Smooth muscle hamartoma of the lungs in a Wistar Hannover rat.

Hamartomas are tumor-like masses comprising disorganized normal tissue elements. To date, spontaneous hamartomas have been reported in several organs and tissues in rodents but not in the lungs. Here, we report the first case of a hamartoma in the lungs of a 108-week-old female Wistar Hannover rat. Grossly, a white spot, 7 mm in diameter, was observed on the costal surface of the left lung. Histopathologically, the nodular lesions adjacent to the bronchioles comprised mature smooth muscle cells. The lesion was not encapsulated and spread along the alveolar walls and ducts without compression of the surrounding tissue. In the nodules, elastic fibers enclosed small lumens lined with factor VIII-related antigen-positive endothelial cells. This structure suggested that the nodule mimicked an artery. Moreover, structural abnormalities were observed within the bronchioles and arterioles owing to the increased number of smooth muscle cells in the surrounding tissues. These features suggested that this was a case of tissue malformation rather than a neoplasm, leading to the diagnosis of a smooth muscle hamartoma of the lung.

Pathological Characterization of Spontaneous AA Amyloidosis in Microminipigs.

The minipig has been used as a non-rodent species in nonclinical toxicology studies, but little is known about amyloid A (AA) amyloidosis in this species. Among domestic pigs, reports of AA amyloidosis have been limited to animals with mutations in the N-terminal residue of serum AA (SAA), which is thought to be a primary etiological factor. In this study, we histologically examined 26 microminipigs aged 0.6 to 10 years and observed amyloid deposition in one 0.6-year-old and six 5-year-old or older microminipigs. The amyloid deposits were identified as AA based on mass spectrometry (MS) and immunohistochemistry (IHC). The 0.6-year-old microminipig showed severe deposition in the renal cortex and spleen, whereas 5-year-old or older animals had severe deposition in the renal medulla. MS and IHC detected serum amyloid P-component (SAP) in amyloid deposits in older animals but not in a 0.6-year-old animals. Based on the proteomic analysis and gene sequencing, amino acid mutations of SAA, previously found in domestic pigs, were not involved in the pathogenesis of AA amyloidosis in microminipigs. This study demonstrates that microminipigs with wild-type SAA develop AA amyloidosis and presents the possibility that differences in the environment surrounding amyloid, such as SAP, may influence differences in the pathological phenotype.

A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays.

Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.

Syphilitic orchitis with long-term imaging follow-up and conservative treatment.

An increased number of patients with syphilis was recently reported in Japan and the United States. Syphilitic orchitis, a late complication of syphilis, is a rare disease that presents as testicular swelling. In most cases, a diagnostic orchiectomy is performed because of the possibility of testicular cancer. We report a case treated conservatively with antibiotics considering the possibility of syphilitic orchitis based on the blood test results and describe long-term changes observed in the imaging findings.

Ectopic pancreatic acinar cell carcinoma in the thoracic cavity of F344 rat.

Ectopic pancreatic tissue can occasionally cause inflammation, hemorrhage, stenosis, and invagination, similar to normal pancreatic tissue; however, tumorigenesis is rare. This case report describes an ectopically observed pancreatic acinar cell carcinoma in the thoracic cavity of a female Fischer (F344/DuCrlCrlj) rat. Histopathologically, polygonal tumor cells with periodic acid-Schiff-positive cytoplasmic eosinophilic granules showed solid proliferation and infrequently formed acinus-like structures. Immunohistochemically, the tumor cells were positive for cytokeratin, trypsin, and human B-cell leukemia/lymphoma 10, which specifically reacted with pancreatic acinar cells, and negative for vimentin and human α-smooth muscle actin. Ectopic pancreas develops in the submucosa of the gastrointestinal tract; however, there are few reports of its development and neoplasia in the thoracic cavity. To the best of our knowledge, this is the first report of ectopic pancreatic acinar cell carcinoma in the thoracic cavity of a rat.

Metastatic small-cell carcinoma of the bladder that maintains a complete response to chemoradiation therapy.

