Description and validation of a new automated surveillance system for Clostridium difficile in Denmark.

The surveillance of Clostridium difficile (CD) in Denmark consists of laboratory based data from Departments of Clinical Microbiology (DCMs) sent to the National Registry of Enteric Pathogens (NREP). We validated a new surveillance system for CD based on the Danish Microbiology Database (MiBa). MiBa automatically collects microbiological test results from all Danish DCMs. We built an algorithm to identify positive test results for CD recorded in MiBa. A CD case was defined as a person with a positive culture for CD or PCR detection of toxin A and/or B and/or binary toxin. We compared CD cases identified through the MiBa-based surveillance with those reported to NREP and locally in five DCMs representing different Danish regions. During 2010-2014, NREP reported 13 896 CD cases, and the MiBa-based surveillance 21 252 CD cases. There was a 99·9% concordance between the local datasets and the MiBa-based surveillance. Surveillance based on MiBa was superior to the current surveillance system, and the findings show that the number of CD cases in Denmark hitherto has been under-reported. There were only minor differences between local data and the MiBa-based surveillance, showing the completeness and validity of CD data in MiBa. This nationwide electronic system can greatly strengthen surveillance and research in various applications.

Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture.

The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine toxigenic culture. In total, 300 faecal samples from Danish hospitalised patients with diarrhoea were included consecutively. Culture was performed in duplicate (routine and 'expanded toxigenic culture': prolonged and/or re-culture) and genotypic toxin profiling by polymerase chain reaction (PCR), PCR ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene C. difficile and PCRFast C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p < 0.05) and significantly more robust to inhibition compared to PCRFast (p < 0.001). Duplicate 'expanded toxigenic culture' increased the culture-positive rate by 29% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95%), and in-house PCR displayed 100% correct identification of PCR ribotypes 066 and 078. Furthermore, the presence of the PCR enhancer bovine serum albumin (BSA) was found to be related to high sensitivity and low inhibition rate. Rapid laboratory diagnosis of toxigenic C. difficile by RT PCR was accurate.

Separate norovirus outbreaks linked to one source of imported frozen raspberries by molecular analysis, Denmark, 2010-2011.

Norovirus outbreaks occur frequently in Denmark and it can be difficult to establish whether apparently independent outbreaks have the same origin. Here we report on six outbreaks linked to frozen raspberries, investigated separately over a period of 3 months. Norovirus from stools were sequence-typed; including extended sequencing of 1138 bp encompassing the hypervariable P2 region of the capsid gene. Norovirus was detected in 27 stool samples. Genotyping showed genotype GI.Pb_GI.6 (polymerase/capsid) with 100% identical sequences. Samples from five outbreaks were furthermore identical over the variable capsid P2 region. In one outbreak at a hospital canteen, frozen raspberries was associated with illness by cohort investigation (relative risk 6·1, 95% confidence interval 3·2-11). Bags of raspberries suspected to be the source were positive for genogroup I and II noroviruses, one typable virus was genotype GI.6 (capsid). These molecular investigations showed that the apparently independent outbreaks were the result of one contamination event of frozen raspberries. The contaminated raspberries originated from a single producer in Serbia and were originally not considered to belong to the same batch. The outbreaks led to consultations and mutual visits between producers, investigators and authorities. Further, Danish legislation was changed to make heat-treatment of frozen raspberries compulsory in professional catering establishments.

A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85-99 complex.

Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II-restricted presentation of central nervous system-derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2-peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2-peptide-specific antibodies from HLA-DR2-transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85-99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2-MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP-specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85-99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2-MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.

Whole-genome sequencing of Campylobacter jejuni isolated from Danish routine human stool samples reveals surprising degree of clustering.

