[New molecular biology detection methods for tick-borne infectious agents]. / Neue molekularbiologische Nachweismethoden am Beispiel zeckengebundener Infektionserreger.

Tick-borne zoonotic pathogens are well known in many areas all over the world. Among the tick-borne transmitted diseases in Switzerland, Lyme disease caused by Borrelia burgdorferi, ehrlichiosis caused by various species of Ehrlichia and tick-borne encephalitis caused by the tick-borne encephalitis virus (TBEV) are the most important zoonotic diseases. Early diagnosis and treatment is necessary to prevent fatal infections and chronic damage to various tissues. Due to the variety of uncharacteristic clinical signs, tick-borne diseases are not easily recognized. Diagnosis is based on clinical findings, a record of tick exposure, and direct or indirect detection of the pathogen. Here we discuss briefly the most important tick-borne infections and their diagnosis with emphasis on a new molecular diagnostic tool--the real-time TaqMan PCR--and its importance for the diagnosis of tick-borne pathogens.

Swiss Army Survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks.

A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.

[Establishing an umbilical cord blood bank for unrelated allogenic stem cell transplantation]. / Aufbau einer Nabelschnurblutbank für unverwandte allogene Stammzelltransplantationen.

We report the establishment of a cord-blood bank in a routine hematological laboratory. Cord-blood collection was performed with placenta in utero by a trained team and immediately sent to the cord-blood bank. There, 6.8 ml cord-blood was used for analysis of nucleated cell counts, counts of CD34-positive cells, CFU's, complete HLA-typing, ABO and Rhesus blood groups, bacteriologic cultures and serology for HIV 1 and 2, HbsAg, HVC, CMV, syphilis and toxoplasmosis. The cord-blood collection was frozen and conserved at -192 degrees C. From each cord-blood vials of DNA, viable cells and plasma were cryopreserved. Between June 1997 and April 1998, 54 cord-bloods were collected. 40 of them were cryo-preserved, and 14 discarded because of low cell counts. The median volume was 109 ml with 1.4 x 10(9) nucleated cells. The in vitro capacity of proliferation of the cord-blood correlated well with the absolute counts of CD34-positive cells (r = 0.93), moderately with the relative counts of CD34 (r = 0.68) as well as the nucleated cells (r = 0.70), poorly with the volume (r = 0.44). Three of the 40 (7.5%) cord-blood products contained a bacterial contamination. This study shows that a cord-blood bank can be organised in a routine hematological laboratory, which is familiar with transplantation products. However, the procedure is time consuming, expensive and requires a highly qualified team and specialised technical equipment.

Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3.

Human calmodulin is encoded by three genes CALM1, CALM2 and CALM3 located on different chromosomes. To complete the characterization of this family, the exon-intron structure of CALM2 was solved by a combination of genomic DNA library screening and genomic PCR amplification. Intron interruptions were found at identical positions in human CALM2 as in CALM1 and CALM3; however, the overall size of CALM2 (16 kb) was almost twice that of the other two human CALM genes. Over 1 kb of the 5' flanking sequence of human CALM2 were determined, revealing the presence of a TATA-like sequence 27 nucleotides upstream of the transcriptional start site and several conserved sequence elements possibly involved in the regulation of this gene. To determine if differential transcriptional activity plays a major role in regulating cellular calmodulin levels, we directly measured and compared the mRNA abundance and transcriptional activity of the three CALM genes in proliferating human teratoma cells. CALM3 was at least 5-fold more actively transcribed than CALM1 or CALM2. CALM transcriptional activity agreed well with the mRNA abundance profile in the teratoma cells. In transient transfections using luciferase reporter genes driven by 1 kb of the 5' flanking DNA of the three CALM genes, the promoter activity correlated with the endogenous CALM transcriptional activity, but only when the 5' untranslated regions were included in the constructs. We conclude that the CALM gene family is differentially active at the transcriptional level in teratoma cells and that the 5' untranslated regions are necessary to recover full promoter activation.

Repression of the candidate tumor suppressor gene S100A2 in breast cancer is mediated by site-specific hypermethylation.

The calcium-binding protein S100A2 is expressed in normal breast tissue but downregulated during breast cancer progression. Hence it was previously identified as a candidate tumor suppressor gene. In this report, we investigated the molecular basis of S100A2 gene expression in normal and tumorigenic human breast epithelial cells. We cloned the gene coding for S100A2 including its 5' flanking region. To locate positively or negatively acting elements responsible for transcriptional regulation, promoter deletion studies were performed. Results from these experiments demonstrate that an enhancer element is located 1.2 kb upstream of the transcription start site. This element contains two AP1-like binding sites suggesting that transcriptional activation of S100A2 might be mediated by immediate early genes. Interestingly, the enhancer stimulates transcription in both normal and tumorigenic cells, indicating that repression of endogenous S100A2 transcription in tumorigenic cells might lie at an epigenetic level. Indeed, the proximal promoter region was found, by genomic sequencing, to be unmethylated in normal but hypermethylated in tumorigenic cells. Hypermethylation of the promoter at the same CpG sites was also found in a breast cancer biopsy. In addition, site specific in vitro methylation led to reduced expression of the S100A2 gene in normal cells. These experiments provide strong evidence that S100A2 repression in tumor cells is mediated by site-specific methylation. Since transcription of a number of known tumor suppressor genes is also repressed by methylation, our observation is consistent with the suggestion that S100A2 might have a tumor suppressor function.

