Lung cancer-derived exosomal miR-132-3p contributed to interstitial lung disease development.

PURPOSE: Interstitial lung diseases (ILDs) have high morbidity and mortality and poor prognosis. The significance of microRNAs (miRNAs) was highlighted in ILDs development. Currently, we attempted to confirm the functions of lung cancer-derived exosomal miR-132-3p and reveal the underlying mechanism. METHOD: Characteristics of exosomes were verified by transmission electron microscope (TEM), nanoparticle tracking analysis, and Western blot assay. Exosome uptake for the normal human lung fibroblasts (NHLF) was assessed using a PKH67 staining assay. MTT and colony formation assays were applied to examine the proliferation abilities of NHLF. The interaction between miR-132-3p and sprouty1 (SPRY1) was confirmed by a luciferase reporter assay. RESULTS: Lung cancer-derived exosomes promoted normal human lung fibroblast activation. Exosome inhibitor GW4869 reversed the effects of Exo on NHLF. Subsequently, miR-132-3p in lung cancer-derived exosomes activated the normal human lung fibroblast and promoted interstitial lung disease development ex vivo. Next, SPRY1 was verified to be the binding protein of miR-132-3p, and sh-SPRY1 abrogated the effects of the miR-132-3p inhibitor on NHLF. CONCLUSION: Exosomal miR-132-3p from A549 cells accelerated the development of interstitial lung disease through binding to SPRY1, which might serve as an important target for ILDs.

Study on the pathogenicity, immunogenicity, endogenous development and drug sensitivity of Eimeria kongi.

Following the discovery of Eimeria kongi, we investigated the pathogenicity, immunogenicity, endogenous development and drug sensitivity of this coccidian. Coccidia-free rabbits were inoculated with 1 × 102 to 5 × 104 sporulated oocysts of E. kongi before challenge 14 days post inoculation. E. kongi was moderately pathogenic and induced good immunity against re-infection. All inoculated doses results in reduced food intake and body weight gain, and an inoculation oocyst dose of 1 × 103 or higher caused various degrees of diarrhea. Except for one death of the highest dose group, all rabbits recovered 12 days post inoculation. An inoculation dose of 1 × 103 or 1 × 104 oocysts conferred the most effective protection from re-infection, which reduced oocyst output by approximately 99% and maintained body weight gain. Four generations of schizogony were observed, and the endogenous development mainly occurred in the jejunum and ileum of rabbits. E. kongi was most sensitive to sulfachloropyrazine sodium, followed by decoquinate; it is resistant to diclazuril. Both decoquinate and sulfachloropyrazine sodium may be effective in the control of E. kongi infection.

Dynamic changes of T-lymphocyte subsets and the correlations with 89 patients with coronavirus disease 2019 (COVID-19).

BACKGROUND: In December 2019, an outbreak of coronavirus disease 2019 (COVID-19), caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), occurred in Wuhan City, Hubei Province, China. The coronavirus has spread throughout the world, posing a severe threat to human health. By using flow cytometry, here we observed the dynamic changes of peripheral blood T lymphocyte subsets in COVID-19 patients, with an attempt to explore their roles in the pathogenesis of COVID-19 and their impacts on prognosis. METHODS: Eighty-nine COVID-19 patients were divided into a moderate group (n=70) and the severe/critical group (n=19) according to the disease severity. Furthermore, the severe/critical patients were divided into the improved group (n=14) and unimproved group (n=5) according to the outcomes. The absolute peripheral blood lymphocytes counts and subsets, including CD45+, CD3+, CD4+, and CD8+, in the acute phase, and flow cytometry measured the recovery phase for all patients. Then, the results were compared with those in the normal control group. RESULTS: The absolute counts of lymphocytes, T lymphocytes, and their subsets decreased during the acute phase in COVID-19 patients, especially in the severe/critical group. The T-lymphocyte count reached the lowest point on the 14th day in the severe/critical group. It rose with fluctuations to the normal level in the improved group as the immune function recovered; in the unimproved group, however, the T-lymphocyte count remained at a low level or even continued to decrease. The percentages of CD4+ and CD8+ T lymphocytes showed no visible change in the improved group; however, the percentage of CD8+ T cells dropped in the unimproved group, resulting in higher CD4+/CD8+ ratio. CONCLUSIONS: T lymphocytes count, and their subsets can be used for monitoring the immune functions and predicting the prognosis of COVID-19 patients.