OBJECTIVES: Outbreaks of Campylobacter are traditionally considered to be rare; however, rather than being the true nature of the disease, this may reflect our present inability to detect them. The aim of this study was to determine the genetic and epidemiological degree of clustering among Campylobacter jejuni isolates from Danish patients. METHODS: Whole-genome sequencing (WGS) was applied to 245 C. jejuni isolates from patients with domestically acquired infection over a 9-month period in 2015 and 2016. RESULTS: WGS demonstrated that 62 of the 245 isolates (25%) clustered genetically. In total, 21 genetic clusters were identified of which four (18%) consisted of five isolates or more. Seventeen (81%) of the 21 genetic clusters were clustered in space and/or time. Of the 245 isolates, 49 (20%) were part of a temporal and/or geographical cluster. The identified clusters included two outbreaks; one which had not been identified through the existing surveillance system. CONCLUSIONS: Using WGS, we show that Campylobacter case clustering and even outbreaks appear to occur more often than previously assumed, providing important new insight into the relatively poorly understood epidemiology of the most important cause of bacterial gastroenteritis in the industrialized world.

Systematic serotyping and riboprinting of Campylobacter spp. improves surveillance: experiences from two Danish counties.

In order to monitor the distribution of subtypes of Campylobacter and to identify clusters, 975 isolates of Campylobacter spp., obtained from human infections occurring in two Danish counties, were studied during a 1-year period. The isolates were characterised by Penner serotyping and automated ribotyping. Pulsed-field gel electrophoresis (PFGE) profiling was used to confirm clustering of identical serotypes and ribotypes. The 975 isolates were divided into 48 serotypes, 210 ribotypes and 277 serotype-ribotype combinations. The overall distribution of serotypes and ribotypes was similar between the two counties. After taking into account the rare or common occurrence of subtypes, a model identified 43 clusters of subtypes during the study period. Clustered isolates represented 28% (273/975) of the study population, with clusters containing between three and 20 isolates. PFGE confirmed the validity of selected clusters identified by serotyping and ribotyping. The observed clustering of Campylobacter isolates, with identical types in time and place, indicates that common-source outbreaks of campylobacteriosis are more common than is usually thought.

Automated surveillance system for hospital-acquired urinary tract infections in Denmark.

BACKGROUND: The Danish Hospital-Acquired Infections Database (HAIBA) is an automated surveillance system using hospital administrative, microbiological, and antibiotic medication data. AIM: To define and evaluate the case definition for hospital-acquired urinary tract infection (HA-UTI) and to describe surveillance data from 2010 to 2014. METHODS: The HA-UTI algorithm defined a laboratory-diagnosed UTI as a urine culture positive for no more than two micro-organisms with at least one at ≥10(4)cfu/mL, and a probable UTI as a negative urine culture and a relevant diagnosis code or antibiotic treatment. UTI was considered hospital-acquired if a urine sample was collected ≥48h after admission and <48h post discharge. Incidence of HA-UTI was calculated per 10,000 risk-days. For validation, prevalence was calculated for each day and compared to point prevalence survey (PPS) data. FINDINGS: HAIBA detected a national incidence rate of 42.2 laboratory-diagnosed HA-UTI per 10,000 risk-days with an increasing trend. Compared to PPS the laboratory-diagnosed HA-UTI algorithm had a sensitivity of 50.0% (26/52) and a specificity of 94.2% (1842/1955). There were several reasons for discrepancies between HAIBA and PPS, including laboratory results being unavailable at the time of the survey, the results considered clinically irrelevant by the surveyor due to an indwelling urinary catheter or lack of clinical signs of infection, and UTIs being considered HA-UTI in PPS even though the first sample was taken within 48h of admission. CONCLUSION: The HAIBA algorithm was found to give valid and valuable information and has, among others, the advantages of covering the whole population and allowing continuous standardized monitoring of HA-UTI.

Functional intron+ and intron- rDNA in the same macronucleus of the ciliate Tetrahymena pigmentosa.

Diallelic clones of Tetrahymena pigmentosa containing equal amounts of intron+ and intron- rDNA in the macronucleus were constructed. The macronucleus of the resulting strains divides amitotically during vegetative growth and the diallelic genotype is therefore unstable. The coexistence of the two alleles was followed in the total culture and in single cells during their vegetative segregation and it was observed that replication was non-preferential with respect to the two alleles. The diallelic clones were also used to demonstrate that intron-containing rDNA was transcribed and the transcript processed in the presence of corresponding intron- rDNA. The results are discussed in the light of the 'non-function' idea for ribosomal RNA introns.

