<Emphasis Type="Italic">Slc11a1</Emphasis> (<Emphasis Type="Italic">Nramp</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) gene modulates immune-inflammation genes in macrophages during pristane-induced arthritis in mice

Objective and design

Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S alleles was used to characterize the influence of Slc11a1 gene polymorphism on immune responses during disease manifestation. Previous reports demonstrated that the presence of the Slc11a1 S allele increased the incidence and severity of PIA in AIRmax ^SS , suggesting that this gene could interact with inflammatory loci to modulate PIA. We investigated the effects of Slc11a1 alleles on the activation of phagocytes during PIA.

Treatment

Mice were injected intraperitoneally with two doses of 0.5 mL of mineral oil pristane at 60-day intervals. Arthritis development was accompanied for 180 days.

Results

AIRmax ^SS mice showed differential peritoneal macrophage gene expression profiles during PIA, with higher expression and production of H₂O₂, NO, IL-1β, IL-6, TNF-α, and several chemokines. The presence of the Slc11a1 R allele, on the other hand, diminished the intensity of macrophage activation, restricting arthritis development.

Conclusion

Our data demonstrated the fine-tuning roles of Slc11a1 alleles modulating macrophage activation, and consequent PIA susceptibility, in those mouse lines.

     

Chitinase 3-Like-1-Deficient Splenocytes Deteriorated the Pathogenesis of Acute Graft-<Emphasis Type="Italic">Versus</Emphasis>-Host Disease <Emphasis Type="Italic">via</Emphasis> Regulating Differentiation of Tfh Cells

Acute graft-versus-host disease (aGVHD) is an intractable complication in transplant patients, limiting the efficacy of this therapy. Chitinase 3-like-1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, plays a critical role in a variety of inflammatory diseases. Here, we investigated the in vitro and in vivo effects of CHI3L1 on the development of aGVHD. In this study, mixed lymphocyte reactions (MLR) in vitro showed that CHI3L1 deficiency in CD4⁺ T cell promoted the production of interferon (IFN)-γ and T follicular helper (Tfh)-related cytokines such as interleukin-6 (IL-6) and interleukin-21 (IL-21). Meanwhile, the inducible Tfh cell population increased remarkably in CHI3L1-KO CD4⁺ T cells’ induction group, compared with WT group. Then, in the murine acute GVHD model, we found that CHI3L1 deficiency in donor splenocytes dramatically increased the severity of aGVHD through enhancing Tfh cell differentiation. Moreover, at mRNA and protein levels, we defined several molecules that may account for the enhanced ability of CHI3L1-KO splenocytes to migrate into target organs and produce IFN-γ and Tfh-related cytokines and chemokines, such as IL-6, IL-21, and CXCL13. Expression of inducible co-stimulator (ICOS) and B cell lymphoma 6 (Bcl6) increased in the skin, the intestine, the lung, and the liver from CHI3L1-KO splenocyte-treated aGVHD mice. Therefore, these results strongly imply that CHI3L1 levels in donor cells may be related to the risk of aGVHD and targeting CHI3L1 represents a novel therapeutic strategy for controlling aGVHD progression.     

Cranberry proanthocyanidins have anti-biofilm properties against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background

Bacteria within a biofilm are phenotypically more resistant to antibiotics, desiccation, and the host immune system, making it an important virulence factor for many microbes. Cranberry juice has long been used to prevent infections of the urinary tract, which are often related to biofilm formation. Recent studies have found that the A-type proanthocyanidins from cranberries have anti-biofilm properties against Escherichia coli.

Methods

Using crystal violet biofilm staining, resazurin metabolism assays, and confocal imaging, we examined the ability of A-type proanthocyanidins (PACs) to disrupt the biofilm formation of Pseudomonas aeruginosa. We used mass spectrometry to analyze the proteomic effects of PAC treatment. We also performed synergy assays and in vitro and in vivo infections to determine whether PACs, alone and in combination with gentamicin, could contribute to the killing of P. aeruginosa and the survival of cell lines and G. mellonella.

Results

Cranberry PACs reduced P. aeruginosa swarming motility. Cranberry PACs significantly disrupted the biofilm formation of P. aeruginosa. Proteomics analysis revealed significantly different proteins expressed following PAC treatment. In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G. mellonella.

Conclusions

Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P. aeruginosa and should be further tested.

     

Initial Host Response to Bacteria in the Murine Lung Differs Between <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>, <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> and <Emphasis Type="BoldItalic">Streptococcus pneumoniae</Emphasis>

Phagocytosis of bacteria is an important process during early host defence. It has been directly observed only ex vivo or in vitro. Here, we report on the observation of phagocytosis under in vivo conditions by using intravital microscopy in the murine lung. Suspensions of fluorescently labelled Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa cells were each instilled intratracheally to anaesthetized mice. After thoracotomy, the alveolar surface was observed for 30 min. Alveolar phagocytes exhibiting ingested bacteria could be detected and counted. The highest numbers were found after the infection with P. aeruginosa. By using intravital microscopy, cellular host defence could be observed in living mice lungs. The initial phagocytic reaction crucially depends on the species of applied bacteria invading the lung.     