Introduction: Small-cell carcinoma of the urinary bladder has a poor prognosis, and no standard treatment has been established. We encountered a case of a patient with metastasis in which complete response and long-term survival were obtained by treating the primary lesion with a combination of irinotecan, carboplatin chemotherapy, and radiation therapy. Case presentation: An 83-year-old man was diagnosed with a bladder tumor with liver metastasis. Small-cell carcinoma was diagnosed via transurethral resection. Second-line chemotherapy with irinotecan and carboplatin and irradiation of the primary lesion were significantly effective. The imaging evaluation showed a complete response. The therapeutic effect was maintained for 1 year, even after the discontinuation of chemotherapy. Conclusion: Irinotecan and carboplatin should be considered for the treatment of small-cell carcinoma of the bladder. Irradiation of the primary lesion may also be useful if the extent of metastasis is low.

Systemic amyloidosis derived from EFEMP1 in a captive Tsushima leopard cat.

In animals, most cases of systemic amyloidosis are of amyloid A type, and the other types of systemic amyloidoses are rare. This study analyzed systemic amyloidosis in a 15-year-old female Tsushima leopard cat. Amyloid deposits strongly positive for Congo red staining were observed in the arterial walls as well as the interstitium in multiple organs. Mass spectrometry-based proteomic analysis with laser microdissection of amyloid deposits identified epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) as a prime amyloidogenic protein candidate. Immunohistochemistry showed that the amyloid deposits were positive for the N-terminal region of EFEMP1. From these results, the present case was diagnosed as EFEMP1-derived amyloidosis. It is the first such case in an animal. EFEMP1-derived amyloidosis in humans has recently been reported as a systemic amyloidosis, and it is known as an age-related venous amyloidosis. The present case showed different characteristics from human EFEMP1-derived amyloidosis, including the amyloid deposition sites and the amyloidogenic region of the EFEMP1 protein, suggesting a different pathogenesis between Tsushima leopard cat and human EFEMP1-derived amyloidosis.

Screening of viral-vectored P. falciparum pre-erythrocytic candidate vaccine antigens using chimeric rodent parasites.

To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.

Creative interior design by Plasmodium falciparum: Lipid metabolism and the parasite's secret chamber.

Malaria parasites conceal themselves within host erythrocytes and establish a necessary logistics system through the three-membrane layered structures of these cells. To establish this system, lipid metabolism is needed for the de novo synthesis of lipids and the recycling of extracellular lipids and erythrocyte lipid components. Cholesterol supply depends on its uptake from the extracellular environment and erythrocyte cytoplasm, but phospholipids can be synthesized on their own. This differential production of lipid species creates unique modifications in the lipid profile of parasitized erythrocytes, which in turn may influence the biophysical and/or mechanical properties of organelles and vesicles and communication among them. Variations in local membrane properties possibly influence the transportation of various molecules such as parasite-derived proteins, because efficiencies in secretion, vesicle fusion and budding are partly determined by the lipid profiles. Comprehensive understanding of the parasite's lipid metabolism and the biophysics of lipid membranes provides fundamental knowledge about these pathogenic organisms and could lead to new anti-malarials.

Plasmodium falciparum SURFIN4.1 forms an intermediate complex with PTEX components and Pf113 during export to the red blood cell.

Plasmodium falciparum malaria parasites export several hundred proteins to the cytoplasm of infected red blood cells (RBCs) to modify the cell environment suitable for their growth. A Plasmodium translocon of exported proteins (PTEX) is necessary for both soluble and integral membrane proteins to cross the parasitophorous vacuole (PV) membrane surrounding the parasite inside the RBC. However, the molecular composition of the translocation complex for integral membrane proteins is not fully characterized, especially at the parasite plasma membrane. To examine the translocation complex, here we used mini-SURFIN4.1, consisting of a short N-terminal region, a transmembrane region, and a cytoplasmic region of an exported integral membrane protein SURFIN4.1. We found that mini-SURFIN4.1 forms a translocation intermediate complex with core PTEX components, EXP2, HSP101, and PTEX150. We also found that several proteins are exposed to the PV space, including Pf113, an uncharacterized PTEX-associated protein. We determined that Pf113 localizes in dense granules at the merozoite stage and on the parasite periphery after RBC invasion. Using an inducible translocon-clogged mini-SURFIN4.1, we found that a stable translocation intermediate complex forms at the parasite plasma membrane and contains EXP2 and a processed form of Pf113. These results suggest a potential role of Pf113 for the translocation step of mini-SURFIN4.1, providing further insights into the translocation mechanisms for parasite integral membrane proteins.

