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PubMed2006

Etiology of tick-borne febrile illnesses in adult residents of North-Eastern Poland: report from a prospective clinical study.

Febrile tick-borne diseases can be caused by different pathogens. The study objective was to prospectively determine the etiology of infection among adults exposed to tick bite and to detect acute human granulocytic anaplasmosis (HGA), a recently emerging infection, in north-eastern Poland. We evaluated 68 patients (34 males and 34 females, mean age 44.2+/-15 years) who had fever within 4 weeks after a tick bite. Paired sera were tested for antibodies against tick-borne encephalitis virus, Borrelia burgdorferi and Anaplasma phagocytophilum. Microscopic examination of EDTA whole blood and nested PCR targeting A. phagocytophilum 16S rDNA gene fragment were carried out at enrolment. Tick-borne infections were diagnosed in 57 individuals (84%). Sixty-four patients were hospitalised. Tick-borne encephalitis (TBE) was revealed in 49 cases; in 39 patients as a single infection, in three patients concurrent with erythema migrans or other Lyme borreliosis symptoms, in five patients concurrent with probable or possible Lyme borreliosis, and in two patients concurrent with A. phagocytophilum infection. Diagnosis of HGA was confirmed in one patient by PCR and in another one by seroconversion. In two further individuals, A. phagocytophilum infection was confirmed by immunofluorescence (antibody titers > or = 1:128), which, however, does not fulfil the ESCAR (European Society of Clinical Microbiology and Infectious Diseases Study Group on Coxiella, Anaplasma, Rickettsia, and Bartonella) criteria for HGA case definition. Lyme borreliosis was diagnosed in 16 patients; in six of them as a single infection. Both confirmed HGA cases and seropositive individuals had A. phagocytophilum infection concurrent either with Lyme borreliosis (two cases) or with TBE (two cases). The clinical course was severe in three and moderate to mild in the remaining TBE cases, as well as in all Lyme borreliosis and HGA cases. TBE was found the most prevalent disease among adults with febrile illnesses occurring after a tick bite in north-eastern Poland. Concurrent tick-borne infections were frequent with multiple pathogens involved. Two confirmed acute HGA cases (fulfilling the European case definition criteria defined by ESCAR) were detected for the first time in a prospective manner in Poland.

Adolescent↗

PubMed2006

Prevalence and molecular characterization of human group C rotaviruses in Hungary.

BACKGROUND: Group C rotaviruses are recognized enteric pathogens of humans and animals. Human group C rotaviruses have been associated with sporadic episodes and large outbreaks of gastroenteritis in children and adults but their epidemiology and ecology are still unexplored. OBJECTIVES: To collect epidemiological data on group C rotavirus infections among children with gastroenteritis in Hungary and perform molecular characterization on the identified strains. STUDY DESIGN: Fecal samples were collected during the 2003 surveillance in Baranya County, Hungary. The presence of group C rotavirus RNA was investigated by polyacrylamide gel electrophoresis and by reverse transcription-nested polymerase chain reaction for the VP6 gene. The identified strains were further characterized by sequencing and phylogenetic analysis of the VP7, VP6, VP4, and NSP4 genes. RESULTS: Three of 472 samples (0.6%) tested positive for group C rotavirus. Two samples were selected for molecular analysis. Strains BaC 6104/03 and BaC 11549/03 displayed an overall identity of >99.8% and 99.3% at the nucleotide and amino acid level, respectively. The VP7 of the strain BaC 6104/03 was most closely related (99.5% aa) to the Nigerian strain Jajeri, while the VP4s of strains BaC 6104/03 and BaC 11549/03 were more similar (98.1% aa) to strains Belem and 208, detected in Brazil and China, respectively. CONCLUSIONS: Based on this 1-year study, we conclude that group C rotaviruses are not of epidemiological relevance in the etiology of childhood acute gastroenteritis in Hungary. The low sequence divergence between the Hungarian strains suggested that a single group C rotavirus strain circulated in this period in the study area.

Adolescent↗

PubMed1998

Cloning, structure, chromosomal localization and promoter analysis of human 2-oxoglutarate dehydrogenase gene.

