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Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China.

Hepatocellular carcinoma (HCC) is a biologically aggressive cancer. Targeted therapy is in need to tackle challenges in the treatment perspective. A growing body of evidence suggests a promising role of pharmacological inhibition of PIM (proviral integration site for Moloney murine leukaemia virus) kinase in some human haematological and solid cancers. Yet to date, the potential application of PIM inhibitors in HCC is still largely unexplored. In the present study we investigated the pre-clinical efficacy of PIM inhibition as a therapeutic approach in HCC. Effects of PIM inhibitors on cell proliferation, migration, invasion, chemosensitivity, and self-renewal were examined in vitro. The effects of PIM inhibitors on tumour growth and chemoresistance in vivo were studied using xenograft mouse models. Potential downstream molecular mechanisms were elucidated by RNA sequencing (RNA-seq) of tumour tissues harvested from animal models. Our findings demonstrate that PIM inhibitors SGI-1776 and PIM447 reduced HCC proliferation, metastatic potential, and self-renewal in vitro. Results from in vivo experiments supported the role of PIM inhibition in suppressing of tumour growth and increasing chemosensitivity of HCC toward cisplatin and doxorubicin, the two commonly used chemotherapeutic agents in trans-arterial chemoembolisation (TACE) for HCC. RNA-seq analysis revealed downregulation of the MAPK/ERK pathway upon PIM inhibition in HCC cells. In addition, LOXL2 and ICAM1 were identified as potential downstream effectors. Taken together, PIM inhibitors demonstrated remarkable anti-tumourigenic effects in HCC in vitro and in vivo. PIM kinase inhibition is a potential approach to be exploited in formulating adjuvant therapy for HCC patients of different disease stages. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Converter smelting medium-low carbon ferrochrome replaces electronic by oxygen and reduces production energy consumption greatly. Medium-low carbon ferrochrome end sulfur content analysis is necessary to determine the product quality. Now we still use the product sampling, laboratory analysis to determine end sulfur content, which can not realize online monitoring. According to this status, a PLSBP prediction model of end sulfur content has been established based on particle swarm algorithm, which combined partial least squares method and BP neural network. At the same time, A feedback compensation model has been established by dichotomy, which avoided the model failure caused by raw material quality change. The simulation results showed that the hit rate of prediction model was 96.67% within the absolute error, 90% within relative error. High precision was achieved, which provided an important theoretical basis to improve product quality and optimize the production process.

AOD furnace Smelting Ferrochrome is a complex process. The discretion of the impurity content of phosphorus is one of the important factors affecting the quality of ferrochrome products. If ferrochrome phosphorus excesses, the product is easy to break. At present, the most detection methods of determination the end phosphorus content are artificial experiments, so efficiency is lagging behind. It is necessary to establish a model to predict end-point phosphorus content for improving production efficiency, reducing costs, saving energy consumption. The important influence factors of AOD furnace smelting ferrochrome end-point phosphorus content were determined based on analyzing response characteristics of hot metal dephosphorization pretreatment and thermodynamic condition, the control variables of the end phosphorus content were fixed. According to Sinosteel Jilin Ferroalloys Co., Ltd 5t AOD furnace smelting medium-low carbon ferrochrome technology and production data, a prediction model for AOD furnace smelting ferrochrome end-point phosphorus content has been established based on RBF artificial neural network in accordance with the ferrochrome smelting process for online prediction of end-point phosphorus content. Results showed that the prediction precision of target shooting is 85.7% within the error 0.003%, which has provided important theoretical basis on improving the smelting process and product quality.

