Utility of metagenomic Next-Generation Sequencing for simultaneously detecting pathogens and neoplasms.

Objectives: Clinicians often face the challenge of differentially diagnosing febrile patients who are suspected of infectious diseases, since the clinical manifestations of infection and cancer may overlap. A single test that can detect both pathogens and tumor could provide timely and accurate diagnostic clues to aid the treatment and management of these patients. Methods: We enrolled eight patients to evaluate the utility of metagenomic Next-Generation Sequencing for simultaneously detecting pathogens and neoplasms using body fluids and tissue samples. Patients were selected by the following criteria: 1) Tumor was not considered upon hospitalization, but mNGS testing indicated neoplasm; 2) Tumor was not excluded, but microbial infection was primarily suspected according to initial clinical assessment. Results: We detected potential pathogens in five patients, three of whom had progressed into critical infections. Moreover, abnormal chromosomal copy numbers were identified in all patients that indicated presence of neoplasms, which were pathologically confirmed. Conclusions: Although copy number variations do not render a definitive cancer diagnosis, it can prompt clinicians to conduct more focused diagnostic testing for cancer, potentially saving time and cost. As a result, integrating copy number analysis with pathogen detection in mNGS may help establish rapid and accurate diagnosis for febrile patients.

Human umbilical cord mesenchymal stem cell-derived TGFBI attenuates streptozotocin-induced type 1 diabetes mellitus by inhibiting T-cell proliferation.

MSCs have been demonstrated to have a great benefit for type 1 diabetes mellitus (T1DM) due to their strong immunosuppressive and regenerative capacity. However, the comprehensive mechanism is still unclear. Our previous study indicated that transforming growth factor beta induced (TGFBI) is highly expressed in human umbilical cord-derived mesenchymal stem or stromal cells (hUC-MSCs), which are also implicated in T1DM. In this study, we found that infusion of TGFBI knockdown hUC-MSCs displayed impaired therapeutic effects in T1DM mice and decreased immunosuppressive capability. TGFBI knockdown hUC-MSCs could increase the proportion of T-cell infiltration while increasing the expression of IFN-gamma and interleukin-17A in the spleen. In addition, we also revealed that hUC-MSC-derived TGFBI could repress activated T-cell proliferation by interfering with G1/S checkpoint CyclinD2 expression. Our results demonstrate that TGFBI plays a critical role in MSC immunologic regulation. TGFBI could be a new immunoregulatory molecule controlling MSC function for new treatments of T1DM. Schematic Representation of the Immunosuppression capacity of hUC-MSC by TGFBI.

Neoantigen cancer vaccines: a new star on the horizon.

Immunotherapy represents a promising strategy for cancer treatment that utilizes immune cells or drugs to activate the patient's own immune system and eliminate cancer cells. One of the most exciting advances within this field is the targeting of neoantigens, which are peptides derived from non-synonymous somatic mutations that are found exclusively within cancer cells and absent in normal cells. Although neoantigen-based therapeutic vaccines have not received approval for standard cancer treatment, early clinical trials have yielded encouraging outcomes as standalone monotherapy or when combined with checkpoint inhibitors. Progress made in high-throughput sequencing and bioinformatics have greatly facilitated the precise and efficient identification of neoantigens. Consequently, personalized neoantigen-based vaccines tailored to each patient have been developed that are capable of eliciting a robust and long-lasting immune response which effectively eliminates tumors and prevents recurrences. This review provides a concise overview consolidating the latest clinical advances in neoantigen-based therapeutic vaccines, and also discusses challenges and future perspectives for this innovative approach, particularly emphasizing the potential of neoantigen-based therapeutic vaccines to enhance clinical efficacy against advanced solid tumors.

Coxiella burnetii and Bartonella Endocarditis Diagnosed by Metagenomic Next-Generation Sequencing.

(1) Background: Culture-negative endocarditis is challenging to diagnose. Here, we retrospectively identified 23 cases of Coxiella burnetii and Bartonella endocarditis by metagenomic next-generation sequencing. (2) Methods: Twenty-three patients with culture-negative endocarditis were retrospectively enrolled from Guangdong Provincial People's Hospital (n = 23) between April 2019 and December 2021. Metagenomic next-generation sequencing was performed on blood (n = 22) and excised cardiac valvular tissue samples (n = 22) for etiological identification, and Sanger sequencing was performed for pathogenic diagnostic verification. The demographic and clinical data of the 23 patients were obtained from hospital electronic health records. (3) Results: A total of 23 male patients (median age, 56 years (interquartile range, 16)) with culture-negative endocarditis were diagnosed with Coxiella burnetii (n = 21) or Bartonella (n = 2) species infection by metagenomic next-generation sequencing. All patients underwent cardiac surgery. The resected tissue exhibited both a significantly higher number of unique suspected pathogen read-pairs and more unique pathogen read-pairs than the blood specimens. The results of Sanger sequencing tests on all remaining tissue and blood specimens were positive. Oral doxycycline was added to the antibiotic regimen for at least 1.5 years according to etiology. A total of 21 patients (91%) were discharged, and 20 patients were healthy at the 21-month (interquartile range, 15) follow-up visit. One patient exhibited endocarditis relapse with the same pathogen from inadequate antibiotic administration. The last 2 patients (9%) developed septic shock and multiple organ dysfunction syndrome postoperatively and died shortly after discharge. (4) Conclusions: CNE caused by C. burnetii and Bartonella species is challenging to diagnose and exhibits poor outcome due to delayed treatment. In response, mNGS, characterized by high sensitivity and rapid results, is an effective alternative for the etiological identification of C. burnetii and Bartonella endocarditis.

hUC-MSCs Attenuate Acute Graft-Versus-Host Disease through Chi3l1 Repression of Th17 Differentiation.