Clinical characteristics of 199 discharged patients with COVID-19 in Fujian Province: A multicenter retrospective study between January 22nd and February 27th, 2020.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has quickly spread throughout the country and the world since first broke out in Wuhan, China. The outbreak that started from January 22, 2020, in Fujian Province has been controlled as the number of indigenous cases has not increased since March. We aimed to describe the clinical characteristics of patients with COVID-19 in Fujian Province, China. METHODS: In this retrospective, multicenter study, we collected and analyzed the epidemiological, clinical, and laboratory data of all cases confirmed by nucleic acid tests in five designated hospitals in Fujian Province between January 22 and February 27, 2020. All patients were followed up until discharge. COVID-19 severity was classified as mild, moderate, severe, or critical. RESULTS: Of 199 discharged patients with COVID-19, 105 patients were male, with a median age of 46.3 years, and 17 patients were severe, and 5 patients were critical on admission. Hypertension and diabetes were the most common comorbidities. The symptoms at illness onset were mainly fever (76.4%), cough (60.8%), and myalgia or fatigue (27.6%). A total of 96.5% of patients had abnormal imaging findings on chest computed tomography. Lymphopenia (37.2%) and hypoxemia (13.6%) were observed. Acute respiratory distress syndrome and respiratory failure occurred in 9 patients (4.5%) and 8 patients (4.0%) respectively. One patient died and the others were cured and discharged with the median hospital stay of 19 days. Old age was negatively correlated with lymphocyte count (r = - 0.296, p < 0.001) and oxygenation index (r = - 0.263, p = 0.001). Bivariate regression analysis revealed that old age (≥ 75 years), hypertension, diabetes, and lymphopenia were correlated with the severity of COVID-19. CONCLUSIONS: Patients in Fujian Province were mostly nonsevere cases with mild or moderate symptoms, and had a lower mortality than patients in Wuhan (4.3%-15%). Older age, hypertension, diabetes, and lymphopenia were risk factors for severity of COVID-19.

Immune protection provided by a precocious line trivalent vaccine against rabbit Eimeria.

A pilot study was conducted to evaluate the pathogenicity and immunogenicity of vaccinated rabbits with different doses of oocysts (5 × 102, 1 × 103, 1 × 104, and 5 × 104) of a precocious line, including Eimeria magna, E. intestinalis or E. media following the challenge with their corresponding parent strains. Our results showed that each precocious line had weak pathogenicity but good immunogenicity in terms of clinical symptoms, average daily weight gains (ADGs), and oocyst outputs. Therefore, a precocious line trivalent vaccine, including E. magna, E. intestinalis, and E. media was formulated. A total of sixty 40-day-old coccidia-free rabbits were allocated to ten treatments with a 2 × 3 factorial arrangement that included 2 vaccination doses (5 × 102 or 1 × 103 oocysts of the precocious line). Groups I to Ⅷ and Unimmunized Challenged Control group were challenged with mixed oocysts of their corresponding parent strains (1 × 104 oocysts of each parent strain) 14 days after vaccination. No clinical symptoms were observed in the immunized groups after vaccination. Average daily weight gains (ADGs) were similar to those of unimmunized unchallenged controls (P > 0.05) after vaccination or after challenge. Oocyst outputs in the vaccinated challenged groups were significantly different from those of unimmunized challenged controls (P < 0.01) after challenge. These results indicated that the trivalent vaccine could provide immune protection against coccidiosis and therefore, it could be used as a candidate vaccine.

Selection and characterization of a precocious line of Eimeria media.