Echinococcus multilocularis causes alveolar echinococcosis primarily in rodents, but also in humans where it represents one of the most lethal helmintic infections. We used a susceptible mouse (C57BL/6) model to demonstrate failure in controlling secondary infection with the E. multilocularis metacestode, even when performed at the lowest possible infection dose. This was achieved by intraperitoneal or intrahepatic inoculation of a single parasite vesicle. In secondary infections, the primary physical barrier between the parasite and the host is constituted by the acellular laminated layer (LL), which is predominantly composed of high-molecular-weight glycans and surrounds the entire metacestode. Only those metacestode structures which exhibited an intact LL were successful in establishing infection, whereas metacestodes which were punctured - thus exhibiting an opened LL and thereby an accessible germinal layer - were no longer infective. Conversely, both types of vesicle survived in vivo maintenance, as assessed by RT-PCR based upon II/3 gene expression. In consequence, the encapsulating LL appears to be one of the key factors that mediates survival and successful proliferation of the parasite metacestode in vivo.

Hepatitis C virus (HCV) is a widespread infectious disease in humans with the negative implication of becoming chronic in most persons. Patients infected with HCV are at risk of liver cirrhosis or hepatocellular carcinoma at later stages. In contrast to hepatitis A and hepatitis B, there is no immunization yet available, neither prophylactic nor therapeutic. Thus, there is an urgent need to develop a safe, protective vaccine against this fatal disease. Developing countries are even more at risk for HCV. There are currently a number of scientific approaches aimed towards solving this problem. Taking both risks and costs of immunization into consideration, a peptide-based vaccine may be a reasonable prophylactic protection. Also, it might be of therapeutic use in already infected patients by increasing a specific CTL response against HCV. In our lab, we are focusing on immunopotentiating reconstituted influenza virosomes (IRIVs) as carriers for immunogenic HLA-A2-restricted core epitopes to induce peptide-specific cytotoxic T lymphocytes (CTLs). The IRIVs are similar to liposomes, but in addition contain influenza-derived hemagglutinin and neuraminidase on their outer surface which makes them fusogenic, thus, permitting antigen delivery to host cells. So far, virosomes have been successfully used for vaccine development and as a result a virosomal vaccine against both influenza virus (Inflexal) BERNA) and hepatitis A virus (HAV) (Epaxal) BERNA) already exist on the market. This paper focuses on the importance of development of a successful vaccine against HCV and, more specifically, we discuss the use, advantages and disadvantages of a peptide-based vaccine. A brief report of our latest findings will be included.

Neosporosis is a disease affecting predominantly fetal development in cattle and dog hosts; and it may cause neuromuscular disfunction in infected newborn calves and pups. Predispositions--including, e.g. transient immunosuppression during pregnancy--may result in an increased dissemination of the parasite within the host or its offspring. Chemotherapeutic treatment of neosporosis may be an issue, provided that an appropriate drug is made available. In this respect, we describe the use of a mouse model for the evaluation of toltrazuril and ponazuril medication as a means of preventing parasite dissemination and subsequent formation of cerebral lesions. Toltrazuril- and ponazuril-treated mice were experimentally infected intraperitoneally (i.p.) with 2 x 10(6) Neospora caninum tachyzoites. The infection was monitored at three levels: clinically, by assessing symptoms, histologically, by assessing the occurrence of cerebral lesions and parasites by immunohistochemistry, and on the molecular level, by detection of parasite DNA using PCR. Chemotherapy using either toltrazuril or ponazuril, both applied in a drinking-water formulation (20 mg toltrazuril or ponazuril kg(-1) body weight day(-1)) completely prevented the formation of cerebral lesions in all treated animals, as assessed by immunohistochemistry. PCR analyses of these treated animals showed that DNA-detectability was reduced by 91% and 90% upon toltrazuril and ponazuril medication, respectively.

