Phosphorus mitigation during springtime runoff by amendments applied to grassed soil.

Permanent grass vegetation on sloping soils is an option to protect fields from erosion, but decaying grass may liberate considerable amounts of dissolved reactive P (DRP) in springtime runoff. We studied the effects of freezing and thawing of grassed soil on surface runoff P concentrations by indoor rainfall simulations and tested whether the peak P concentrations could be reduced by amending the soil with P-binding materials containing Ca or Fe. Forty grass-vegetated soil blocks (surface area 0.045 m, depth 0.07 m) were retrieved from two permanent buffer zones on a clay and loam soil in southwest Finland. Four replicates were amended with either: (i) gypsum from phosphoric acid processing (CaSO × 2HO, 6 t ha), (ii) chalk powder (CaCO, 3.3 t ha), (iii) Fe-gypsum (6 t ha) from TiO processing, or (iv) granulated ferric sulfate (Fe[SO], 0.7 t ha), with four replicates serving as untreated controls. Rainfall (3.3 h × 5 mm h) was applied on presaturated samples set at a slope of 5% and the surface runoff was analyzed for DRP, total dissolved P (TDP), total P (TP), and suspended solids. Rainfall simulation was repeated twice after the samples were frozen. Freezing and thawing of the samples increased the surface runoff DRP concentration of the control treatment from 0.19 to 0.46 mg L, up to 2.6-3.7 mg L, with DRP being the main P form in surface runoff. Compared with the controls, surface runoff from soils amended with Fe compounds had 57 to 80% and 47 to 72% lower concentrations of DRP and TP, respectively, but the gypsum and chalk powder did not affect the P concentrations. Thus, amendments containing Fe might be an option to improve DRP retention in, e.g., buffer zones.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Prevalence of antibodies against Chlamydia trachomatis and incidence of C. trachomatis-induced reactive arthritis in an early arthritis series in Finland in 2000.

OBJECTIVE: To study the prevalence of different serotypes of Chlamydia trachomatis antibodies and the incidence of C. trachomatis-induced reactive arthritis (ReA) among patients with early arthritis in a defined population. METHODS: Serum samples were collected from a cohort of 122 adult patients in the age group 18-65 years included in the Kuopio 2000 Arthritis Survey. Antibodies against C. trachomatis serotypes C, E, and G were studied using enzyme immunoassay (EIA) tests among patients and in a control cohort of 78 adults without any joint symptoms. The incidence assessment for Chlamydia-induced ReA was based on a ligase chain reaction (LCR) test in urine and clinical symptoms and signs appropriate for ReA. RESULTS: Of 122 patients, with the baseline diagnosis of rheumatoid arthritis (RA) in 11, spondyloarthropathy (SpA) in 28, and undifferentiated arthritis (UA) in 83 cases, 42 (34%) showed immunoglobulin (Ig)G or IgA antibodies against at least one serotype C, E, or G. Among the patients with UA the prevalence was significantly increased compared with the controls (p = 0.010). C. trachomatis-induced ReA arthritis was diagnosed in only three patients with the LCR test. On this basis the incidence of C. trachomatis-induced arthritis was 5.4/100 000 [95% confidence interval (CI) 1.1-15.7] in the age group 18-65 years. CONCLUSION: Antibodies against C. trachomatis were most common in patients with UA reflecting the fact that cases with chlamydia-induced ReA are included in this subgroup.

The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes.

