BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium.

Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17 mm; AdLacZ: 3.07 ± 1.33 mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD.

SproutAngio: an open-source bioimage informatics tool for quantitative analysis of sprouting angiogenesis and lumen space.

Three-dimensional image analyses are required to improve the understanding of the regulation of blood vessel formation and heterogeneity. Currently, quantitation of 3D endothelial structures or vessel branches is often based on 2D projections of the images losing their volumetric information. Here, we developed SproutAngio, a Python-based open-source tool, for fully automated 3D segmentation and analysis of endothelial lumen space and sprout morphology. To test the SproutAngio, we produced a publicly available in vitro fibrin bead assay dataset with a gradually increasing VEGF-A concentration ( https://doi.org/10.5281/zenodo.7240927 ). We demonstrate that our automated segmentation and sprout morphology analysis, including sprout number, length, and nuclei number, outperform the widely used ImageJ plugin. We also show that SproutAngio allows a more detailed and automated analysis of the mouse retinal vasculature in comparison to the commonly used radial expansion measurement. In addition, we provide two novel methods for automated analysis of endothelial lumen space: (1) width measurement from tip, stalk and root segments of the sprouts and (2) paired nuclei distance analysis. We show that these automated methods provided important additional information on the endothelial cell organization in the sprouts. The pipelines and source code of SproutAngio are publicly available ( https://doi.org/10.5281/zenodo.7381732 ).

Molecular identification of Taenia spp. in the Eurasian lynx (Lynx lynx) from Finland.

Cestodes of the genus Taenia are parasites of mammals, with mainly carnivores as definitive and herbivores as intermediate hosts. Various medium-sized cats, Lynx spp., are involved in the life cycles of several species of Taenia. The aim of the present study was to identify Taenia tapeworms in the Eurasian lynx (Lynx lynx) from Finland. In total, 135 tapeworms from 72 lynx were subjected to molecular identification based on sequences of 2 mtDNA regions, the cytochrome c oxidase subunit 1 and the NADH dehydrogenase subunit 1 genes. Available morphological characters of the rostellar hooks and strobila were compared. Two species of Taenia were found: T. laticollis (127 samples) and an unknown Taenia sp. (5 samples). The latter could not be identified to species based on mtDNA, and the rostellar hooks were short relative to those described among other Taenia spp. recorded in felids from the Holarctic region. In the phylogenetic analyses of mtDNA sequences, T. laticollis was placed as a sister species of T. macrocystis, and the unknown Taenia sp. was closely related to T. hydatigena and T. regis. Our analyses suggest that these distinct taeniid tapeworms represent a putative new species of Taenia. The only currently recognized definitive host is L. lynx and the intermediate host is unknown.

Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

Kinship, dispersal and hantavirus transmission in bank and common voles.

Hantaviruses are among the main emerging infectious agents in Europe. Their mode of transmission in natura is still not well known. In particular, social features and behaviours could be crucial for understanding the persistence and the spread of hantaviruses in rodent populations. Here, we investigated the importance of kinclustering and dispersal in hantavirus transmission by combining a fine-scale spatiotemporal survey (4 km2) and a population genetics approach. Two specific host-hantavirus systems were identified and monitored: the bank vole Myodes, earlier Clethrionomys glareolus--Puumala virus and the common vole Microtus arvalis--Tula virus. Sex, age and landscape characteristics significantly influenced the spatial distribution of infections in voles. The absence of temporal stability in the spatial distributions of viruses suggested that dispersal is likely to play a role in virus propagation. Analysing vole kinship from microsatellite markers, we found that infected voles were more closely related to each other than non-infected ones. Winter kin-clustering, shared colonies within matrilineages or delayed dispersal could explain this pattern. These two last results hold, whatever the host-hantavirus system considered. This supports the roles of relatedness and dispersal as general features for hantavirus transmission.

Serological survey for viral pathogens in Turkish rodents.

Wild rodents (n = 330) were trapped around the villages of Altindere and Cosandere (Maçka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihemsin, Rize Province), and Bozdag (Odemis, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.

Is there an association of Pneumocystis infection with the presence of arena-, hanta-, and poxvirus antibodies in wild mice and shrews in Finland?

As part of studies on the nature of the endemic virus infections in natural rodent hosts, the possible association of cyst forms of Pneumocystis spp. with the presence of hanta-, cowpox-, and arenavirus antibodies in wild mice (Apodemus flavicollis, N=105; Apodemus agrarius, N=63; Micromys minutus, N=50) and the common shrew (Sorex araneus, N=101) was studied in south-central Finland. One hantavirus (Saaremaa virus, SAAV) seropositive A. agrarius, and 2 cowpoxvirus (CPXV) seropositive S. araneus were detected, and antibodies against an arenavirus (Lymphocytic choriomeningitis virus, LCMV) were found in all 3 mouse species but not in shrews. Cyst forms of Pneumocystis spp. were detected in all species except A. agrarius. There was no significant association between virus antibodies (LCMV in mice, and CPXV in shrews) and cyst forms of Pneumocystis in any of the species. Concurrent presence of virus antibodies (LCMV) and cyst forms of Pneumocystis were detected only in 1 M. minutus. In conclusion, we found no evidence of any association between Pneumocystis and antibodies to any of the viruses tested.