Human 2-oxoglutarate dehydrogenase (OGDH) is an E1-component of the OGDH multi-enzyme complex and catalyzes both the ThDP-dependent decarboxylation of 2-oxoglutarate and the subsequent reductive succinylation of the lipoyl moiety which is covalently bound to the E2 component, dihydrolipoamide succinyltransferase. The cDNA and genomic DNA encoding human OGDH has been cloned and sequenced. The cDNA contains a 3006-bp open reading frame encoding a 40-amino acid leader peptide and a 962-amino acid mature OGDH protein (Mr=108878). The gene contains 22 exons spanning approximately 85 kb. The putative ThDP-binding sequence motif is identified in both DNAs. The gene is localized to chromosome 7 at p13-p14 by fluorescence in situ hybridization. With the TATA- and CAAT-less 5'-flanking region (wild type, -3276/+212) of the OGDH gene-luciferase reporter vector construct and its nested deletion or linker-scanning mutant constructs the transient reporter expression assays in BHK-21 cells reveal the existence of two 10-bp cis-acting elements (-53/-44 and -33/-24) and two trans-acting elements (-536/-496 and -93/-84). A nuclear factor that binds to the region from -63 to -24 including two cis-acting elements.

Amino Acid Sequence↗

PubMed2002

Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction.

OBJECTIVE: Quantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations. MATERIALS AND METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques. RESULTS: Validation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)CD38(+) cells (p = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)CD38(-) compared to CD34(+)CD38(+) cells. CONCLUSIONS: Real-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis.

ADP-ribosyl Cyclase↗

PubMed2003

Epidemiological survey of Anaplasma platys and Ehrlichia canis using ticks collected from dogs in Japan.

Detection of Anaplasma platys and Ehrlichia canis in ticks recovered from dogs in Japan was attempted using a species-specific nested PCR based on the 16S rRNA gene. A total of 1211 ticks recovered from 1211 dogs from all over Japan were examined for A. platys and E. canis. Four tick samples from Fukushima, Miyazaki and Kagoshima Prefectures recovered from four different dogs showed a positive reaction for A. platys. Although the four dogs did not show any clinical signs and no blood examination data were available, it is possible that A. platys has already been spread widely in Japan. No positive reactions were observed in any ticks examined for E. canis.

Anaplasma↗

PubMed2001

CYP21 mutations and congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder caused mainly by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene on chromosome 6p21.3. The wide range of CAH phenotypes is associated with multiple mutations known to affect 21-hydroxylase enzyme activity. To date, 56 different CYP21 mutations have been reported, mostly point mutations, but small deletions or insertions have been described too, as well as complete gene deletions. Fifteen mutations, constituting 90-95% of alleles, are derived from intergenic recombination of DNA sequences between the CYP21 gene and the highly homologous CYP21P pseudogene, while the remaining are spontaneous mutations. A reliable and accurate detection of CYP21 mutations is not only important for clinical diagnosis, but also for carrier detection as there is a high variability in the basal level of 17-hydroxyprogesterone between normal and heterozygous individuals. Several strategies based on polymerase chain reaction (PCR)-driven amplification with allele-specific oligonucleotides to the CYP21 gene have been developed. It has been demonstrated that one reaction for PCR amplification of the CYP21 gene and the chimeric CYP21P/CYP21 gene using mixed primers in combination with nested PCR and single-strand conformation polymorphism is considered highly efficient and accurate for molecular diagnosis of CAH due to 21-hydroxylase deficiency.

Adrenal Hyperplasia, Congenital↗

PubMed1998

Detection of chimaeric transcripts of the immunoglobulin heavy chain and BCL6 genes by reverse-transcriptase polymerase chain reaction in B-cell non-Hodgkin's lymphomas.

T(3;14)(q27;q32) is frequently detected in B-cell non-Hodgkin's lymphomas, especially the diffuse large cell type and the follicular type. The BCL6 gene encoding a putative transcriptional factor which resides on 3q27 rearranges to the immunoglobulin heavy chain (IgH) gene on 14q32 in this chromosomal translocation. The upstream regulatory region of the BCL6 gene is replaced by the IgH gene. Deregulation of the BCL6 gene may contribute to tumourigenesis of these diseases. The rearrangement between the IgH and BCL6 genes generates chimaeric transcripts in which the joining (J) region of the IgH gene fuses to exon 3 of the BCL6 gene. We established a method to detect these chimaeric transcripts by reverse transcriptase polymerase chain reaction (RT-PCR) using the consensus sequence of the J region and the sequence of exon 3 of the BCL6 gene as primers. Using the semi-nested RT-PCR method and a cell line carrying t(3;14)(q27;q32), we detected one lymphoma cell among 10,000 background cells. We detected these chimaeric transcripts in two out of 13 clinical samples by this method. This method can detect t(3;14)(q27;q32) easily, whereas this alteration is frequently overlooked by routine karyotype analysis. Since this technique is sensitive enough to detect a small number of lymphoma cells with this genetic abnormality, it could be employed to detect contaminating lymphoma cells in bone marrow and peripheral blood and minimal residual diseases.