BACKGROUND: Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing. OBJECTIVE: Knowing the T cell epitopes in allergens is a prerequisite for developing novel preparations for allergen immunotherapy. METHODS: Specific T cell lines were generated with recombinant Equ c 1 from the peripheral blood mononuclear cells (PBMCs) of 10 horse-allergic subjects. For determining T cell epitopes, the lines were stimulated with 16mer synthetic Equ c 1 peptides overlapping by 14 amino acids. The binding capacity of Equ c 1 peptides to human leucocyte antigen class II molecules was determined by the competitive ELISA. RESULTS: The major horse allergen Equ c 1 resembles two other lipocalin allergens, the major cow allergen Bos d 2 and the major dog allergen Can f 1, in that it is weakly stimulatory for the PBMCs of sensitized subjects. Moreover, the T cell epitopes of Equ c 1 are clustered in a few regions along the molecule, as is the case with Bos d 2 and Can f 1. Similar to Bos d 2, Equ c 1 contains one immunodominant epitope region at the carboxy-terminal end of the molecule. The T cell lines of eight horse-allergic subjects out of 10 showed strong reactivity to one or both of the two overlapping peptides, p143-158 and p145-160, in this region. The region probably contains two overlapping epitopes. CONCLUSION: The 18mer peptide p143-160 from the immunodominant region of Equ c 1 is a potential candidate for the peptide-based immunotherapy of horse-sensitized subjects.

SPECT/CT imaging of baculovirus biodistribution in rat.

Non-invasive imaging provides essential information regarding the biodistribution of gene therapy vectors and it can also be used for the development of targeted vectors. In this study, we have utilized micro Single-photon emission computed tomography to visualize biodistribution of a (99m)Tc-polylys-ser-DTPA-biotin-labelled avidin-displaying baculovirus, Baavi, after intrafemoral (i.f.), intraperitoneal (i.p.), intramuscular (i.m.) and intracerebroventricular (i.c.v.) administration. The imaging results suggest that the virus can spread via the lymphatic network after different administration routes, also showing accumulation in the nasal area after systemic administration. Extensive expression in the kidneys and spleen was seen after i.p. administration, which was confirmed by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Additionally, transduction of kidneys was seen with i.m. and i.f. administrations. We conclude that baculovirus may be beneficial for the treatment of kidney diseases after i.p. administration route.

Magnetic resonance imaging of viral particle biodistribution in vivo.

We describe here a technique for the visualization of viral vector delivery by magnetic resonance imaging (MRI) in vivo. By conjugating avidin-coated baculoviral vectors (Baavi) with biotinylated ultra-small superparamagnetic iron oxide particles (USPIO), we are able to produce vector-related MRI contrast in the choroid plexus cells of rat brain in vivo over a period of 14 days. Ten microlitres of 2.5 x 10(10) PFU/ml nuclear-targeted LacZ-encoding Baavi with bUSPIO coating was injected into rat brain ventricles and visualized by MRI at 4.7 T. As baculoviruses exhibit restricted cell-type specificity in the rat brain, altered MRI contrast was detected in the choroid plexus of the injected ventricles. No specific signal loss was detected when wild-type baculoviruses or intact biotinylated USPIO particles were injected into the lateral ventricles. Cryosectioned brains were stained for nuclear-targeted beta-galactosidase gene expression, which was found to colocalize with MRI contrast. This study provides the first proof of principle for robust and non-invasive viral vector MRI by using avidin-displaying viruses in vivo. Considering the widespread use of MRI in current medical imaging, the approach is likely to provide numerous future applications in imaging of therapeutic gene transfer.

Effect of temporin A modifications on its cytotoxicity and antimicrobial activity.

Temporin A (TA), a short alpha-helical antimicrobial peptide isolated from the skin of the frog Rana temporaria, is effective against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains. TA interacts directly with the cell membrane of the microorganism and it has been reported to be non-toxic to erythrocytes at concentrations that are antimicrobial. Less is known about the effects on the viability and growth of nucleated eukaryotic cells. In this study we have tested antibacterial and growth-inhibitory properties of TA, its dimeric analogue (TAd), and all-L (TAL L512) and all-D (TAD L512) enantiomeric derivatives of modified TA towards S. aureus and cultured human keratinocytes, respectively. All molecules were antibacterial at concentrations from 1.5 microM to 10 microM. In keratinocyte cultures, TAD L512, as well as TAd, showed cytotoxicity. The original TA and TAL L512 did not affect the viability of the cells at their bacteriolytic concentrations. The growth of keratinocytes in low- and high-calcium media was only slightly inhibited by temporins at concentrations which were antibacterial to S. aureus. This suggests that original TA and its modification, TAL L512, are promising molecules against multiresistant bacterial infections.

Rapid field test for detection of hantavirus antibodies in rodents.