Hantavirus and arenavirus antibody prevalence in rodents and humans in Trentino, Northern Italy.

The spatial and temporal distribution of hantavirus and arenavirus antibody-positive wild rodents in Trentino, Italy, was studied using immunofluorescence assays (IFA) in two long-term sites trapped in 2000-2003, and six other sites trapped in 2002. The overall hantavirus seroprevalence in the bank voles, Clethrionomys glareolus (n=229) screened for Puumala virus (PUUV) antibodies was 0.4%, and that for Apodemus flavicollis mice (n=1416) screened for Dobrava virus (DOBV) antibodies was 0.2%. Antibodies against lymphocytic choriomeningitis virus (LCMV) were found in 82 (5.6%) of the 1472 tested rodents; the seroprevalence being 6.1% in A. flavicollis (n=1181), 3.3% in C. glareolus (n=276), and 14.3% in Microtus arvalis (n=7). Of the serum samples of 488 forestry workers studied by IFA, 12 were LCMV-IgG positive (2.5%) and one DOBV-IgG positive (0.2%), however, the latter could not be confirmed DOBV-specific with a neutralization assay. Our results show a widespread distribution but low prevalence of DOBV in Trentino, and demonstrate that the arenavirus antibodies are a common finding in several other rodent species besides the house mouse.

Pneumocystosis in wild small mammals from California.

Cyst forms of the opportunistic fungal parasite Pneumocystis carinii were found in the lungs of 34% of the desert shrew, Notiosorex crawfordi (n = 59), 13% of the ornate shrew, Sorex ornatus (n = 55), 6% of the dusky-footed wood rat, Neotoma fuscipes (n = 16), 2.5% of the California meadow vole, Microtus californicus (n = 40), and 50% of the California pocket mouse, Chaetodipus californicus (n = 2) caught from southern California between February 1998 and February 2000. Cysts were not found in any of the harvest mouse, Reithrodontomys megalotis (n = 21), California mouse, Peromyscus californicus (n = 20), brush mouse, Peromyscus boylii (n = 7) or deer mouse, Peromyscus maniculatus (n = 4) examined. All infections were mild; extrapulmonary infections were not observed. Other lung parasites detected were Hepatozoon sp./spp. from M. californicus and Notiosorex crawfordi, Chrysosporium sp. (Emmonsia) from M. californicus, and a nematode from S. ornatus.

Haemogregarines of the genus Hepatozoon (Apicomplexa: Adeleina) in rodents from northern Europe.

We studied the prevalence and distribution of Hepatozoon infections in small rodents from Finland and other areas in northern Europe. Hepatozoon infections were more common in voles (Arvicolinae) than mice (Murinae) and more prevalent in voles of the genus Clethrionomys than in voles of the genus Microtus. Transmission electron microscopical examination of Hepatozoon erhardovae Krampitz, 1964 from bank voles Clethrionomys glareolus (Schreber) showed that intracellular lung meronts were located in alveolar septa. Meronts consisted of varying numbers of merozoites packed with amylopectin vacuoles inside electron-lucent parasitophorous vacuole. The size of the meronts was approximately 19 x 14 microm. Monozoic or dizoic cysts were frequent findings in the lung alveoles; the size of cysts was approximately 10 x 6 microm. Gametocytes were found inside eosinophilic granulocytes in the capillaries of lung tissue. Ultrastructurally, micronemes, microtubules, mitochondria, nuclei and lipid droplets were visible.

Molecular evolution of puumala hantavirus in Fennoscandia: phylogenetic analysis of strains from two recolonization routes, Karelia and Denmark.

Like other members of the genus HANTAVIRUS: in the family BUNYAVIRIDAE:, Puumala virus (PUUV) is thought to be co-evolving with its natural host, the bank vole Clethrionomys glareolus. To gain insight into the evolutionary history of PUUV in northern Europe during the last post-glacial period, we have studied wild-type PUUV strains originating from areas along two postulated immigration routes of bank voles to Fennoscandia. Full-length sequences of the S RNA segment and partial sequences (nt 2168-2569) of the M segment were recovered by RT-PCR directly from bank vole tissues collected at three locations in Russian Karelia and one location in Denmark. Phylogenetic analysis showed that strains from Karelia and Finland belong to the same genetic lineage, supporting the hypothesis that PUUV spread to present Finland via a Karelian land-bridge. The Danish PUUV strains showed no particularly close relatedness to any of the known PUUV strains and formed a distinct phylogenetic lineage on trees calculated for both S and M segment sequences. Although no direct link between the Danish PUUV strains and those of the southern Scandinavian lineage was found, within the S segment of Danish PUUV strains, two regions with higher similarity to either northern Scandinavian or - to a less extent - southern Scandinavian genetic lineages were revealed, suggesting evolutionary connections of their precursors.

Ultrastructure of Frenkelia sp. from a Norwegian lemming in Finland.