Comparison of Flow Estimators for Rotary Blood Pumps: An <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Study

Various approaches for estimating the flow rate of a rotary blood pump have been proposed for monitoring and control purposes. They have been evaluated under different test conditions and, therefore, a direct comparison among them is difficult. Furthermore, a limited performance has been reported for the areas where the pump flow and motor current present a non-monotonic relationship. In this regard, we selected most approaches that have been presented in literature and added a modified one, resulting in four estimators, which are either non-invasive or invasive, i.e., inlet and outlet pump pressure sensors are used. Data from in vitro and in vivo studies with the Deltastream pump DP2 were used to compare the estimators under the same test conditions. These data included both constant and varying pre- and afterload, contractility, viscosity, as well as pump speed settings. Bland–Altman plots were used to evaluate the performance of the estimators. The mean error of the overall estimated flow in vitro ranged from 0.002 to 0.38 L/min and the limits of agreement (LoA) between?±?2 L/min. During negative flows the mean error decreased by about 25% when the pump inlet pressure was added as an input. In vivo, the mean errors increased, while the LoA remained in the same range. An estimator based on pump pressure difference improves the reliability in areas where flow and current relationship is not monotonic. A trade-off between estimation accuracy and number of sensors was identified. The estimation objective and the potential errors should be considered when selecting an estimation approach and designing the pump systems.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

Electrospun PLGA Nanofiber Scaffolds Release Ibuprofen Faster and Degrade Slower After <Emphasis Type="Italic">In Vivo</Emphasis> Implantation

While delayed delivery of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with improved tendon healing, early delivery has been associated with impaired healing. Therefore, NSAID use is appropriate only if the dose, timing, and mode of delivery relieves pain but does not impede tissue repair. Because delivery parameters can be controlled using drug-eluting nanofibrous scaffolds, our objective was to develop a scaffold for local controlled release of ibuprofen (IBP), and characterize the release profile and degradation both in vitro and in vivo. We found that when incubated in vitro in saline, scaffolds containing IBP had a linear release profile. However, when implanted subcutaneously in vivo or when incubated in vitro in serum, scaffolds showed a rapid burst release. These data demonstrate that scaffold properties are dependent on the environment in which they are placed and the importance of using serum, rather than saline, for initial in vitro evaluation of biofactor release from biodegradable scaffolds.     

Luteoloside Protects the Uterus from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>-Induced Inflammation,Apoptosis, and Injury

Luteoloside is a flavonoid extracted from several natural herbs that exhibits anti-microbial and anti-inflammation properties. Our study mainly identified the anti-inflammatory mechanism of action of luteoloside in Staphylococcus aureus-induced endometritis. Histopathological observations and myeloperoxidase (MPO) activity showed that luteoloside could protect the uterus from S. aureus-induced damage and ameliorate the infiltration of inflammatory cells. Quantitative PCR (qPCR) and ELISA analysis also revealed that luteoloside could decrease the expression of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and increase the expression of the anti-inflammatory cytokine IL-10 both in vivo and in vitro. However, western blot analysis revealed that luteoloside inhibited the expression of TLR2 and IL-8 and inhibited the phosphorylation of IκBα and NF-κB p65. Moreover, luteoloside inhibited the apoptosis of endometrial epithelial cells (EECs), suppressed the phosphorylation of p53, and decreased the expression of caspase-3 induced by S. aureus. Furthermore, this study showed that luteoloside inhibited the expression of Bax but increased the expression of Bcl-2. These results indicate that luteoloside has anti-inflammatory properties by inhibiting the TLR2 and NF-κB signaling pathways and plays an anti-apoptotic role in S. aureus-induced endometritis, which may be valuable for the clinical treatment of S. aureus-induced inflammation.     

Tripartite Motif 8 (TRIM8) Positively Regulates Pro-inflammatory Responses in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>-Induced Keratitis Through Promoting K63-Linked Polyubiquitination of TAK1 Protein

Pseudomonas aeruginosa (PA)-induced keratitis is a rapidly progressive ocular infectious disease that often leads to inflammatory epithelial edema, stromal infiltration, tissue destruction, corneal ulceration, and vision loss. In this study, we investigate the role of tripartite motif 8 (TRIM8) in regulating the inflammatory process of PA-induced keratitis. The expression of TRIM8 was increased in mouse corneas and in vitro-cultured macrophages after PA infection. Knockdown of the expression of TRIM8 significantly inhibited the activation of NF-κB signaling and decreased the production of pro-inflammatory cytokines both in vivo and in vitro after infected with PA. Furthermore, we investigated the potential mechanism and we found after PA infection that TRIM8 could promote K63-linked polyubiquitination of transforming growth factor β-activated kinase 1 (TAK1), leading to the activation of TAK1 and enhanced inflammatory responses. Taken together, we demonstrated that TRIM8 has pro-inflammatory effect on PA-induced keratitis and suggest TRIM8 as a potential therapeutic target for keratitis.     