Puumala virus (PUUV) is the causative agent of nephropathia epidemica, a mild form of haemorrhagic fever with renal syndrome. PUUV is transmitted to humans via aerosolized excreta of the infected bank vole (Clethrionomys glareolus). Current methods for screening of the PUUV prevalence among bank vole populations are laborious, combining sampling in the field and subsequent analyses in the laboratory. In order to facilitate animal testing, a new serological immunochromatographic rapid test was developed. The test uses PUUV nucleocapsid protein as antigen, and it detects anti-PUUV IgG antibodies in rodents. With fresh and undiluted bank-vole blood samples (n = 105) the efficacy of the test was 100%, and with frozen and diluted samples (n = 78) the efficacy was 91%. The test was also shown to detect related hantavirus infections in Norway lemmings and sibling voles (n = 31) with 99% efficacy. The test provides an applicable tool for studying PUUV and related hantavirus infections in arvicoline rodents.

Patient evaluation of the care and rehabilitation process in geriatric hospital care.

PURPOSE: To gain a deeper understanding of how elderly persons experience and evaluate the care and rehabilitation process. METHOD: Qualitative interview data from elderly patients were analysed using a grounded theory approach. The patients were interviewed twice, at the beginning of geriatric hospital care and some weeks after discharge. RESULTS: The patient-perceived outcome of the care and rehabilitation process reflected two dimensions; the effect on their health and the quality of the process, i.e. how their needs were met. The analysis revealed that the patients' needs differed during the care and rehabilitation process. It also indicated that patients perceived their needs and the care differently based on their previous experience of the care unit, their perceived trajectory of illness and their "patient character" which represented the patient's definition of himself/herself and the situation. A hypothetical model of the patients' evaluation process has been derived. CONCLUSION: The results indicate the importance of using a process perspective in the assessment and the interpretation of patient-perceived outcome of care and rehabilitation, and that patient expectations, trajectories of illness and the patient character must be taken into consideration.

Use of a peptide based enzyme immunoassay in diagnosis of Chlamydia trachomatis triggered reactive arthritis.

OBJECTIVE: To assess the presence of circulating IgA and IgG antibodies to Chlamydia trachomatis in sera of patients with reactive arthritis (ReA) and other arthritides. METHODS: A peptide based enzyme immunoassay (EIA) was used to study 132 patients divided into 5 groups: C. trachomatis triggered ReA, uroarthritis, enteroarthritis, oligoarthritis, and rheumatoid arthritis (RA). Followup sera were available from 19 patients. RESULTS: An increased prevalence of C. trachomatis antibodies was observed in patients with ReA triggered by C. trachomatis; 18/23 (78%) had IgA and 19/23 (83%) had IgG antibodies. In patient groups with uroarthritis (n = 12), enteroarthritis (n = 56), oligoarthritis (n = 16), and RA (n = 25), C. trachomatis IgA/IgG antibodies were detected in 58%/75%, 27%/21%, 25%/31%, and 20%/32% of patients, respectively. Both the IgA and IgG antibodies were positive in 74%, 50%, 16%, 25%, and 12% of the patients with C. trachomatis triggered ReA, uroarthritis, enteroarthritis, oligoarthritis, and RA, respectively. Based on positivity of both isotypes the sensitivity of the assay was 74% and specificity 84%. In the followup sera, an association between circulating C. trachomatis-specific antibody concentrations and clinical disease outcome of the arthritis was seen in patients with culture-positive C. trachomatis triggered ReA. CONCLUSION: C. trachomatis species-specific peptide EIA correlates well with conventional diagnosis of primary C. trachomatis infection in patients with ReA. This assay may be a valuable contribution to the diagnosis of C. trachomatis triggered ReA.

Comparison of a new immunochromatographic rapid test with a commercial EIA for the detection of Puumala virus specific IgM antibodies.