Characterization of the human S100A12 (calgranulin C, p6, CAAF1, CGRP) gene, a new member of the S100 gene cluster on chromosome 1q21.

Here, we report the characterization of a human cDNA coding for the recently published amino acid sequence of a calcium-binding S100 protein, S100A12 (CGRP, calgranulin C, CAAF1, p6). The exon/intron structure of the S100A12 gene is similar to most other S100 genes. It is composed of three exons which are divided by two introns of 900 bp and 400 bp. The protein is encoded by sequences in exons 2 and 3, with exon 2 coding for the N-terminal 45 amino acids and exon 3 coding for the C-terminal 46 amino acids. So far, ten S100 genes are known to be located on human chromosome 1q21 in a clustered organization. Hence, we investigated whether S100A11 (S100C, calgizzarin) and S100A12 are also localized in the S100 gene cluster. We found both genes within the cluster, with S100A11 being close to S100A10 and S100A12 between the genes S100A8 and S100A9. Therefore, the S100 gene cluster now is composed of 12 differentially expressed family members.

Characterization of the human and mouse cDNAs coding for S100A13, a new member of the S100 protein family.

Here we report the characterization of the human S100A13 cDNA coding for a novel calcium-binding protein belonging to the S100 protein family. The predicted S100A13 protein shows sequence homologies to other S100 proteins between 50.5% (to S100A5) and 59.3% (to S100A12). High mRNA amounts were found in skeletal muscle, heart, kidney, ovary, small intestine and pancreas. Since twelve S100 genes are clustered on human chromosome 1q21, we determined the chromosomal localization of the human S100A13. It co-localizes with S100A1 on the cluster. Furthermore, we characterized the cDNA sequence coding for the mouse homolog of S100A13. Similar to the putative human protein, mouse S100A13 is composed of 98 amino acids displaying a homology of 86.7% compared to human S100A13.

Isolation of a YAC clone covering a cluster of nine S100 genes on human chromosome 1q21: rationale for a new nomenclature of the S100 calcium-binding protein family.

S100 proteins are low-molecular-weight calcium-binding proteins of the EF-hand superfamily and appear to be involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. More than 10 members of the S100 protein family have been described from human sources so far. We have now isolated a YAC clone from human chromosome 1q21, on which 9 different genes coding for S100 calcium-binding proteins could be localized. Moreover, we have mapped the gene coding for S100P to human chromosome 4p16 and thereby completed the chromosomal assignments of all known human S100 genes. The clustered organization of S100 genes in the 1q21 region allows us to introduce a new logical nomenclature for these genes, which is based on the physical arrangement on the chromosome. The new nomenclature should facilitate and further the understanding of this protein family and be easily expandable to other species.

Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2.

The human CALM1 calmodulin gene has been isolated and characterized. The gene contains six exons spread over about 10 kb of genomic DNA. The exon-intron structure is identical to that of the human CALM3 and of the rat CALM1 and CALM3 genes. A cluster of transcription-start sites was identified 200 bp upstream of the ATG translation-start codon, and several putative regulatory elements were found in the 5' flanking region as well as in intron 1. Sequence comparison with the rat CALM1 gene revealed significant similarities in the promoter regions of the two genes and an even more striking degree of identity (70%) in the available intron 1 sequences. A short CAG trinucleotide repeat region was identified in the 5' untranslated region of the human CALM1 gene; this sequence is not conserved in the rat counterpart. Expression of the CALM1 gene was detected in all human tissues tested, although at varying levels. A 1.7-kb mRNA was uniformly present at comparable levels, whereas a 4.2-kb mRNA species was particularly abundant in brain and skeletal muscle. Clones for two different CALM1-related pseudogenes CALM1P1 and CALM1P2 were also isolated and characterized. Both pseudogenes are intronless and non-functional as judged from the presence of mutations abolishing the open reading frame. Genomic Southern analysis indicates that the human CALM1 gene/pseudogene subfamily comprises at least three but probably no more than four members. The entire family consists of three bona fide CALM genes, at least one expressed calmodulin-like CALML gene as well as at least five pseudogenes.