Inflammatory protein response in <Emphasis Type="Italic">CDKL5</Emphasis>-Rett syndrome: evidence of a subclinical smouldering inflammation

Background

Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT.

Methods

Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated.

Results

CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status.

Conclusion

For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.

     

Liver cancer with concomitant <Emphasis Type="Italic">TP53</Emphasis> and <Emphasis Type="Italic">CTNNB1</Emphasis> mutations: a case report

Background

In the spectrum of molecular alterations found in hepatocellular carcinoma (HCC), somatic mutations in the WNT/β-catenin pathway and the p53/cell cycle control pathway are among the most frequent ones. It has been suggested that both mutations occur in a mutually exclusive manner and they are used as molecular classifiers in HCC classification proposals.

Case presentation

Here, we report the case of a treatment-naïve mixed hepatocellular/cholangiocellular carcinoma (HCC/CCC) with morphological and genetic intratumor heterogeneity. Within the predominant part of the tumor with hepatocellular differentiation, a p.D32V mutation in exon 3 of the CTNNB1 gene occurred concomitantly with a TP53 intron 7/exon 8 splice site mutation.

Conclusion

Intratumor heterogeneity challenges the concept of CTNNB1 and TP53 gene mutations being mutually exclusive molecular classifiers in HCC, which has implications for HCC classification approaches.

     

Development of <Emphasis Type="Italic">T</Emphasis><Subscript><Emphasis Type="Italic">m</Emphasis></Subscript>-shift genotyping method for detection of cat-derived <Emphasis Type="Italic">Giardia lamblia</Emphasis>

To develop T _m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5′-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T _m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T _m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10^?5 and 4.88 × 10^?5 ng/μL samples of assemblage A and F standard plasmids. The T _m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T _m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T _m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.     

Clinical Impact of <Emphasis Type="Italic">p27</Emphasis><Superscript><Emphasis Type="Italic">Kip1</Emphasis></Superscript> and CaSR Expression on Primary Hyperparathyroidism

We aimed to investigate the expressions of p27 kinase inhibitory protein 1 (p27^Kip1) and calcium sensing receptor (CaSR) in adenomas and normal parathyroid tissue and to evaluate the relationship of these molecules with clinical and biochemical parameters in primary hyperparathyroidism (PHPT). Fifty-one patients with histopathologically confirmed parathyroid adenomas and 20 patients with normal parathyroid glands (which were removed incidentally during thyroid resection) were included. Immunohistochemical stainings of CaSR and p27^Kip1 were performed in surgical specimens. Clinical features, biochemical parameters, and BMD measurements of patients with PHPT were evaluated retrospectively. Expressions of p27^Kip1 and CaSR were decreased in parathyroid adenomas, compared to normal glands (p <?0.05). High intensity of CaSR staining (3+) was more frequent in normal parathyroid tissue (75%) than adenomas (12%) (p <?0.01). Hypertension was not observed in patients with high staining intensity of CaSR (p =?0.032). There was a negative association between CaSR expression and body mass index (BMI) (p =?0.027, r =???0.313). There was no significant relationship between p27^Kip1 and CaSR expressions, serum calcium, plasma parathormone, 25-hydroxy vitamin D levels, and bone density (p >?0.05). The expressions of p27^Kip1 and CaSR were decreased in PHPT patients. This reduction may play an important role in the pathogenesis of PHPT. However, neither p27^Kip1 nor CaSR expression was found to be useful in predicting prognosis or severity of disease.     

Switch of Steady-State to an Accelerated Granulopoiesis in Response to <Emphasis Type="Italic">Androctonus australis hector</Emphasis> Venom

Although several studies have shown that scorpion venoms cause a systemic inflammatory response syndrome, little is known about the contribution of the hematopoietic organs. The aim of this study was to investigate the effect of Androctonus australis hector venom on the bone marrow and on local inflammatory mediators, in concordance with the systemic inflammatory reaction elicited in mice. The consequences of a direct interaction of venom with murine bone marrow cells were also assessed by in vitro study. Obtained results showed that the early systemic neutrophilia correlated with a rapid granulocyte mobilization. This response was followed by an accelerated granulopoiesis that was supported by TNF-α and IL-6 signals. In vitro data revealed that the venom exerted a proliferative effect on murine hematopoietic cells and stimulated their differentiation towards granulocyte lineage mainly through cytokine secretion. In conclusion, this study indicated that the bone marrow rapidly exerts its activity in response to the experimental envenomation via the granulopoiesis process and inflammatory mediators in concert with the development of a systemic response. The ability of venom to directly switch steady-state granulopoiesis to an accelerated state in vitro could aggravate the disturbance caused by the venom. Better understanding of the mechanisms involved may lead to the emergence of new targets to avoid cell spreading and accumulation by acting on the very early stage of the systemic inflammatory response.     