Structural organization of the genes encoding the small nuclear RNAs U1 to U6 of Tetrahymena thermophila is very similar to that of plant small nuclear RNA genes.

We report the sequences of the genes encoding the small nuclear RNAs (snRNAs) U1 to U6 of the ciliate Tetrahymena thermophila. The genes of the individual snRNAs exist in two to six slightly different copies per haploid genome. Sequence analyses of the gene-flanking regions indicate that there are two classes of snRNA genes. Both classes are characterized by several conserved sequence elements, some of which are unique to each class and some of which are found in both classes. Comparison of the promoter structure of the snRNA genes of T. thermophila with the promoter structures of snRNA genes of other organisms revealed several similarities to plant snRNA genes. These similarities include the overall promoter architecture as well as specific sequence elements. The structural organization of the 3' flanking region of some of the T. thermophila snRNA genes is not observed in other organisms. This finding is discussed in relation to a possible role in snRNA 3'-end formation.

Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions.

We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T. thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two of these occur between U2 and U6, whereas the third occurs between U1 and U2. The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6. We envisage that these interactions may facilitate the first step of pre-mRNA splicing.

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon.

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.

Campylobacter concisus: an evaluation of certain phenotypic and genotypic characteristics.

The clinical relevance of Campylobacter concisus in gastrointestinal disease has not been determined definitively. This study investigated the phenotypic and genotypic characteristics of 39 C. concisus isolates from Danish patients with diarrhoea, three isolates from healthy individuals and the type strain. A cytolethal distending toxin (CDT)-like effect on Vero cells was observed in 35 (90%) isolates from patients with diarrhoea, in all three isolates from healthy individuals and in the type strain. Analysis of SDS-PAGE protein profiles and PCR amplification of 23S rDNA assigned the isolates into two distinct, but discordant groups. Automated ribotyping (RiboPrinting) identified 34 distinct patterns among the 43 isolates, but cluster analysis did not separate isolates from patients with diarrhoea from isolates from healthy patients. Random amplified polymorphic DNA (RAPD) analysis with three primers identified 37 unique profiles, but requires further evaluation. The isolates obtained from healthy carriers were distinguished by cluster analysis from the isolates obtained from patients with diarrhoea. All the isolates were susceptible to 11 antimicrobial agents tested. Overall, there was considerable variability between the C. concisus isolates, but there were no clear phenotypic or genotypic differences between isolates from patients with diarrhoea and isolates from healthy carriers. Further evidence is needed to support the possible role of C. concisus as a human enteric pathogen.

Invasive listeriosis in Denmark 1994-2003: a review of 299 cases with special emphasis on risk factors for mortality.

Listeriosis is a rare, but serious, foodborne infection which, in the invasive form, presents as bloodstream (BS) infection, an infection of the central nervous system (CNS), a maternofetal infection or a focal infection. The disease is notifiable in Denmark. This paper reviews the results of the Danish surveillance of invasive listeriosis from 1994 to 2003, excluding maternofetal cases. In total, 299 invasive cases of listeriosis were reported. Two-thirds of the cases were caused by isolates of serogroup 1/2, and one-third by serogroup 4. Most (70%) cases had conditions known to predispose to listeriosis. More patients with BS infection were predisposed because of concurrent underlying illness than were patients with CNS infection. Half of the patients were aged > 70 years, and 21% died of the disease. There was no change in the case fatality rate (CFR) during the 10-year period. The CFR was identical for men and women. BS and CNS infection caused the same incidence of mortality, but no mortality was observed in patients with focal infections at normally sterile body sites. In a multivariate analysis, isolates belonging to serogroup 4 were associated with a higher CFR than were isolates of serogroup 1/2. In patients aged < 70 years, underlying conditions predisposing to disease were related strongly to mortality, which was not the case in patients aged > 70 years. The underlying conditions associated most strongly with mortality in the younger age group were non-haematological malignancies.

The ribosomal RNA genes of Tetrahymena: structure and function.