An apparently healthy Norwegian lemming (Lemmus lemmus) caught in northern Finland was observed to have a whitish body 0.5 to 1.0 mm in diameter in the external layer of the cerebral cortex. By light microscopy a highly lobulated cyst of Frenkelia sp. was observed. By transmission electron microscopy lemmus) collected in the cyst wall was seen consisting of a parasitophorous vacuolar membrane, an underlying electron-dense layer and a granular layer. The membrane was only slightly convoluted. The protrusions of the cyst wall appeared round but were often not distinctive. A very thin septum divided the interior of the cyst into compartments packed with bradyzoites and maturing zoites. The bradyzoites were elongate measuring 5-8 x 1.5-2 microm. This is the first electron microscopical study of Frenkelia sp. from L. lemmus.

Disease patterns in field and bank vole populations during a cyclic decline in central Finland.

Declining field vole (Microtus agrestis) and bank vole (Clethrionomys glareolus) populations were sampled (117 field voles and 34 bank voles) in south-central Finland during the winter of 1988-89. The last surviving field voles were caught in April and bank voles in February. A subsample (16) of the April field voles were taken live to the laboratory for immunosuppression. The histopathology of the main internal organs and the presence of aerobic bacteria and certain parasites were studied. In the lungs, an increase in lymphoid tissue, probably caused by infections, was the most common finding (52% of all individuals). The prevalences in the voles, in the whole material, of Chrysosporium sp. and Pneumocystis carinii in lungs were 13 and 10% in field voles, and 9 and 0% in bank voles, respectively. Cysts of Taenia mustelae (9 and 27%) were the most common pathological changes in the liver. Enteritis was also rather common (14 and 34%). In field voles the prevalences of Frenkelia sp. in the brain and Sarcocystis sp. in leg muscles were low (both 6%). Bordetella bronchiseptica was commonly (31%) isolated from field vole lungs and Listeria monocytogenes from the intestines (34%). Salmonella spp. could not be found. The dynamics and abundance of inflammations in the lungs and intestines, as well as B. bronchiseptica isolations from the lungs, indicate that obvious epidemics took place in declining vole populations. Of the Luhanka subsample of 16 field voles brought to the laboratory in April, one died of listeriosis, two of Bordetella, and five died for unknown reasons. Even if small mustelids are the driving force in microtine cycles, it is possible that diseases also contribute to the decline.

Seasonal dynamics of Pneumocystis carinii in the field vole, Microtus agrestis, and in the common shrew, Sorex araneus, in Finland.

Seasonal dynamics of Pneumocystis carinii in the field vole, Microtus agrestis, and in the common shrew, Sorex araneus, were investigated in southern and central Finland by microscopical examination of methenamine silver-stained tissue sections. In both host species at both localities the number of P. carinii cyst forms was highest in late autumn (November). In S. araneus, prevalence was higher than in M. agrestis during all seasons. None of the animals was heavily infected or apparently ill, and neither species showed any extrapulmonary dissemination. In this study covering an increase phase and 4 peak host-density phases of the vole cycle, the occurrence of P. carinii seemed to be related to the population density of M. agrestis.

Isolation and characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus agrarius) in Estonia.

Dobrava hantavirus (DOB) was isolated from the striped field mouse (Apodemus agrarius) trapped on Saaremaa Island, Estonia, and its genetic and antigenic characteristics were subsequently analysed. Phylogenetic analysis showed that the Estonian DOB strain, together with several wild strains carried by Apodemus agrarius, forms a well-supported lineage within the DOB clade. The topography of the trees calculated for the S, M and L nucleotide sequences of the Estonian DOB suggests a similar evolutionary history for all three genes of this virus and, therefore, the absence of heterologous reassortment in its evolution. A cross-neutralization comparison of the Estonian virus with the prototype DOB, isolated from a yellow-necked mouse (A. flavicollis) in Slovenia, revealed 2- to 4-fold differences in the end-point titres of rabbit and human antisera. When studied with a panel of 25 monoclonal antibodies (MAbs), the Estonian and Slovenian DOB isolates showed similar antigenic patterns that could be distinguished by two MAbs. Genetic comparison showed sequence differences in all three genome segments of the two DOB isolates, including an additional N-glycosylation site in the deduced sequence of the G2 protein from the Estonian virus. Whether any of these mutations relates to the different rodent hosts rather than to the distant geographical origin of the two isolates remains to be resolved. Taken together, our observations suggest that A. agrarius, which is known to harbour Hantaan virus in Asia, carries another hantavirus, DOB, in north-east Europe.

Lung parasites of least weasels in Finland.

Because of their constant exposure to normal rodent definitive hosts, least weasels (Mustela nivalis) were trapped in southern Finland in late fall 1994 and examined for lung parasites. Histological examination showed that 46% of the weasels (n = 46) were infected with adiaspores identified as Chrysosporium sp. Granulomas surrounding the adiaspores consisted of mostly unorganized layers of mononuclear cells. The adiaspores from least weasels were much smaller than those reported from their prey animals. Infection with Pneumocystis carinii also was found in two weasels.