Alexandra Berger

Tachykinins in the immune system.

Until recently, the mammalian tachykinins included substance P, neurokinin A and neurokinin B. Following the discovery of the fourth member of this family, hemokinin 1, a diverse group of novel tachykinins and tachykinin gene-related peptides have been identified in mammals. These newly identified members are preferentially expressed in peripheral tissues. Currently, the impact of these new tachykinin peptides on the immune system remains unclear. Some data imply an important role for hemokinin 1 in the generation of lymphocytes. Tachykinins are traditionally viewed as neuropeptides with well-defined functions as neurotransmitters. Many studies however, indicate that they may also be produced by non-neuronal cells, and exert profound influence on inflammatory responses by affecting multiple aspects of immune cell function. It is of great importance to determine whether the new tachykinin peptides have similar effects. A more detailed understanding of the interactions between tachykinins and immune cells may provide the basis for the development of new therapies for inflammatory and immune-mediated diseases.

Severe depletion of mitochondrial DNA in spinal muscular atrophy

Abstract. Spinal muscular atrophy (SMA) is a neuromuscular disorder in childhood leading to a dramatic loss of muscle strength. Functional investigations with high-resolution polarography and enzyme measurements of the respiratory chain revealed lowered activities in muscle tissue of SMA patients. To gain a better understanding of this low energy supply we analyzed the amount of mitochondrial DNA (mtDNA) in skeletal muscle of 20 unrelated children with genetically proven SMA and 31 controls. Quantitative Southern blot analysis revealed a severe and homogeneous decrease in the content of muscle mtDNA in relation to nuclear DNA in SMA patients (90.3±7.8%), whereas by immunofluorescence no decrease in the number of mitochondria was detected. In addition, a two- to threefold reduction of the nuclear-encoded complex II (succinate dehydrogenase) activity was detected in SMA muscle tissue. Western blot analysis showed a significant reduction of both mitochondrial- and nuclear-encoded cytochrome c oxidase subunits. Our results indicate that mtDNA depletion in SMA is a consequence of severe atrophy, and has to be differentiated by measurement of complex II from an isolated reduction of mtDNA as found in patients with mitochondriocytopathies and the so-called mtDNA depletion syndrome.

Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists

The hypothalamic galanin-like peptide (GALP) was isolated by its ability to activate galanin receptors. The mature porcine GALP is a 60-amino acid neuropeptide proteolytically processed from a 120-amino acid precursor protein. It contains a region identical to the N-terminal 13-amino acids of the neuropeptide galanin. Within the sequence of human GALP (1-60) a potential proteolytic cleavage site between two basic amino acids is present at position 33, which might lead to a shorter C-terminally amidated peptide. In addition, the first two amino acids could be potentially removed via the action of dipeptidase IV. Ligand binding assays using the human neuroblastoma cell line SH-SY5Y transfected with the respective galanin receptors revealed that human GALP (1-60) displayed the highest affinity for the galanin receptor subtype GalR3 (IC50 = 10 nM) followed by GalR2 (IC50 = 28 nM) and GalR1 (IC50 = 77 nM). Ligand binding assays and functional studies showed that the human GALP (3-32) fragment was at least as potent as full length GALP (1-60). Other studies have shown that shorter fragments like human GALP (1-21) and GALP (22-60) were not effective on feeding responses in mice as compared to the full length peptide. Taken together these data suggest that the putative fragment GALP (3-32) might represent the strongest mediator of biological GALP activity. Furthermore it might be a useful tool to study the affinity of GALP to galanin receptors and to search for specific GALP receptors.

Inflammation rapidly reorganizes mouse bone marrow B cells and their environment in conjunction with early IgM responses

Systemic inflammation perturbs the bone marrow environment by evicting resident B cells and favoring granulopoiesis over lymphopoiesis. Despite these conditions, a subset of marrow B cell remains to become activated and produce potent acute immunoglobulin M (IgM) responses. This discrepancy is currently unresolved and a complete characterization of early perturbations in the B-cell niche has not been undertaken. Here, we show that within a few hours of challenging mice with adjuvant or cecal puncture, B cells accumulate in the bone marrow redistributed away from sinusoid vessels. This response correlates with enhanced sensitivity to CXC chemokine ligand 12 (CXCL12) but not CXCL13 or CC chemokine ligand 21. Concurrently, a number of B-cell survival and differentiation factors are elevated to produce a transiently supportive milieu. Disrupting homing dynamics with a CXC chemokine receptor 4 inhibitor reduced the formation of IgM-secreting cells. These data highlight the rapidity with which peripheral inflammation modifies the marrow compartment, and demonstrate that such modifications regulate acute IgM production within this organ. Furthermore, our study indicates that conversion to a state of emergency granulopoiesis is temporally delayed, allowing B cells opportunity to respond to antigen.

Galanin and galanin receptors in human cancers

The increasing interest in peptides and peptide receptors in cancer is based on the possibility of receptor targeting, because peptide receptors are often expressed in different human tumors. The neuropeptide galanin has also been suggested to be involved in the development of neuroendocrine tumors based on the development of estrogen-induced tumors in estrogen-sensitive rats. This study summarizes our current knowledge on the expression of galanin peptide and galanin receptors in different human neuroendocrine tumors. The expression of both, peptide and corresponding receptor, seems to be a common feature of human gliomas, pheochromocytomas, pituitary and neuroblastic tumors. The co-expression of galanin and its receptors supports a role for galanin in tumor cell pathology via autocrine/paracrine mechanisms.