Using interleukin-10 (IL-10)-deficient (IL-10−/−) mice, previous studies revealed a pathological immune response after infection withTrypanosoma cruzithat is associated with CD4+T cells and overproduction of proinflammatory cytokines. In this study we further investigate the pathology and potential mediators for the mortality in infected animals.T. cruzi-infected IL-10−/−mice showed reduced parasitemia accompanied by increased systemic release of gamma interferon (IFN-γ), IL-12, and reactive nitrogen intermediates and overproduction of tumor necrosis factor alpha (TNF-α). Despite this early resistance, IL-10−/−mice died within the third week of infection, whereas all control mice survived acute infection. The clinical manifestation with weight loss, hypothermia, hypoglycemia, hyperkalemia, and increased liver-derived enzymes in the blood together with hepatic necrosis and intravascular coagulation in moribund mice indicated a toxic shock-like syndrome, possibly mediated by the systemic TNF-α overproduction. Indeed, high production of systemic TNF-α significantly correlated with mortality, and moribund mice died with critically high TNF-α concentrations in the blood. Consequent treatment with anti-TNF-α antiserum attenuated pathological changes inT. cruzi-infected IL-10−/−mice and significantly prolonged survival; the mice died during the fourth week postinfection, again with a striking correlation between regaining high systemic TNF-α concentrations and the time of death. Since elevated serum IL-12 and IFN-γ concentrations were not affected by the administration of antiserum, these studies suggest that TNF-α is the direct mediator of this toxic shock syndrome. In conclusion, induction of endogenous IL-10 during experimentally induced Chagas' disease seems to be crucial for counterregulating an overshooting proinflammatory cytokine response resulting in TNF-α-mediated toxic shock.

NK cells are believed to play a mandatory role during the early phases of Listeria monocytogenes infection by producing IFN-γ, which is required for the activation of macrophage effector functions. Mice deficient in the common cytokine receptor γ-chain (γc−/−), which completely lack NK cells, were used to examine whether NK cells were essential for resistance to Listeria infection in vivo. Surprisingly, infected γc−/− mice showed normal innate immunity and macrophage responses against sublethal Listeria infection 2 days postinfection. At this time point, γc−/− mice showed increased blood IFN-γ levels compared with those in noninfected controls, demonstrating an NK-independent source of IFN-γ, which explains early resistance. Listeria-infected γc−/− × recombinase-activating gene-2−/− double-deficient mice were unable to produce IFN-γ and were highly susceptible to L. monocytogenes. Since T cells, but not B cells, are major IFN-γ producers, and γc−/− T cells were found to be efficient IFN-γ producers in vitro, we conclude from these results that T cells functionally replace NK cells for the early IFN-γ production that is necessary for activating the innate immune system following infection with L. monocytogenes. This novel observation in listeriosis underscores how the adaptive immune response can maintain and influence innate immunity.

Virus specific T cell responses play an important role in resolving acute hepatitis C virus (HCV) infections. Using the HLA-A2.1 transgenic mouse model we investigated the potential of a liposomal peptide vaccine to prime a CD8(+) T cell response against 10 different HCV epitopes, relevant for human applications. We were able to demonstrate the induction of strong cytotoxic T cell responses and high numbers of IFN-gamma-secreting cells, which persisted at high levels for at least 3 months. Co-integrating CpG oligonucleotides into liposomes further increased the number of IFN-gamma-secreting cells by 2-10-fold for most epitopes tested. The frequency of specific cells was further analysed with chimeric A2 tetramers bearing the NS31073-1081 epitope and was estimated at 2-23% of the CD8(+) T cell population. Importantly, mouse effector cells, specific for this epitope, were also capable of lysing a human target cell line expressing HCV proteins. This finding and the specific protection observed in challenge experiments with recombinant vaccinia virus expressing HCV sequences emphasise the biological relevance of the vaccine-induced immune response. In conclusion, such liposome formulations represent a safe and promising strategy to stimulate the CD8(+) T cell against HCV.