Coccidiosis, caused by the infection of Eimeria parasites, is one of the most common diseases in domestic rabbits. Live anticoccidial vaccine formulated with attenuated precocious lines of pathogenic eimerian parasites is expected to be valuable for the control of rabbit coccidiosis as a similar strategy to produce anticoccidial vaccines against chicken coccidiosis has being used for several decades. Eimeria media, moderate pathogenic, is widespread in China. Therefore, attenuated anticoccidial vaccines against rabbit coccidiosis should contain vaccine strain(s) of E. media. In this study, a precocious line of E. media (Empre) was selected by collecting and propagating the early excreted oocysts with 16 successive generations. The prepatent period of Empre reduced from 108 h of its parental strain (Emwt) to 70 h. The fecundity of Empre was about 1/10 to 1/3 lower than that of Emwt. Each sporocyst of Empre sporulated oocyst contained only one large refractile body instead of two smaller ones seen in the parental strain. When vaccinated with 1 × 103 or 1 × 104 precocious line oocysts, the rabbits were completely protected against homologous challenge with the parental strain 14 days post challenge by terms of body weight gain and oocyst output counting, indicating the efficacy of Empre. Meanwhile, all immunized rabbits showed no clinical sign post immunization, indicating the safety of Empre. For co-immunization, 1 × 103Empre oocysts and 5 × 102 oocysts of a precocious line of E. intestinalis (EIP8) were inoculated to each rabbit in a trial. No diarrhea or mortality was found after vaccination, and the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group, while the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group (P > 0.05), but significantly higher than that of UCC group (P < 0.01) after challenge, indicating it is safe and effective when using co-immunization. These results together show that Empre, as a precocious line, is a good candidate of precocious line of E. media for anticoccidial vaccine development.

A new species of Eimeria (Apicomplexa: Eimeriidae) from Californian rabbits in Hebei Province, China.

Rabbit coccidiosis is caused by infection with one or usually several Eimeria species, parasitizing in hepatobiliary ducts or intestinal epithelium of rabbits. To date, 11 species of rabbit coccidia have been well documented. Here we report a new species of Eimeria from rabbits. Sporulated oocysts were ellipsoidal to slightly ovoidal, 37.4 (32.6-41.2) µm in length, 23.5 (20.9-25.5) µm in width, with a shape index (length/width) 1.6 (1.43-1.91) and smooth, bilayered, homogeneously thick wall. The micropyle was obvious and with an inner diameter of 6.2 (5.0-7.5) µm. Both oocyst residuum and polar granule were absent. Sporocysts were ellipsoidal to elongate, 17.2 (13.2-20.0) µm long and 8.4 (7.5-9.1) µm wide, with a shape index (length/width) of 2.1 (1.74-2.21) and the presence of Stieda body and sporocyst residuum. The prepatent period was 132h. Phylogenetic analysis showed that 18S rDNA sequence of the new species clustered together with the 11 rabbit Eimeria species into a clade. However, ITS-1 sequence of the new species shared low similarities (27.1%-30%) with those of 11 rabbit Eimeria species. As the data above supported the erection of a new species, we named it as Eimeria kongi n. sp., in honor of Fanyao Kong, a Chinese parasitologist. The finding of the new species has important implications for the diagnosis and prevention of rabbit coccidiosis.

High pathogenicity and strong immunogenicity of a Chinese isolate of Eimeria magna Pérard, 1925.

Coccidia infection of rabbits with one or several species of parasites of the genus Eimeria causes coccidiosis, a disease leading to huge economic losses in the rabbit industry. Eimeria magna, one of the causal agents of rabbit coccidiosis, was characterized as mildly pathogenic and moderately immunogenic in previous studies. In this study, we identified a Chinese isolate of E. magna by testing its biological features (oocyst morphology and size, prepatent time) and sequencing its internal transcribed spacer 1 (ITS-1) DNA fragment. This isolate is highly pathogenic; infection of rabbits with only 1×102 oocysts caused a 55% reduction in weight gain in 14days. In addition, immunization with 1×102 oocysts prevented body weight loss against re-infection with 5×104 oocysts, indicating the high immunogenicity of this isolate. Our study described the distinctive phenotype of the Chinese isolate of E. magna and contributed to the research of geographic variation of rabbit coccidia.

Genetic variability among Hymenolepis nana isolates from different geographical regions in China revealed by sequence analysis of three mitochondrial genes.

Hymenolepis nana is a common tapeworm that parasitizes in the small intestine of rodent animals and humans. The present study examined the sequence diversity of three mitochondrial (mt) genes namely NADH dehydrogenase subunits 5 (nad5), small subunit ribosomal RNA (rrnS), and ATPase subunit 6 (atp6) of H. nana from mice in different geographical regions of China. A part of the nad5 (pnad5), complete rrnS and atp6 genes were amplified separately from individual H. nana isolates using polymerase chain reaction (PCR) and then sequenced. The sequences of pnad5, rrnS, and atp6 were 710 bp, 704-711 bp, and 516 bp in length, respectively. The A + T contents of the sequences were 70.1-73.5% (pnad5), 70.1-71.7% (rrnS), and 76.6-77.9% (atp6). Sequence variation within H. nana was 0-1.4% for atp6, 0-1.7% for rrnS, and 0-0.7% for pnad5. The inter-specific sequence differences between H. nana and Hymenolepis diminuta were significantly higher, which was 31.6-31.7% (pnad5), 16.1-17.6% (rrnS), and 26.5-27.1% (atp6). Phylogenetic analysis based on the combined three sequences using the maximum parsimony (MP) method supported that H. nana is a species complex or "cryptic" species. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics and systematic studies of H. nana of human and animal health significance.