Base Sequence↗

PubMed2001

Population structure and biogeography of migratory freshwater fishes (Prochilodus: Characiformes) in major South American rivers.

Mitochondrial DNA (mtDNA) sequences were used to infer the phylogenetic relationships of Prochilodus species in the Paraná, Amazonas, Orinoco, and Magdalena basins. Sequences of ATPase subunits 6 and 8 (total 840 bp) were obtained for 21 Prochilodus specimens from the four river systems. Using Semaprochilodus as an outgroup, phylogenetic analyses showed that: (i) each river basin contains a monophyletic group of mtDNA lineages; and (ii) the branching order places Magdalena in a basal position with subsequent branching of Orinoco, Amazon and Paraná. The mitochondrial control region was sequenced for 26 P. lineatus (from the Paraná basin) and six other Prochilodus specimens from the Magdalena, Orinoco and Amazon. All 26 control region haplotypes were unique with sequence divergence ranging from 0.3 to 3.6%. The control region phylogeny is well resolved but phylogenetic structure is not associated with geography. For example, mtDNA haplotypes from the upper Paraná (Mogui Guassú) and the upper Bermejo, separated by at least 2600 km, have close genealogical ties. Phylogeographic analyses, including nested clade analysis, suggest high levels of gene flow within this basin.

Animals↗

PubMed2006

Domain structure and protein interactions of the silent information regulator Sir3 revealed by screening a nested deletion library of protein fragments.

Transcriptional silencing in yeast is mediated by the interactions of silent information regulator (Sir) proteins with chromatin and with one another. The stable association of Sir3 with Sir4 is mediated by a C-terminal region of Sir3 that has additional functions including the dimerization of Sir3. We have developed a simple, robust expression screening methodology that allows for the unbiased identification of functional protein domains expressed from nested-deletion libraries of full-length genes. Using these methodologies, Sir3 dimerization was shown to be mediated by two separate domains. One of these domains also binds cooperatively to the C-terminal coiled-coil motif of Sir4 and dimerization further increases the affinity of Sir3 for Sir4. The resulting Sir3-Sir4 complexes form progressively higher order assemblies with increasing protein concentration, with implications for the mechanism of gene silencing.

Base Sequence↗

PubMed1999

Genetic analysis reveals ongoing HIV type 1 evolution in infected human placental trophoblast.

To provide a better understanding of the role of placenta in vertical human immunodeficiency virus (HIV) transmission, we have studied the infection of placental trophoblast in a group of 15 mother-neonate pairs. By nested PCR amplification of the C2V3 env gene region, HIV-1 has been found to infect the placenta in five cases (33%). Phylogenetic analysis of the cloned sequences showed that all recovered maternal variants were of the B subtype. Further investigation into the ancestral relationships at the nucleotide level revealed that the trophoblast sequences evolved into a quasispecies population clearly distant from that observed in the mother. As expected, the populations transmitted to the trophoblast were also found to be more homogeneous than those in the mothers when characterized on the basis of pairwise nucleotide sequence distances. With regard to the predicted biological properties, the primary amino acid structure of the V3 loop domain was consistent, with a macrophage-tropic, non-syncytium-inducing phenotype in all patients. We also attempted to determine if any of a number of selected maternal or viral factors was associated with trophoblast infection. However, none of the followed parameters, including maternal age, disease stage, antiretroviral therapy, CCR5delta32 deletion status of the infant, and viral genotype, could be associated with viral transmission. Moreover, in one pair with proven trophoblast infection, HIV was also detected in the cord blood. Taken together, our data suggest that the productive trophoblast infection by HIV-1 in vivo is a relatively frequent event that may bear direct implications for a further transplacental propagation of the virus.

Adult↗

PubMed2005

Babesia DNA detection in canine blood and Dermacentor reticulatus ticks in southwestern Siberia, Russia.