This report reviews the structural and functional characteristics of the ribosomal RNA genes (rDNA) of the ciliate protozoan, Tetrahymena. The study of rDNA was initiated some 10 years ago in order to establish a model system for rRNA gene expression in lower eukaryotes. The system proved very useful for studying several aspects of rRNA gene expression so that a considerable amount of information on rDNA structure and function has accumulated during the past years as a result of studies done in several laboratories. The initial finding that starved Tetrahymena cells, upon refeeding, preferentially replicated their rDNA for up to 90 min before bulk macronuclear DNA synthesis resumed, greatly facilitated the first studies. This observation permitted the rDNA to be labeled selectively with isotopes and in this way it was possible to isolate rDNA in pure form, to study replicative intermediates of the rDNA and to study rDNA containing chromatin in intact nuclei without resorting to chromatin fractionation. As a result of these studies it was found that the macronuclear rDNA of all Tetrahymena species consisted of a homogeneous population of extrachromosomal, linear molecules with a size of about 20 kilobase-pairs (kb) containing two identical transcription units for pre-rRNA arranged in a reverse repeat orientation (palindromic symmetry). The origins of replication were localized to the central non-transcribed spacer region and it was shown that this region has a chromatin structure which is different from that of the transcribed region. The primary DNA sequence is now known in many parts of the rDNA molecule, including the central part (containing the replication origins), the telomeric parts and the regions containing the sites of transcription initiation and termination. Transcription studies demonstrated the presence of an intervening sequence (intron) in the 26S rRNA coding region in some strains of Tetrahymena. Interestingly, the intron is transcribed and later removed from the primary transcript as a result of a rather unusual reaction which can take place in vitro in the absence of added protein factors. The finding of interbreeding strains of the intron+ and intron- rDNA genotype provided physical markers on the genes and have made possible a description of the inheritance and allelic assortment of the Tetrahymena rDNA. These studies proved that the free, palindromic rDNA molecules of the macronucleus arise from a chromosomally integrated, micronuclear rDNA copy as a result of a conjugational dependent amplification event, and that the intron is inherited as a neutral character during sexual and vegetative reproduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus.

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.

A novel class of nucleolar RNAs from Tetrahymena.

We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate, Tetrahymena. The snoRNAs are 120-140 nucleotides long, moderately AU-rich and contain no modified nucleotides. Their 5' ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single-stranded AU-rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases.

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA.

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D.

A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.

Equipotent in vitro actions of alpha- and beta-CGRP on guinea pig basilar artery are likely to be mediated via CRLR derived CGRP receptors.

The aim of the present study was to investigate and compare specific in vitro pharmacological actions of human alpha- and beta-CGRP applied as single concentrations to prostaglandin F2alpha precontracted segments of guinea pig basilar artery. To support the suggestion of a possible link between the pharmacological actions of alpha- and beta-CGRP and a specific receptor, we wished to determine whether mRNAs required for the expression of calcitonin receptor-like receptor (CRLR) derived CGRP receptors were present in the guinea pig basilar artery. In the pharmacological experiments we demonstrated an increase in the cAMP content by 2.5-fold and a concomitant significant vasorelaxation of the precontracted basilar artery segments following 1 min of stimulation by 10(-7) M alpha- or beta-CGRP. In another set of experiments, the time course of alpha- and beta-CGRP induced vasodilatation was investigated and concentration dependent responses of the two peptides were demonstrated. No significant differences between the actions of alpha- and beta-CGRP regarding induction of cAMP formation, amount of vasodilatation, time course of vasodilatation and mode of inhibition by the CGRP receptor antagonist, human alpha-CGRP(8-37), could be detected. The presence of mRNA encoding the guinea pig CRLR and the guinea pig CGRP receptor component protein (RCP) in the guinea pig basilar artery was demonstrated by RT-PCR methods. Furthermore, a partial sequence of mRNA encoding the guinea pig CRLR was determined. The expression in this tissue of a CRLR derived CGRP receptor and a functional RCP is therefore likely, and the equipotent pharmacological actions of alpha- and beta-CGRP might be mediated via CRLR derived CGRP receptors.