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Human umbilical cord blood cells do not improve sensorimotor or cognitive outcome following transient middle cerebral artery occlusion in rats

The present study investigated effects of human umbilical cord blood (HUCB) cells on sensorimotor, cognitive, and histological outcome in rats subjected to transient middle cerebral artery occlusion (MCAO). Halothane anesthetized adult male Wistar rats were subjected to transient MCAO for 2 h. HUCB cells (mononuclear 1-5x10(7) or Lin(-) cells 1-5x10(5)) were administered intravenously after 24 h recovery. The limb-placing test was performed on postoperative days 2, 4, 6, 9, 12, 16, and 20. In addition, beam-walking and cylinder tests were used to assess sensorimotor function at baseline, and on postoperative days 4, 12, and 20. Morris water-maze was used to assess cognitive performance on postoperative days 22-24. Subsequently, rats were perfused for measurement of infarct volumes and detection of HUCB cells by immunohistochemistry (MAB1281). MCAO rats showed a partial spontaneous recovery in sensorimotor function during the follow-up. However, the recovery profile was similar in MCAO controls and in MCAO rats that received HUCB cells. HUCB did not affect impaired water-maze performance of MCAO rats. Only few human nuclei-specific MAB1281-positive cells were detected in the ipsilateral hemisphere in MCAO rats that received HUCB cells. Infarct volumes did not differ between the experimental groups. A group of additional rats were used to further study biodistribution of intravenously given (111)In-oxine-labelled mononuclear HUCB cells in MCAO and sham-operated rats. SPECT imaging data indicated a high tracer uptake in the lung, liver, spleen, and kidney, but not in the brain immediately after administration or 24 h post-administration. The present study suggests that HUCB cells do not improve functional recovery or histological outcome in MCAO rats after systemic administration because of limited migration of cells in the ischemic brain.

The SPECT imaging shows the accumulation of neural progenitor cells into internal organs after systemic administration in middle cerebral artery occlusion rats

The regenerative potential of stem cells from various sources has been under intense investigation in the experimental models of cerebral ischemia. To end up with a restorative therapeutic treatment, it is crucial to get the cell transplants to the site of injury. Here, we evaluated the feasibility of small animal SPECT/CT in assessing the definite accumulation of (111)In-oxine-labeled human embryonic stem (ES) cell-derived neural progenitors and rat hippocampal progenitors after intravenous or intra-arterial administration (femoral vein vs. common carotid artery) in middle cerebral artery occlusion (MCAO) and sham-operated rats. Cell detection was carried out immediately and 24h after the infusion using a SPECT/CT device. The results showed that after intravenous injections both cell types accumulated primarily into internal organs, instead of brain. In contrast, after intra-arterial injection, a weak signal was detected in the ischemic hemisphere. Additional studies showed that the detection sensitivity of SPECT/CT device was approximately 1000 (111)In-oxine-labeled cells and labeling did not affect the cell viability. In conclusion, a small animal SPECT is powerful technique to study the whole body biodistribution of cell-based therapies. Our data showed that intravenous administration is not an optimal route to deliver neural progenitor cell-containing transplants into the brain after MCAO in rats.

Antigenic regions of poliovirus type 3/sabin capsid proteins recognized by human sera in the peptide scanning technique

We used the peptide scanning technique to identify regions of poliovirus type 3/Sabin capsid proteins that bind antibodies from human immune sera. Several reactive regions were seen in VP1, VP2, and VP3 while peptides resembling VP4 did not bind antibodies. Peptides derived from sequences of the previously known antigenic sites 1 and 3 were recognized to a moderate degree. Peptides imitating the four loops in the closed ends of the beta barrels or the alpha helical CD insertions of VP1, VP2 or VP3, whether exposed in the crystal structure or not, all represented major reactivity in the scans. In VP1 several additional reactive regions were found in the amino terminal quarter of the protein, which is buried in the crystal structure, and in a partially exposed region close to but separated from the carboxy terminus. In VP2 the nonexposed peak activities clustered in a bridge-like structure spanning from the outer to the inner surface of the capsid shell. Likewise, most of the novel antigenic regions of VP3 clustered in an internal location and partially composed of beta sheets with a conserved amino acid sequence. Whether any of the novel antigenic sites is capable of inducing neutralizing antibodies is not known.

Diagnostic rapid tests for acute hantavirus infections: specific tests for Hantaan, Dobrava and Puumala viruses versus a hantavirus combination test.