The complete mitochondrial genomes of five Eimeria species infecting domestic rabbits.

Rabbit coccidiosis caused by members of the genus Eimeria can cause enormous economic impact worldwide, but the genetics, epidemiology and biology of these parasites remain poorly understood. In the present study, we sequenced and annotated the complete mitochondrial (mt) genomes of five Eimeria species that commonly infect the domestic rabbits. The complete mt genomes of Eimeria intestinalis, Eimeria flavescens, Eimeria media, Eimeria vejdovskyi and Eimeria irresidua were 6261bp, 6258bp, 6168bp, 6254bp, 6259bp in length, respectively. All of the mt genomes consist of 3 genes for proteins (cytb, cox1, and cox3), 14 gene fragments for the large subunit (LSU) rRNA and 11 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA (tRNA) genes. The gene order of the mt genomes is similar to that of Plasmodium, but distinct from Haemosporida and Theileria. Phylogenetic analyses based on full nucleotide sequences using Bayesian analysis revealed that the monophyly of the Eimeria of rabbits was strongly statistically supported with a Bayesian posterior probabilities. These data provide novel mtDNA markers for studying the population genetics and molecular epidemiology of the Eimeria species, and should have implications for the molecular diagnosis, prevention and control of coccidiosis in rabbits.

The complete mitochondrial genome sequence of Eimeria magna (Apicomplexa: Coccidia).

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequence of Eimeria magna from rabbits for the first time, and compared its gene contents and genome organizations with that of seven Eimeria spp. from domestic chickens. The size of the complete mt genome sequence of E. magna is 6249 bp, which consists of 3 protein-coding genes (cytb, cox1 and cox3), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, without transfer RNA genes, in accordance with that of Eimeria spp. from chickens. The putative direction of translation for three genes (cytb, cox1 and cox3) was the same as those of Eimeria species from domestic chickens. The content of A + T is 65.16% for E. magna mt genome (29.73% A, 35.43% T, 17.09 G and 17.75% C). The E. magna mt genome sequence provides novel mtDNA markers for studying the molecular epidemiology and population genetics of Eimeria spp. and has implications for the molecular diagnosis and control of rabbit coccidiosis.

Sequence variability in four mitochondrial genes among rabbit pinworm (Passalurus ambiguus) isolates from different localities in China.

Passalurus ambiguus is a common pinworm which parasitizes in the caecum and colon of rabbits. This study examined genetic variability among P. ambiguus isolated from naturally infected rabbits in four different provinces in China. The partial mitochondrial (mt) cytochrome c oxidase subunit 1 (pcox1), cytochrome b (pcytb) and NADH dehydrogenase subunits 1 and 5 (pnad1 and pnad5) were amplified separately from individual nematodes by PCR and sequenced. The results showed that pcox1, pcytb, pnad1 and pnad5 were 714, 663, 645 and 546 bp in length, respectively. The intra-specific sequence variations within P. ambiguus were 0-1.1% for pcox1, 0-1.2% for pcytb, 0-0.6% for pnad1 and 0-1.3% for pnad5, whereas inter-specific sequence differences with other members of the Oxyuridae were 16.2-17.3% for pcox1, 27.8-30.4% for pcytb, 20.2-24.0% for pnad1 and 27.1-30.3% for pnad5. Phylogenetic analyses using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods, based on the combined sequences of the four partial mtDNA sequences, revealed that all the P. ambiguus samples form monophyletic groups. This study demonstrated the existence of low-level intra-specific variation in cox1, cytb, nad1 and nad5 genes among P. ambiguus isolates from different geographic regions in China, and these four mtDNA sequences can be used as genetic markers for the population genetic studies of P. ambiguus, as well as the differentiation of P. ambiguus from other oxyurid nematodes.