Jonas Blomberg

Activation of type I interferon system in systemic lupus erythematosus correlates with disease activity but not with antiretroviral antibodies

The objective was to investigate the relation between serum levels of interferon-α (IFN-α), the activity of an endogenous IFN-a inducing factor (SLE-IIF), clinical and immunological disease activity as well as serum levels of antiretroviral antibodies in SLE. Serum levels of IFN-α were measured in serial sera from 30 patients sampled at different stages of disease activity (SLEDAI score). The SLE-IIF activity was measured by its ability to induce IFN-α production in cultures of normal peripheral blood mononuclear cells. Both serum IFN-α and SLE-IIF increased markedly at flare in serially followed patients. The SLEDAI score, levels of anti-dsDNA antibodies and IL-10 correlated positively, and complement components Clq, C3 and leukocytes correlated inversely with serum concentrations of IFN-α. The extent of multiple organ involvement correlated with serum IFN-α. No relation between concentrations of retroviral peptide binding antibodies and IFN-α or SLE-IIF activity was found. The close relationship between disease activity in SLE patients and IFN-α serum levels suggests that activation of the type I IFN system might be of importance in the disease process.

Expression of human endogenous retroviral sequences in peripheral blood mononuclear cells of healthy individuals.

The polymerase chain reaction was used to detect expression of retroviral sequences with oligonucleotide primers derived from conserved regions of the retroviral genome. Four primer pairs derived from gag and one from pol were used in amplification of reverse-transcribed total RNA prepared from peripheral blood mononuclear cells of seven blood donors. The amplification pattern was the same from each of the seven samples. Sequencing of cloned amplification products revealed that at least three subclasses of sequences related to the human endogenous retroviruses (HERV) RTVL-H, HERV-E and HERV-K, are expressed in peripheral blood mononuclear cells of healthy individuals. This has not been previously reported.

Automated recognition of retroviral sequences in genomic data—RetroTector©

Eukaryotic genomes contain many endogenous retroviral sequences (ERVs). ERVs are often severely mutated, therefore difficult to detect. A platform independent (Java) program package, RetroTector© (ReTe), was constructed. It has three basic modules: (i) detection of candidate long terminal repeats (LTRs), (ii) detection of chains of conserved retroviral motifs fulfilling distance constraints and (iii) attempted reconstruction of original retroviral protein sequences, combining alignment, codon statistics and properties of protein ends. Other features are prediction of additional open reading frames, automated database collection, graphical presentation and automatic classification. ReTe favors elements >1000-bp long due to its dependence on order of and distances between retroviral fragments. It detects single or low-copy-number elements. ReTe assigned a ‘retroviral’ score of 890–2827 to 10 exogenous retroviruses from seven genera, and accurately predicted their genes. In a simulated model, ReTe was robust against mutational decay. The human genome was analyzed in 1–2 days on a LINUX cluster. Retroviral sequences were detected in divergent vertebrate genomes. Most ReTe detected chains were coincident with Repeatmasker output and the HERVd database. ReTe did not report most of the evolutionary old HERV-L related and MalR sequences, and is not yet tailored for single LTR detection. Nevertheless, ReTe rationally detects and annotates many retroviral sequences.

Etiology of community-acquired pneumonia in patients requiring hospitalization

The etiology of community-acquired pneumonia was studied in 127 patients with roentgenologically verified pneumonia who needed hospitalization. Etiology was determined on the basis of a positive blood culture and/or a significant antibody titer increase.Streptococcus pneumoniae was the probable etiological agent in 69 patients, nontypeableHaemophilus influenzae in five patients,Streptococcus pyogenes in two patients, andLegionella pneumophila andStaphylococcus aureus in one patient each. Evidence ofMycoplasma pneumoniae infection was found in 18 patients and ofChlamydia psittaci infection in three patients. Influenza virus type A was the cause of infection in 15 patients. One patient had infection with influenza virus type B, one patient with parainfluenza virus type 1, and three patients with respiratory syncytial virus. In 20 patients there was evidence of infection with more than one microorganism. No etiological agent was found in 27 patients. SinceStreptococcus pneumoniae was the predominant etiological agent penicillin should be drug of first choice in patients with pneumonia who need treatment in hospital. In young adults, however, the high frequency ofMycoplasma pneumoniae infection would justify the use of erythromycin or doxycycline as drug of first choice.

Evolution of human endogenous retroviral sequences: a conceptual account.