BACKGROUND: Hantaviruses are associated with two human diseases: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV), which is one of the Hantaviruses, is a causative agent of nephropathia epidemica (NE), a mild form of HFRS. OBJECTIVE: a new 5 min rapid test, POC PUUMALA (Erilab Ltd, Finland), for detecting IgM antibodies to PUUV was evaluated and compared with the commercially available Hantavirus (Puumala) IgM ELISA test (Progen, Germany). Discrepant test results between the two tests were confirmed by a mu-capture reference EIA. STUDY DESIGN: Two hundred and thirty five serum samples, which had earlier been analyzed with the Progen IgM ELISA, were assayed with the POC PUUMALA rapid test. Five persons, without knowing the Progen IgM ELISA test results, interpreted independently the rapid test results. In addition, a panel of 48 serum samples was analyzed in parallel with the rapid test and the Progen IgM ELISA by one technician in daily routine diagnostics in a clinical microbiology laboratory. RESULTS: the agreement between the results of the five interpreters was 95%, and the congruence of the results between individual readers and commercial ELISA test varied from 93 to 96%. Diagnostic efficacy of the rapid test varied between 98 and 99% compared with 96% of the Progen IgM ELISA. The POC PUUMALA rapid test showed higher or similar sensitivity compared with the Progen IgM ELISA, whereas both the tests had similar levels of specificity. CONCLUSIONS: the analytical performance of the POC PUUMALA rapid test was found to be as good or even slightly better than the analytical performance of the Progen IgM ELISA. In addition, the rapid and straightforward procedure makes the POC PUUMALA a feasible tool for the diagnosis of the acute PUUV infection.

Synthesis and antimicrobial activity of the symmetric dimeric form of Temporin A based on 3-N,N-di(3-aminopropyl)amino propanoic acid as the branching unit.

Dimeric derivative of antimicrobial peptide amide Temporin A (TA) was synthesized by using a new branching unit 3-N,N-di(3-aminopropyl)amino propanoic acid (DAPPA), which allows building of the parallelly symmetric alpha-helical structures. Antimicrobial effect of the original peptide amide, its monomeric carboxy (TAc) and novel dimeric (TAd) analogues were tested against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). Both TA and TAd completely inhibited the growth of S. aureus at the concentrations of 5 and 10 microM, respectively, whereas TAc did not show any inhibitory activity. The activities of TAc, TA and TAd correlate directly with the net charges of the molecules, +1, +2 and +4, respectively. Interestingly, TAd displayed antibacterial effect against E. coli at a concentration of 10 microM, where as monomeric TA did not show any activity at concentration as high as 20 microM. The results indicate that the novel structural modification improves the antibacterial properties of Temporin A especially towards Gram-negative bacteria.

New immunochromatographic rapid test for diagnosis of acute Puumala virus infection.

A new immunochromatographic rapid test, POC PUUMALA (Erilab Ltd., Kuopio, Finland), for detection of acute-phase Puumala virus (PUUV) infection was developed based on a highly purified baculovirus-expressed PUUV nucleocapsid protein antigen and lateral immunodiffusion techniques. After addition of sample (5 microl of serum, plasma, or fingertip blood) and buffer, PUUV-specific immunoglobulin M (IgM) antibodies, if present, together with the gold-conjugated anti-human IgM, formed a specific colored line in 5 min. The sensitivity and specificity of the test were evaluated with 200 serum samples and 30 fingertip blood samples. The reference method for the serum samples was a micro-capture enzyme immunoassay (EIA) for IgM and an immunofluorescence assay (IFA) for IgG antibodies. The analytical sensitivity and specificity of the rapid test were 100 and 99%, respectively, for unfrozen serum samples (n = 103; 12 PUUV IgM-positive samples). When freeze-thawed serum samples were used, the sensitivity and specificity were each 97.1% (n = 70; 35 PUUV IgM-positive samples). The specificity of the test was 96.2% for 27 serum samples with nonspecific IgM antibodies or rheumatoid factor (RF). The fingertip blood samples (n = 30) were negative, but they gave clear positive results when spiked with IgM-positive sera (n = 20). The results were in good agreement with the standard diagnostic methods. The rapid performance, the lack of need for refined laboratory equipment, and the high specificity with fresh serum and fingertip blood samples indicate that the developed POC PUUMALA rapid test is a useful tool for fast diagnosis of acute PUUV infection.

Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma.

CONTEXT: Human papillomavirus (HPV) infection has been established as a cause of cervical cancer. Epidemiologic studies suggest that Chlamydia trachomatis infection also confers increased risk for cervical squamous cell carcinoma (SCC). Whether this risk is serotype-specific is unknown. OBJECTIVE: To study the association between exposure to different C trachomatis serotypes and subsequent development of cervical SCC. DESIGN AND SETTING: Longitudinal, nested case-control study within a cohort of 530 000 women who provided samples to serum banks in Finland, Norway, and Sweden. The data files were linked to respective national cancer registries. SUBJECTS: One hundred twenty-eight women who had developed invasive cervical SCC at least 12 months following serum donation. Each case had 3 matched controls. MAIN OUTCOME MEASURE: Risk for the development of cervical SCC by IgG antibodies to 10 different C trachomatis serotypes, adjusted for antibodies to HPV types 16, 18, and 33 and for serum cotinine levels. RESULTS: Of specific C trachomatis serotypes, serotype G was most strongly associated with SCC (adjusted odds ratio [OR], 6.6; 95% confidence interval [CI], 1. 6-27.0). Other serotypes associated with SCC were I (OR, 3.8; 95% CI, 1.3-11.0) and D (OR, 2.7; 95% CI, 1.3-5.6). Presence of serum IgG antibodies to more than 1 serotype increased the adjusted ORs for SCC (P<.001 for trend). CONCLUSIONS: Chlamydia trachomatis serotype G is most strongly associated with subsequent development of cervical SCC. Increasing numbers of exposures to different C trachomatis serotypes also increases risk. Our results strengthen the evidence that there is a link between past C trachomatis infection and cervical SCC.

Chlamydia trachomatis seropositivity is associated both with stillbirth and preterm delivery.

The cause of stillbirth and preterm delivery is often unknown. We studied the prevalence of Chlamydia trachomatis antibodies in mothers with stillbirth and preterm labor. Serum specimens from 72 mothers with stillbirth after the 21st gestational week, and from 48 mothers with preterm delivery between gestational weeks 23 and 29, both from the greater Helsinki area, and cord blood from 96 consecutive liveborn deliveries at the Department of Obstetrics and Gynecology, the University of Helsinki, were studied for antibodies to C. trachomatis immunotypes CJHI, GFK and BED by microimmunofluorescence test. The prevalence of C. trachomatis antibodies was highest, 33.3%, in mothers with stillbirth, 18.8% in mothers with preterm delivery, and 10.4% in cord blood. The IgM seropositivity rate was high among mothers with preterm delivery (8.3%). We conclude that C. trachomatis IgG antibodies are frequently detected in sera from mothers with stillbirth, suggesting past infection, while mothers with preterm delivery often have serum IgM antibodies, suggesting of acute infection.

Development of novel peptide ligands modulating the enzyme activity of prostate-specific antigen.

Prostate-specific antigen (PSA) is a serine proteinase produced mainly by epithelial cells of the prostate. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. The major problem of the PSA determination in early diagnosis is the high false positive rate due to benign prostatic hyperplasia, but the clinical accuracy can be improved by determining the proportions of various molecular forms of PSA. The main biological function of PSA is liquefaction of the seminal gel formed after ejaculation, but PSA has also been suggested to regulate invasiveness and metastatic potential of prostatic tumors. Thus, agents binding to and affecting the function of PSA have the potential to be used for diagnosis and therapy of prostate cancer. We have developed peptides specific for PSA by using cyclic phage display peptide libraries. After deducing the amino acid sequence of the peptides by sequencing the relevant part of phage genome, the peptides were expressed as glutathione-S-transferase (GST) fusion proteins or produced by chemical synthesis. The peptides were shown to bind to PSA specifically as indicated by lack of binding to other related serine proteinases. The binding of the peptides with PSA was strongly inhibited by monoclonal antibodies specific for free PSA and they did not bind to PSA-inhibitor complexes indicating that they bind close to the active site of the enzyme. Most of the peptides enhanced the enzyme activity of PSA against a chromogenic substrate. The affinity of the peptides could be increased by including Zn2+ in the reaction mixture. These results show that peptides that bind to PSA and modulate its enzyme activity can be developed by phage display techniques. These peptides have the potential to be used for targeting of prostatic tumors and diagnostics of prostate cancer.