Morphological characterization and <Emphasis Type="Italic">HSP70</Emphasis>-, <Emphasis Type="Italic">IGS</Emphasis>-based phylogenetic analysis of two microsporidian parasites isolated from <Emphasis Type="Italic">Antheraea pernyi</Emphasis>

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 μm, and the other type was short-axis-oval, measuring 3.64 × 2.17 μm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 μm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.     

Differentiation of Mouse Primordial Germ Cells into Functional Oocytes <Emphasis Type="Italic">In Vitro</Emphasis>

Various complex molecular events in oogenesis cannot be observed in vivo. As a bioengineering technique for female reproductive tissues, in vitro culture systems for female germ cells have been used to analyze oogenesis and preserve germ cells for over 20 years. Recently, we have established a new methodological approach for the culture of primordial germ cells (PGCs) and successfully obtained offspring. Our PGC culture system will be useful to clarify unresolved mechanisms of fertility and sterility from the beginning of mammalian oogenesis, before meiosis. This review summarizes the history of culture methods for mammalian germ cells, our current in vitro system, and future prospects for the culture of germ cells.     

Distinct Early Inflammatory Events during Ear Tissue Regeneration in Mice Selected for High Inflammation Bearing S<Emphasis Type="Italic">lc11a1 R</Emphasis> and <Emphasis Type="Italic">S</Emphasis> Alleles

High inflammatory AIRmax mice homozygous for Slc11a1 R and S alleles were produced. AIRmax ^SS mice showed faster ear tissue regeneration than AIRmax ^RR mice, suggesting that the S allele favored tissue restoration. Here, we investigated the gene expression profiles and the inflammatory reactions of AIRmax ^RR and AIRmax ^SS mice during the initial phase of ear tissue regeneration. We observed superior levels of analysis of wound myeloperoxidase and edema in AIRmax ^SS mice, although similar cell influx was verified in both lines. Of the genes, 794 were up- and 674 down-regulated in AIRmax ^RR , while 735 genes were found to be up- and 1616 down-regulated in AIRmax ^SS mice 48 h after punch. Both mouse lines showed significant over-represented genes related to cell proliferation; however AIRmax ^SS displayed up-regulation of inflammatory response genes. Quantitative PCR experiments showed higher expressions of Tgfb1, Dap12 and Trem1 genes in AIRmax ^SS mice. These results indicate that Slc11a1 gene modulated the early inflammatory events of ear tissue regeneration.     

<Emphasis Type="Italic">Mycoplasma hominis</Emphasis> and <Emphasis Type="Italic">Gardnerella vaginalis</Emphasis> display a significant synergistic relationship in bacterial vaginosis

Gardnerella vaginalis plays an important role in bacterial vaginosis (BV,) while the role of genital Mollicutes is less obvious. The diagnosis of BV by use of the current Gram stain Nugent score is also suboptimal for defining the role of Mollicutes that lack a cell wall. Since bacterial load and diversity is an important prerequisite for BV, real-time quantitative polymerase chain reaction (qPCR) assays enable these to be assessed. The purpose of this study was to define the role of genital Mollicutes and potential patterns of synergy with G. vaginalis in women with BV. Vaginal swabs from 130 women categorised by Nugent score as BV (n?=?28), intermediate (n?=?22) and non-BV (n?=?80) were tested against four qPCR TaqMan assays targeting G. vaginalis, Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum and U. parvum. Statistical analyses were used to compare bacterial prevalence and load between the three groups of women. Mycoplasma hominis and G. vaginalis co-infection was significantly more common in BV (60.7 %) compared to intermediate (36.4 %) and non-BV (8.8 %) Nugent scores (p?<?0.001). Significantly higher loads of M. hominis (p?=?0.001) and G. vaginalis (p?<?0.001) were detected in women with BV and the respective loads in M. hominis and G. vaginalis co-infections displayed a significant positive correlation (p?<?0.001; r?=?0.60). No significant associations were seen with the other Mollicutes. The findings strengthen the evidence of a role for M. hominis in BV and a potential synergy with G. vaginalis. This synergy could be an important trigger of the condition and sexual contact the conduit for the transmission of an otherwise commensal bacterium that could initiate it.