The role of specific B lymphocytes and T-cell populations in the control of experimental Echinococus multilocularis infection was studied in micro MT, nude, T-cell receptor (TCR)-beta(-/-), major histocompatibility complex (MHC)-I(-/-) and MHC-II(-/-) mice. At 2 months postinfection, the parasite mass was more than 10 times higher in nude, TCR-beta(-/-) and MHC-II(-/-) mice than in infected C57BL/6 wild-type (WT) mice, and these T-cell-deficient mice started to die of the high parasite load at this time-point. In contrast, MHC-I(-/-) and micro MT mice exhibited parasite growth rates similar to those found in WT controls. These findings clearly point to the major role that CD4(+) alphabeta(+) T cells play in limiting the E. multilocularis proliferation, while CD8(+) T and B cells appeared to play a minor role in the control of parasite growth. In the absence of T cells, especially CD4(+) or alphabeta(+) T cells, the cellular immune response to infection was impaired, as documented by the lack of hepatic granuloma formation around the parasite and by a decreased splenocyte responsiveness to concanavalin A (Con A) and parasite antigen stimulation. Surprisingly, in T-cell-deficient mice, the ex vivo expression of interferon-gamma (IFN-gamma) and other inflammatory cytokines (except for interleukin-6) were increased in association with a high parasite load. Thus, the relative protection mediated by CD4(+) alphabeta(+) T cells against E. multilocularis infection seemed not be IFN-gamma dependent, but rather to rely on the effector's function of CD4(+) alphabeta(+) T cells. The local restriction of parasite germinal cell proliferation was reflected by a regulatory effect on the expression of 14-3-3 protein within the parasite tissue in T-cell-deficient mice. These results provide a strong indication that the CD4(+) alphabeta(+) T-cell-mediated immune response contributes to the control of the parasite growth and to the regulation of production of the parasite 14-3-3 protein in metacestode tissues.

In the present study, interleukin-6 (IL-6)-deficient mice were infected withGiardia lambliaclone GS/M-83-H7. Murine IL-6 deficiency did not affect the synthesis of parasite-specific intestinal immunoglobulin A. However, in contrast to wild-type mice, IL-6-deficient animals were not able to control the acute phase of parasite infection. Reverse transcription-PCR-based quantitation of cytokine mRNA levels in peripheral lymph node cells exhibited a short-term up-regulation of IL-4 expression in IL-6-deficient mice that seemed to be associated with failure in controlling the parasite population. This observation suggests a further elucidation of IL-4-dependent, Th2-type regulatory processes regarding their potential to influence the course ofG. lambliainfection in the experimental murine host.

An estimated 400 million people are chronically infected with the hepatitis B virus (HBV). Chronic viral hepatitis infection incurs serious sequelae such as liver cirrhosis and hepatocellular carcinoma. Prevention and treatment, thus, represent an important target for public health. Preventive vaccines using HBsAg alone or combined with other antigens allow for the generation of neutralizing antibodies which effectively prevent infection in immunocompetent individuals. Cell-mediated immunological mechanisms are thought to be crucial in determining viral persistence or viral elimination. Therapeutic approaches aiming to shift cellular immunity towards viral elimination have been on the research agenda for many years. This paper summarizes pre-clinical and clinical results obtained with the use of immunogenic peptides formulated as vaccines to selectively boost cellular immune responses. Such vaccines are capable of generating cellular immune responses in animal models as well as in humans and represent an important step towards the development of a therapeutic vaccine against chronic hepatitis.

We have studied IL-12p35-deficient (IL-12p35(-/-)) mice to evaluate the role of IL-12 in resistance against Listeria monocytogenes. In the absence of bioactive IL-12p75, mutant mice acquired higher bacterial organ burden than wild-type mice and died during the first week following infection with normally sublethal doses of Listeria. Moreover, blood IFN-gamma levels were strikingly reduced in mutant mice at day 2 post-infection. These results suggest that in IL-12p35-deficient mice impaired production of IFN-gamma which is crucial for activation of listericidal effector functions of macrophages leads to defective innate immunity against Listeria. In contrast to mice deficient for IFN-gamma or IFN-gamma receptor which are unable to resist very low infection doses of Listeria, IL-12p35(-/-) mice resisted up to 1000 c.f.u. and were able to eliminate Listeria. Spleen cells from mutant mice re-stimulated with heat-killed Listeria produced considerable amounts of IFN-gamma, suggesting that at low dose infection sufficient IFN-gamma is produced independently of IL-12. Subsequent challenge of these immunized mice with high doses of L. monocytogenes resulted in sterile elimination demonstrating efficient memory responses. These results demonstrate for the first time that at low doses of Listeria IL-12 is neither critical for innate immunity nor for the development of protective T cell-dependent acquired immunity.