Hemokinin-1 has Substance P-like function in U-251 MG astrocytoma cells: a pharmacological and functional study.

Substance P (SP) triggers responses in astrocytoma cells, which are considered important for proliferation and neuroimmunomodulatory activity. In this study, we compared the effects of SP with those of the novel tachykinin Hemokinin-1 (HK-1) in the human astrocytoma cell line U-251 MG. We show that U-251 MG cells express high levels of Neurokinin-1 (NK-1) receptors. The binding affinities of 125I-SP and 125I-mHK-1 to these receptors were in a similar, subnanomolar range. HK-1 and SP stimulated Ca2+ mobilization and induced increased cytokine mRNA expression. A specific NK-1 receptor antagonist blocked the observed effects. We conclude that there are no qualitative differences in SP and HK-1-evoked responses, suggesting that both peptides act through NK-1 receptors in U-251 MG cells. Moreover, we show TAC4 mRNA expression in gliomas, indicating a possible involvement of HK-1 in glioma biology.

Elevated Expression of Galanin Receptors in Childhood Neuroblastic Tumors

The neuropeptide galanin (GAL) has been shown to be present in certain brain tumors. In order to learn more about GAL and its receptors in human tumors of the peripheral nervous system, we investigated the expression of the GAL peptide and the GAL receptors in tumor tissue from childhood neuroblastic tumors. GAL peptide concentrations up to 674 ± 166 fmol/mg of tissue were detected by radioimmunoassay, but no significant correlation with standard tumor markers or the prognosis of the 14 patients investigated was observed. Ligand binding experiments showed different levels of GAL binding in all 28 primary neuroblastomas and 7 ganglioneuromas investigated. All three human GAL receptor subtypes cloned to date could be detected, with the GALR1 receptor subtype being expressed most prominently. GAL binding did not significantly correlate with genetic markers such as unfavorable DNA ploidy, amplification of the oncogene MYCN and allelic loss of chromosome 1p. However, low galanin binding was significantly correlated with survival (p = 0.021) in this limited analysis of neuroblastic tumor samples. These results raise the possibility that the expression of GAL binding sites may play a role in neuroblastic tumor biology and behavior.

Galanin and galanin receptors in human gliomas.

Galanin-like immunoreactivity (GAL-LI) and specific GAL binding sites have been shown to be widely distributed in the central nervous system (CNS) and in CNS tumors. GAL and its receptors have also been shown to be present in glial cells, but to date it is still unknown whether human gliomas produce GAL and express GAL receptors. In this study 20 brain tumors consisting of 15 glioblastomas, 4 meningiomas and 1 gliosarcoma were investigated for the presence of GAL-LI and GAL receptors. Immunofluorescence analysis revealed a dense network of GAL-LI positive cellular processes and cell bodies in 18 of the 20 tumors. In contrast, in vitro 125I-labeled GAL receptor autoradiography showed substantial GAL binding in only 6 glioblastoma tissues. Reverse transcription-PCR analysis detected mRNA of all three known galanin receptors in the tumor tissues, with most tumors expressing multiple receptor subtypes. Pharmacological analysis of tumor membrane homogenates with GAL and the specific GAL receptor GalR2 agonist, AR-M1896, revealed that the GAL receptor GalR1 is most likely the receptor responsible for the observed GAL binding in the glioblastomas. No correlation could be found between GAL-LI, the level of GAL binding and proliferative activity as determined by immunostaining with the cell proliferation marker Ki-67.

Poor storage and handling of tissue mimics mitochondrial DNA depletion.

Analysis of the mitochondrial DNA (mtDNA) is an important part in the diagnosis of mitochondrial disorders. Besides point mutations and deletions in the mitochondrial genome a reduction in the amount of mtDNA molecules (mtDNA depletion) can also be the reason for mitochondrial defects. The DNA stability in clinical samples is essential for proper performance and interpretation of DNA based diagnosis. The stability of mtDNA was compared with that of nuclear DNA under poor handling and storage conditions. Fresh and thawed muscle tissue specimens were kept at different temperatures for a certain period of time before DNA isolation. Quantitative Southern blot analysis revealed a time-dependent decrease in the amount of mtDNA compared with nuclear DNA in thawed tissue specimens. Therefore, the current study demonstrates that proper specimen storage is a critical issue in quantitative mtDNA analysis and that poor handling and storage of tissue may mimic a severe mtDNA depletion.

Co-regulated decrease of Neurokinin-1 receptor and Hemokinin-1 gene expression in monocytes and macrophages after activation with pro-inflammatory cytokines

Hemokinin-1 (HK-1), a potent ligand for the Neurokinin-1 receptor (NK-1) is thought to play a role in the immune system. To investigate the regulation of this receptor-ligand pair, we examined the effects of pro-inflammatory cytokines on their expression in the monocyte/macrophage cell lines Wehi-3 and RAW264.7. We demonstrate co-expression of NK-1 and HK-1 mRNA in both lines, as well as functional NK-1 receptor protein in Wehi-3 cells. Stimulation with IFN-gamma, IL-1beta and TNF-alpha markedly decreased NK-1 and HK-1 mRNA as well as NK-1 receptor protein, which coincided with monocytic differentiation. A co-regulated decrease could also be observed in differentiating primary bone marrow macrophages, suggesting that this receptor-ligand pair may be controlled by cytokine networks and may serve a developmental role in the immune system.