Kinetics of Bacterial Inactivation by Peroxynitric Acid in the Presence of Organic Contaminants.

Low-temperature atmospheric-pressure plasma has been studied for disinfection purposes. When plasma is exposed to water, reactive oxygen and nitrogen species are generated and preserved in the water fraction (plasma-treated water [PTW]), which consequently exhibits bactericidal activity. At low temperatures, one of the bactericidal components of PTW is peroxynitric acid (PNA). Importantly, PNA can also be synthesized by chemical reaction, without exposure to plasma. In this study, we evaluated the bactericidal properties of PNA based on reaction kinetics in comparison with other disinfectants. The analysis, based on dose-dependent effects, showed that PNA exhibited about 1 and 10 times the bactericidal activity of hypochlorous acid (HOCl) and peracetic acid, respectively. In addition, we evaluated the influence of organic contaminants on the bactericidal effects of PNA and HOCl. The bactericidal potential of both disinfectants was reduced by bovine serum albumin (BSA); however, PNA showed about 30-times-higher resistance against BSA inhibition than HOCl. Analysis of the dose-dependent effects of PNA revealed that the inhibition of bactericidal effect was caused by its consumption. Further experiments using model substrates containing particular amino acid residues (Met, Cys, Lys, and Leu) suggested that the bacterial inactivation by PNA is less affected by BSA due to the low reactivity and narrow reactivity spectrum of PNA for amino acid residues. Overall, our results suggest that PNA has a great disinfection potential, especially in the presence of organic contaminants (e.g., on the surface of the human body and on medical instruments contaminated with biological fluids).IMPORTANCE A good disinfectant for the human body should have various properties, such as strong bactericidal activity, harmlessness to living tissues, and resistance against biological fluids (or other organic contaminants). Peroxynitric acid (PNA) showed a bactericidal effect that was several tens up to several hundred times higher per unit of molarity than that of sodium hypochlorite and peracetic acid, which are used as general disinfectants for medical equipment. Moreover, the high resistance of PNA to organic load was confirmed, indicating that PNA will inactivate bacteria effectively even on contaminated surfaces, such as used medical devices or the human body surface. Therefore, we propose that PNA can be used as a strong disinfectant for the human body.

Novel Babesia bovis exported proteins that modify properties of infected red blood cells.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

Artemisinin exposure at the ring or trophozoite stage impacts Plasmodium falciparum sexual conversion differently.

Malaria transmission is dependent on the formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on sexual conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~ fourfold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications.

Pathology and Proteomics-Based Diagnosis of Localized Light-Chain Amyloidosis in Dogs and Cats.

Amyloidosis is classified according to the amyloid precursor protein, and accurate diagnosis of the amyloidosis type may guide appropriate treatment. Immunohistochemistry and Congo red staining are the most frequently used methods used to distinguish types of amyloidosis, but problems with specificity and sensitivity indicate the need for an alternative diagnostic method. In this study, we evaluated laser microdissection-liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS) for the diagnosis of amyloid light-chain (AL) amyloidosis in animals. Plasmacytomas with amyloid deposits from 15 dogs and 2 cats were subjected to Congo red staining with or without potassium permanganate pretreatment, immunohistochemistry for kappa and lambda light chains, and LMD-LC-MS/MS. Congo red staining was diagnostic in 12 of 17 cases based on resistance to potassium permanganate pretreatment, but in 5 of 17 cases the pretreatment unexpectedly reduced Congo red staining or abrogated the birefringence and a definitive diagnosis could not be reached. Immunohistochemistry detected kappa or lambda light chains in 6 of 17 cases. With LMD-LC-MS/MS, immunoglobulin lambda light chain was detected in all 17 cases. The amyloid signature proteins ApoA-I, ApoA-IV, and ApoE were detected in 9, 1, and 3 of the 15 canine cases by LMD-LC-MS/MS, but not in the feline cases. In conclusion, LMD-LC-MS/MS consistently determined the amyloid type in all examined specimens, while Congo red staining after potassium permanganate treatment and immunohistochemistry were less sensitive tests.