Babesia infection was studied in 21 blood samples of dogs with symptoms of babesiosis and among 72 Dermacentor reticulatus and 70 Ixodes persulcatus ticks from southwestern Siberia, Russia. Babesia DNA was detected by hemi-nested PCR based on the 18S rRNA gene with subsequent direct sequencing. All of the analyzed canine blood samples and three D. reticulatus, but none from I. persulcatus ticks studied were shown to contain Babesia DNA. Nucleotide sequences of the Babesia 18S rRNA gene fragment of 354 bp long for all 24 positive samples appeared to belong to the subspecies Babesia canis canis and differed only at three positions. The Babesia nucleotide sequences from 17 canine blood samples and from one D. reticulatus tick were identical to each other and to previously known B. canis canis from canine blood in Slovenia. Four canine blood samples and the second tick sample contained a mixture of two nucleotide sequences previously found in canine blood. B. canis canis nucleotide sequence from the third tick differed in the unique nucleotide transition and could correspond to a new genetic variant. Thus, the main etiological agent of canine babesiosis in Novosibirsk region is B. canis canis, and D. reticulatus, but not I. persulcatus, ticks could serve as a vector of this infectious agent. To our knowledge, this is the first report of the B. canis canis nucleotide sequences from ticks.

Animals↗

PubMed1991

The differentiation of human pro-thymocytes along the TCR-alpha/beta pathway in vitro is accompanied by the site-specific deletion of the TCR-delta locus.

The nested location of the TCR-delta gene within the TCR-alpha locus is a common feature of both mouse and human. Yet alpha/beta and gamma/delta T cells represent two separate lineages. We have previously proposed that a specific rearrangement event (delta Rec--psi J alpha) resulting in the specific deletion of the TCR-delta gene could be responsible for the independent usage of these two loci. We used an in vitro model of T cell differentiation to test this hypothesis. We show that in culture conditions (IL-1 + sCD23) which promote the development of alpha/beta expressing T cells exclusively, the specific deletion of the TCR-delta locus occurs very rapidly, probably before the productive rearrangement of the TCR-alpha gene. These results clearly demonstrate that the specific deletion of the TCR-delta gene could be the initial regulatory event that imprints the irreversible commitment of T cell differentiation along the alpha/beta pathway.

Base Sequence↗

PubMed2002

Alternative transcripts of presenilin-1 associated with frontotemporal dementia.

We have analyzed the expression of Alzheimer's disease-associated presenilin 1 (PS1) in various neurodegenerative disorders. Western blotting identified PS1 N- and C-terminal fragments similarly in the cortex of controls, Parkinson, Huntington and schizophrenia subjects. Additional PS1 immunoreactive species of 42 and 46 kDa were present in six out of seven cases of sporadic frontotemporal dementia (FTD) and these were particularly prominent in two cases. RT-PCR analysis using nested primers showed the presence of PS1 gene products with deletions within the exon 4-8 region. Our results suggest that alternative transcription of PS1 may be associated with FTD.

Alternative Splicing↗

PubMed2002

Alternative transcripts of presenilin-1 associated with frontotemporal dementia.

Adult↗

PubMed2005

Instability of clonality in gastric lymphoid infiltrates: a study with emphasis on serial biopsies.

The evolution of low-grade B-cell mucosa-associated lymphoid-tissue (MALT) lymphoma of the stomach is a multistage process, reflected in the histologic continuum from Helicobacter pylori-chronic gastritis, to low-grade and high-grade lymphoma. Interestingly, in daily gastric biopsy sign-out, the authors observed that some biopsies showed monoclonality on polymerase chain reaction (PCR) even though there were no definite histologic features of malignancy and vice versa. To address the question, the authors studied the endoscopic gastric biopsies at first presentation of 46 patients to examine any clonality differences among various histologic patterns within the spectrum of MALT lymphoma evolution. The gastric biopsies were reviewed histologically and graded according to the Wotherspoon-Isaacson histologic scoring system from grade 0 (normal) to grade 5 (MALT lymphoma). The clonality of cases in each grade was determined by performing nested PCR for immunoglobulin heavy chain (IgH) gene rearrangement using FR2/JH and FR3/JH primer sets. The monoclonality rates among different grades were as follows: grade 2, 6.3% (1 of 16); grade 3, 27.3% (3 of 11); grade 4, 83.3% (5 of 6); grade 5, 69.2% (9 of 13). Statistically significant difference of monoclonality rate is demonstrated in histologic grade 4 versus grades 2 and 3, and grade 5 versus grade 2 (P < 0.05, Fisher exact test). The authors went on to examine the progress of disease by following up the clinical status, histologic changes, and clonality fluctuation of these cases. Four of the 8 patients with monoclonality on PCR, but no definite lymphoma at first presentation later progressed to frank MALT lymphoma. Our study shows that, during the progression to MALT lymphoma, there is an instability of clonality. Clonality can fluctuate between polyclonality, oligoclonality, and monoclonality, none of which defines an irreversible stage for progression to MALT lymphoma. Monoclonality is a risk factor for development of MALT lymphoma. Those cases with dense gastric mucosal lymphoid infiltrate (without definite MALT lymphoma) and monoclonality on PCR need to be closely monitored and Helicobacter infection promptly treated if present. In combination with clinicohistologic examination, PCR can serve as a complementary tool in arriving at a definite diagnosis of MALT lymphoma in cases with borderline histologic features.