Detection of Chlamydia trachomatis antibodies by 2 novel tests: rELISA and peptide EIA.

The performance of 2 newly developed enzyme immunoassays (EIAs) intended for the routine serological diagnosis of chlamydial infections was evaluated. rELISA is based on a recombinant lipopolysaccharide antigen which detects chlamydia genus-specific antibodies, and Chlamydia trachomatis EIA is based on a peptide derived from major outer membrane protein and is therefore species-specific. Both tests distinguished patients with tubal factor infertility (TFI) or pelvic inflammatory disease (PID) from the controls. The prevalence of IgA antibodies was higher for the PID patients than for the TFI patients; the finding indicates a more active state of infections for the PID patients. Furthermore, C. trachomatis EIA detected more IgG antibodies in the TFI patients than in patients with non-tubal factor infertility. In conclusion, rELISA detected chlamydial antibodies in general, and C. trachomatis EIA detected species-specific antibodies. These EIA tests may be useful in the serodiagnosis of chlamydial infection.

Detection of Antibodies to Chlamydia trachomatis With Peptide-Based Species-Specific Enzyme Immunoassay.

OBJECTIVE: We have evaluated the sensitivity and specificity of a new synthetic peptide-based species-specific enzyme immunoassay (EIA) for detection of Chlamydia trachomatis IgG and IgA antibodies. METHODS: Synthetic peptides derived from variable domain IV of major outer membrane protein (MOMP) were used as antigen in indirect EIA. IgG and IgA antibodies were measured in parallel with serum samples from C. trachomatis culture positive, culture negative, and antigen positive patients, and women with suspected C. trachomatis infection and blood donors. Sera from children under 15 years of age were used as controls. RESULTS: Culture positive women, culture positive men, and antigen positive women had positive peptide serology in 84.2%, 61.3%, and 93.1% of the cases, respectively. Among C. trachomatis suspected women, the antibody prevalence was 63.6%. Randomly collected blood donors showed a prevalence of 21.5%. Children with C. pneumoniae antibodies determined with the microimmuno-fluorescence (MIF) method did not show any reactivity in the C. trachomatis peptide EIA. CONCLUSIONS: The results suggest that the new EIA test is highly specific for C. trachomatis, and C. pneumoniae antibodies do not interfere. Both IgG and IgA antibodies appear within at least 2 weeks in acute phase of infection among both culture positive and culture negative patients.

Stillbirths and maternal antibodies to Chlamydia trachomatis. A new EIA test for serology.

OBJECTIVE: The relationship between stillbirths and infections during pregnancy. METHODS: Antibodies to 13 different viruses and to Mycoplasma pneumoniae, Treponema pallidum, Toxoplasma gondii, and Chlamydia trachomatis, psittaci, and pneumoniae were studied from sera of 42 mothers with stillbirth and of matched case-control pregnancies. RESULTS: Elevated antibody levels to Chlamydia trachomatis and human parvovirus B19 were observed in seven mothers with stillbirth as compared with one in matched case controls (p-0.03 and p < 0.06, Fisher's exact one- and two-tailed p values). By a novel EIA test for C. trachomatis, based on synthetic polypeptide, 15 positive cases were detected in patients compared to seven in controls. Placental calcifications and fetal growth retardation appeared more often (p < 0.001 and p < 0.05, respectively) in the C. trachomatis- associated cases than in the others. There was no significant relation between stillbirth and the other antibodies. CONCLUSIONS: We suggest C. trachomatis as a hitherto unrecognized possible cause of still-births besides human parvovirus B19. A new EIA test for C. trachomatis, one covering all strains, is promising and makes the future assays more convenient, and may thus make it possible to reduce the rate of stillbirths.

Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response.

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the endpoint titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%, and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pin-bound peptides was needed to be recognised by most NE sera studied.