (T)] and at codon 171 (TAC-->CAC), resulting in an amino acid change from tyrosine (Y) to histidine (H).

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 D2 and E2 and of thromboxane A2 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).

A novel HLA-A*29 allele (A*2916) of a Caucasian relative to an ALL patient containing a point mutation in a putatively conserved region which was identified through the loss of a specific amplificate in a low-resolution SSP-PCR kit

A novel HLA‐A29 (HLA‐A*2916) allele has been identified in a Caucasian man from Middle Europe using single‐allele‐specific sequencing strategy. This allele is identical to the HLA‐A*29010101 allele except for one point mutation in the putatively conserved codon 128 (GAG→CAG) resulting in an amino acid exchange from glutamic acid (E) to glutamine (Q).

Systemic Lupus Erythematosus Leading to Terminal Renal Failure and Excluding Patients from Kidney Allocation Due to Inadequate CDC-based Cross-Matching: Is There a Diagnostic Way out?

Systemic lupus erythematosus (SLE) is known to proceed to clinically relevant nephritis in more than 50% of patients and, in about 20% of these patients, to terminal renal failure. Thus, renal replacement therapy including kidney allografting is required for a considerable number of SLE-patients. For allografting patients’ donor-specific antibodies against HLA molecules of given donors (DSA) have to be excluded as preformed antibodies against these molecules represent the main cause for hyper-acute or acute rejections. In order to select recipients without these deleterious antibodies the complement-dependent cytotoxicity crossmatch (CDC-XM) assay was developed about forty years ago. Its negative pre-transplant outcome is currently regarded as the most important requirement for a successful kidney graft survival. During the last years several disadvantages of the CDC-based procedure have increasingly been discussed with respect to this assay’s high susceptibility to disruptive factors mainly leading to false positive outcomes. As is clearly shown by our case series this holds also true for the underlying disease SLE. We here present the data of SLE-patients initially destined for cadaver kidney donations. They all exhibited positive CDC-XM outcomes for the most part without known historical immunizing events. Furthermore, solid phase-based antibody screening and specification analyses did generally not show anti-HLA antibodies or antibodies directed against HLA-phenotypes of the corresponding donors thus leading to negative results of the so-called virtual crossmatch. Since exclusive virtual cross-matching is not allowed for the acceptance of allografting all of the positive CDC-XM assays were performed as reruns using alternative solid phase- (ELISA-) based crossmatch assays. In best accordance with virtual cross-matching solid phase-based cross-matching did not exhibit DSA. Our data clearly demonstrate the benefit of alternative ELISA-based cross-matching to circumvent CDC-based artefacts and point on the urgent requirement to substitute the historical CDC-based procedure at least for this group of patients.