Adult↗

PubMed2005

Quantification and detection of bacteria from postoperative maxillary cyst by polymerase chain reaction.

BACKGROUND/AIMS: Postoperative maxillary cyst (POMC) is known to occur as a delayed complication of radical maxillary sinus surgery, such as Caldwell-Luc surgery. The cyst gradually expands with no symptoms over a period of years, and then occasionally causes swelling and pain in the buccal region and/or the mucogingival fold. It is probable that bacterial infection affects the progression of POMC symptoms. The aims of this study were to determine the bacterial density and to examine the presence of 20 oral bacteria in POMC fluids. METHODS: POMC fluids (4 purulent, 2 mucous and 4 serous) were sampled from 10 subjects (aged 43-77 years). Bacterial quantification and detection were performed by real-time polymerase chain reaction (PCR) and nested PCR based on bacterial 16S rRNA genes, respectively. RESULTS: Bacterial DNA was detected in all samples and the average concentrations of bacterial DNA were 5.9 (purulent), 0.5 (mucous), and 0.7 (serous) ng/mg of sample. Twelve bacterial species, including anginosus streptococci, known to be associated with abscess formation, were detected in the purulent fluids, while two and five species were detected in the mucous and serous fluids, respectively. CONCLUSION: Purulent fluids contained numerous bacteria of various types, thus suggesting that oral bacteria may cause symptoms such as pain in POMC with purulent fluids. Mucous and serous fluids also contained bacteria, although their numbers were small, thus suggesting an association between bacteria and progression of POMC.

Abscess↗

PubMed2006

Pneumocystis jirovecii colonisation in patients with interstitial lung disease.

A prospective study was conducted to determine the prevalence of colonisation by Pneumocystis jirovecii in 80 consecutive patients who required bronchoscopy and bronchoalveolar lavage (BAL) following suspicion of interstitial lung disease (ILD). The mtLSU rRNA gene of P. jirovecii was identified by nested PCR in BAL samples. Patients with ILDs were divided into three groups: group A comprised those with idiopathic interstitial pneumonias; group B comprised those with sarcoidosis; and group C comprised those with other ILDs. The overall prevalence of P. jirovecii carriage was 33.8%, with colonisation rates of 37.8%, 18.8% and 37% in groups A, B and C, respectively (p not significant). There were more smokers among the carriers, but there were no other significant differences between carriers and non-carriers. The high prevalence of P. jirovecii carriers found among immunocompetent patients with ILDs in Spain suggests a possible role of P. jirovecii in the natural history of these diseases.

Adult↗

PubMed1994

Bovine T cells specific for Trypanosoma brucei brucei variant surface glycoprotein recognize nonconserved areas of the molecule.

The specificity of bovine CD4+ T-cell responses to Trypanosoma brucei variant surface glycoprotein (VSG) has been examined by using a panel of seven T-cell clones and nested deletions of the ILTat 1.3 VSG gene expressed in Escherichia coli. All clones recognized the polymorphic N-terminal domain of the antigen, and the recognition sites of five of the clones were resolved to three areas with lengths of 14, 18, and 21 amino acids. Comparison of these regions with corresponding areas of other VSG molecules, including those derived from the same trypanosomal serodeme, has shown that the sites are not conserved. In the light of recent observations that VSG-specific T-cell responses are induced in mice infected with T. brucei, these results confirm with the belief that immune pressure from T cells may contribute to the generation of antigenic diversity on the surface of African trypanosomes.

Amino Acid Sequence↗