Hantaviruses infecting humans in Eurasia include Hantaan, Seoul, Puumala and the closely related Dobrava and Saaremaa viruses. These viruses are causative agents of hemorrhagic fever with renal syndrome (HFRS), which is recognized as a severe health care problem in several countries. Diagnostics of hantavirus infections relies on serology, performed principally with enzyme immunoassay (EIA) or immunofluorescence assay (IFA). We developed four 5-min immunochromatographic IgM-antibody tests for diagnostics of acute Puumala, Dobrava and Hantaan virus infections and a similar combination test to detect all Eurasian pathogenic hantavirus infections. We evaluated the assays using 100 fingertip blood samples collected randomly from Finnish volunteers, 28 confirmed hantavirus IgM-negative sera, and 77 sera from patients with acute infections of various hantaviruses. The specificities and sensitivities of the Puumala-, Dobrava- and Hantaan virus -specific tests varied from 96 to 100%, whereas, the combination test showed 96% specificity and 80 to 93% sensitivity. Cross-reactions were observed commonly between the Dobrava and the Hantaan virus tests, but only rarely between the Puumala and the Hantaan virus, or the Puumala and the Dobrava virus, tests. Altogether, the rapid tests showed less cross-reactivity than the respective EIA tests. According to the results, the performance of these tests meets well the requirements for diagnostic use. Nevertheless, the specific one-antigen tests were markedly more sensitive than the combination test. However, if optimized, a combination test would be suitable for regions where several hantaviruses circulate.

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.

Novel Peptide Inhibitors of Human Kallikrein 2

Human kallikrein 2 (hK2) is a serine protease produced by the secretory epithelial cells in the prostate. Because hK2 activates several factors participating in proteolytic cascades that may mediate metastasis of prostate cancer, modulation of the activity of hK2 is a potential way of preventing tumor growth and metastasis. Furthermore, specific ligands for hK2 are potentially useful for targeting and imaging of prostate cancer and for assay development. We have used enzymatically active recombinant hK2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. Using libraries expressing 10 or 11 amino acids long linear peptides, we identified six different peptides binding to hK2. Three of these were shown to be specific and efficient inhibitors of the enzymatic activity of hK2 toward a peptide substrate. Furthermore, the peptides inhibited the activation of the proform of prostate-specific antigen by hK2. Amino acid substitution analyses revealed that motifs of six amino acids were required for the inhibitory activity. These peptides are potentially useful for treatment and targeting of prostate cancer.

T Cell Epitope-Containing Peptides of the Major Dog Allergen Can f 1 as Candidates for Allergen Immunotherapy

One prerequisite for developing peptide-based allergen immunotherapy is knowing the T cell epitopes of an allergen. In this study, human T cell reactivity against the major dog allergen Can f 1 was investigated to determine peptides suitable for immunotherapy. Seven T cell epitope regions (A–G) were found in Can f 1 with specific T cell lines and clones. The localization of the epitope regions shows similarities with those of the epitopes found in Bos d 2 and Rat n 1. On average, individuals recognized three epitopes in Can f 1. Our results suggest that seven 16-mer peptides (p15–30, p33–48, p49–64, p73–88, p107–122, p123–138, and p141–156), each from one of the epitope regions, show widespread T cell reactivity in the population studied, and they bind efficiently to seven HLA-DRB1 molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominant in Caucasian populations. Therefore, these peptides are potential candidates for immunotherapy of dog allergy.

Functionalization of mesoporous silicon nanoparticles for targeting and bioimaging purposes

Carboxylic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi-NP) were synthesized, and their opsonization and targeting properties were studied in vitro alongside with in vivo biodistribution. The carboxyl groups on UnTHCPSi were utilized to further functionalize the nanoparticles. In order to reduce the opsonization of the UnTHCPSi-NPs, different sized polyethylene glycol (PEG) were conjugated or adsorbed to the NPs surface. The latter approach, based on hydrophobic interaction, turned out to be more effective in reducing the opsonization and improving the stability of the nanoparticle suspension. The most abundant opsonins after plasma incubation were fibrinogen precursors and IgG. Furthermore, the targeting properties of UnTHCPSi-NPs were studied in vitro with antibodies against glutathione S-transferase (anti-GST). PEGylated NPs conjugated with anti-GST bound to GST-agarose in human plasma nearly 35-fold compared to control NPs, indicating that UnTHCPSi-NPs are suitable for targeting in physiological environment. The in vivo biodistribution inmice revealed that PEGylated UnTHCPSi-NPs, accumulate fast into the liver and the spleen, regardless of the reduced opsonization in vitro. However, autoradiography and transmission electron microscopy showed that majority of the NPs still remained in hepatic blood vessels and sinusoids suggesting a possibility to utilize them as a sustained release platform for payload molecules.

New Immunochromatographic Rapid Test for Diagnosis of Acute Puumala Virus Infection

ABSTRACT 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 μl 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 μ-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.

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.