Abstract.Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic ‘junk’.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a Multi-author Review)

Classification and nomenclature of endogenous retroviral sequences (ERVs): Problems and recommendations

The genomes of many species are crowded with repetitive mobile sequences. In the case of endogenous retroviruses (ERVs) there is, for various reasons, considerable confusion regarding names assigned to families/groups of ERVs as well as individual ERV loci. Human ERVs have been studied in greater detail, and naming of HERVs in the scientific literature is somewhat confusing not just to the outsider. Without guidelines, confusion for ERVs in other species will also probably increase if those ERVs are studied in greater detail. Based on previous experience, this review highlights some of the problems when naming and classifying ERVs, and provides some guidance for detecting and characterizing ERV sequences. Because of the close relationship between ERVs and exogenous retroviruses (XRVs) it is reasonable to reconcile their classification with that of XRVs. We here argue that classification should be based on a combination of similarity, structural features, (inferred) function, and previous nomenclature. Because the RepBase system is widely employed in genome annotation, RepBase designations should be considered in further taxonomic efforts. To lay a foundation for a phylogenetically based taxonomy, further analyses of ERVs in many hosts are needed. A dedicated, permanent, international consortium would best be suited to integrate and communicate our current and future knowledge on repetitive, mobile elements in general to the scientific community.

Cancer morbidity in blood recipients—Results of a cohort study

Blood transfusions involve the transfer of relatively large volumes of body fluids and cellular material between individuals. A variety of pathogens like viruses, some of which are associated with development of certain tumours, are known to be transmitted by this route. Blood recipients were identified during 1981-1982 in the register of the hospital blood centre, and in-patients by the in-patient and discharge register of the hospital. Tumour occurrence and vital status were determined by means of the population-based regional tumour register. Age, gender and calendar-year specific rates from the general population were used to calculate expected values. In a cohort study of 3177 blood recipients, increased numbers of malignant lymphomas [13 vs. 4.8 expected, standard morbidity ratio (SMR) 2.70 95% confidence interval (CI) 1.44-4.62] and skin cancers [12 vs. 5.2 expected, SMR 2.29, 95% CI 1.19-4.01] were seen 3 to 9 years after transfusion. In a second cohort study of 29,910 hospitalised patients, a total of 37 (29.8 expected) malignant lymphomas was found in 28,338 patients with no transfusion and 10 (2.73 expected) in 1572 patients with a transfusion, 3 to 9 years after the hospitalisation. The incidence rate ratio between these groups was 3.11 (95% CI 1.56-6.20) using a Mantel-Haenszel estimator with age stratification. Non-melanomatous skin cancers had an incidence ratio of 2.74 (95% CI 1.25-6.00). We conclude that, in the cohorts discussed here, malignant lymphomas and skin cancer occur more often in blood recipients than in controls. It remains to be established whether this is due to factors covariating with transfusion or by the transfusion itself. Further studies on these putative associations are warranted, as are analytical studies of the epidemiology of malignant lymphomas, especially non-Hodgkins lymphoma, whose aetiology is still poorly understood.

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis

BackgroundStreptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.ResultsThe analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria.ConclusionsThe PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.

SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses

Summary.Coronaviruses are etiologic agents of respiratory and enteric diseases in humans and in animals. In this study, a one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on SYBR Green chemistry and degenerate primers was developed for the generic detection of coronaviruses. The primers, designed in the open reading frame 1b, enabled the detection of 32 animal coronaviruses including strains of canine coronavirus, feline coronavirus, transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). A specific amplification was also observed with the human coronaviruses (HCoV) HCoV-NL63, HCoV-OC43, HCoV-229E and severe acute respiratory syndrome coronavirus (SARS-CoV). The real-time RT-PCR detected down to 10 cRNA copies from TGEV, BCoV, SARS-CoV and IBV. In addition, the assay exhibited a high sensitivity and specificity on clinical samples from different animal species. The developed assay represents a potential tool for laboratory diagnostics and for detecting still uncharacterized coronaviruses.

Identification of Novel Porcine Endogenous Betaretrovirus Sequences in Miniature Swine

ABSTRACT PCR amplification of genomic DNA from miniature swine peripheral blood lymphocytes, using primers corresponding to highly conserved regions of the polymerase (pol) gene, allowed the identification of two novel porcine endogenous retrovirus (PERV) sequences, PMSN-1 and PMSN-4. Phylogenetic analyses of the nucleotide sequences of PMSN-1 and PMSN-4 revealed them to be most closely related to betaretroviruses. The identification of PERVs belonging to theBetaretrovirus genus shows that endogenous retroviruses of this family are more broadly represented in mammalian species than previously appreciated. Both sequences contained inactivating mutations, implying that these particular loci are defective. However, Southern blot analysis showed additional copies of closely related proviruses in the miniature swine genome. Analyses of fetal and adult miniature swine tissues revealed a broad mRNA expression pattern of both PMSN-1 and PMSN-4. The most abundant expression was detected in whole bone marrow c-kit +(CD117+) progenitor bone marrow cells, fetal liver, salivary gland, and thymus. It appears unlikely that functional loci encoding these novel PERV sequences exist, but this remains to be established. The betaretrovirus sequences described in this report will allow such investigations to be actively pursued.