Generation of Novel Plasmodium falciparum NF135 and NF54 Lines Expressing Fluorescent Reporter Proteins Under the Control of Strong and Constitutive Promoters.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1α promoter (GFP@ef1α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum.

Kinetics Analysis of the Reactions between Peroxynitric Acid and Amino Acids.

Plasma disinfection using low-temperature atmospheric pressure plasma is widely studied in many applications, and the use of plasma-treated water (PTW) for disinfection is being developed by many researchers. Exposing plasma to water supplies and preserves reactive oxygen and nitrogen species (RONS) in the water, and this PTW exhibits bactericidal activity. In our previous study, it was revealed that peroxynitric acid (O2NOOH, PNA) was the dominant bactericidal component in PTW. PNA can be easily synthesized without plasma treatment, and the physicochemical properties of PNA have been well-analyzed. As the application of PNA in fields related to medicine and biology has not been reported, a basic study on the behavior of PNA is required. In this study, the bactericidal activity of PNA and its reactivities with 20 naturally occurring amino acids were evaluated to understand its reaction mechanism with biomolecules. Interestingly, PNA exhibited 10-6 times lower reactivities with amino acids when compared with hypochlorous acid and other RONS, although its bactericidal activity was 310 times higher than that of sodium hypochlorite. In addition, the reactivity of PNA with methionine was over 100 times higher than that with other amino acids, indicating that the reactions of PNA with amino acids are highly specific. No other oxidants have been reported to react selectively with only methionine. As methionine is involved in specific activities in cells, the unique reaction profile of PNA was examined in the context of biological systems.

Generation of a Genetically Modified Chimeric Plasmodium falciparum Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Malaria Vaccine Development.

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.

A P. falciparum NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages.

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line mCherry-luc@etramp10.3). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The mCherry-luc@etramp10.3 parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.

Multiple charged amino acids of Plasmodium falciparum SURFIN4.1 N-terminal region are important for efficient export to the red blood cell.

Plasmodium falciparum, an obligate intracellular protozoan parasite which causes the severe form of human malaria, exports numerous proteins to the infected red blood cell that are important for its survival and of severe pathological effect to its host. These proteins and their export mechanisms are candidates for drug and vaccine development, and among them is the Plasmodium SURFIN family of proteins. Previously we showed that the N-terminal region along with the sequence surrounding the transmembrane domain of SURFIN4.1 is essential for its export to Maurer's clefts in the red blood cell cytoplasm. We proposed that this region is recognized by a machinery responsible for protein translocation across the parasitophorous vacuole membrane surrounding the parasite. To understand the export mechanism further, we utilized a fluorescent protein-tagged mini-SURFIN4.1 consisting of the minimum essential components for export. Alanine scanning of all charged amino acids within the N-terminal region revealed that replacement of 3 glutamic acid and 2 lysine residues significantly impairs the export efficiency of this protein across the parasitophorous vacuole membrane. In addition, N-terminally Myc-tagged mini-SURFIN4.1 and mini-SURFIN4.2 with similar architectures were detected with anti-Myc antibody at Maurer's clefts, indicating that elements required for export to Maurer's clefts are conserved between SURFIN4.1 and SURFIN4.2, and that N-terminal sequences of these SURFIN members are not cleaved during export. Our results implicate a conserved nature of SURFIN export to the red blood cell, particularly an important role of multiple glutamic acid and lysine residues in the SURFIN N-terminal region.