Mesenchymal stem cells (MSCs) have already demonstrated definitive evidence of their clinical benefits in acute graft-versus-host disease (aGvHD) and other inflammatory diseases. However, the comprehensive mechanism of MSCs' immunomodulation properties has not been elucidated. To reveal their potential immunosuppressive molecules, we used RNA sequencing to analyze gene expression in different tissue-derived MSCs, including human bone marrow, umbilical cord, amniotic membrane, and placenta, and found that chitinase-3-like protein 1 (Chi3l1) was highly expressed in human umbilical cord mesenchymal stem cells (hUC-MSCs). We found that hUC-MSCs treated with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) exhibited increased expression of Chi3l1 and concurrently repressed T-helper 17 cell (Th17) differentiation through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, Chi3l1 knockdown hUC-MSCs exhibited impaired therapeutic efficacy in aGvHD mice with an increased inflammatory response by promoting Th17 cell differentiation, including an increase in IL-17A in the spleen, intestine, and serum. Collectively, these results reveal a new immunosuppressive molecule, Chi3l1, in hUC-MSCs in the treatment of aGvHD that regulates Th17 differentiation and inhibits STAT3 activation. These novel insights into the mechanisms of hUC-MSC immunoregulation may lead to the consistent production of hUC-MSCs with strong immunosuppressive functions and thus improved clinical utility.

Wip1 regulates the immunomodulatory effects of murine mesenchymal stem cells in type 1 diabetes mellitus via targeting IFN-α/BST2.

Mesenchymal stem cells (MSCs) show significant therapeutic effects in type 1 diabetes mellitus (T1DM) as regulating the inflammatory processes. However, little is known about the detailed process of MSCs immunosuppression in T1DM. In this study, we investigated the effects of wild-type p53-induce phosphatase 1 (Wip1) on regulating MSCs immunosuppressive capacities in T1DM mice. We found that Wip1 knockout (Wip1-/-) MSCs had lower therapeutic effects in T1DM mice, and displayed weaker immunosuppressive capability. In vivo distribution analysis results indicated thatWip1-/-MSCs could home to the damaged pancreas and increase the expression of tumor necrosis factor-α (TNF-α), interleukin-17a (IL-17a), interferon-α(IFN-α), IFN-ß, and IFN-γ, while decrease the expression of IL-4 and IL-10. Moreover, we confirmedWip1-/-MSCs exhibited weaker immunosuppressive capacity, as evidenced by enhanced expression of bone marrow stromal cell antigen 2(BST2) and IFN-α. In conclusion, these results revealed Wip1 affects MSCs immunomodulation by regulating the expression of IFN-α/BST2. Our study uncovered that Wip1 is required to regulate the therapeutic effects of MSCs on T1DM treatment, indicating a novel role of Wip1 in MSCs immunoregulation properties.

Maternal vitamin D deficiency increases the risk of obesity in male offspring mice by affecting the immune response.

OBJECTIVES: Recently, many epidemiologic and animal studies have indicated that obesity has its origin in early stages of life, including the inappropriate balance of some nutrients. So the objectives of this study were to determine the risk of obesity in male offspring mice as a consequence of maternal vitamin D (VD) deficiency mediating the disordered immune response. METHODS: C57BL/6J female mice 4 wk old were fed VD-deficient or normal reproductive diets during pregnancy and lactation. Their male offspring were given control and high-fat diets for 16 wk after weaning and then weighed and euthanized. The serum was collected for biochemical analyses. Epididymal (eWAT) and inguinal white adipose tissue (iWAT) were excised for histologic examination, immunohistochemistry, gene expression of inflammatory factors, and determination by flow cytometry of the proportions of immune cells. RESULTS: Insufficient maternal VD intake exacerbated the development of obesity in male offspring mice that were both obese and non-obese, as evidenced by larger adipose cells and abnormal glucose and lipid metabolisms. Also, the expressions of proinflammatory cytokines were increased and that of anti-inflammatory cytokines was decreased in eWAT and/or iWAT in the maternal VD-deficient group, accompanied by higher levels of tumor necrosis factor-α and/or interferon-γ and lower levels of interleukin-4 and interleukin-10. Insufficient maternal VD intake was also observed to induce a shift in the profiles of immune cells in the eWAT and/or iWAT of male offspring that were both obese and non-obese, resulting in increased percentages of M1 macrophages, adipose tissue dendritic cells, and CD4+ and CD8+ T cells but a significant decrease in the percentages of M2 macrophages. All these changes in the immune cell profile were more obvious in the eWAT than those in the iWAT. CONCLUSIONS: Maternal VD deficiency might promote the development of obesity in male offspring mice partly by modulating the immune cell populations and causing a polarization in the adipose depots.

Mesenchymal stem cell exosome-derived miR-223 alleviates acute graft-versus-host disease via reducing the migration of donor T cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have been utilized in treating acute graft-versus-host disease (aGvHD) as they show strong immunosuppressive capacity through the release of various mediators, including immunosuppressive molecules, growth factors, chemokines, and exosomes. MicroRNAs (miRNAs) derived from MSC exosomes (MSCs-Exo) play a critical role in the regulation of immune responses. However, the function of miRNAs in treating aGvHD remains unknown. Here, we performed expression profiling of exosome-miRNAs from human umbilical cord MSCs (huc-MSCs) and murine compact bone MSCs (mb-MSCs) to investigate their immunoregulation effects in aGvHD. METHODS: Huc-MSCs-Exo and mb-MSCs-Exo were isolated and constructed MSCs-Exo-derived miRNA expression profiling using high-throughput sequencing. High expression of miR-223 was identified in both kinds of MSCs-Exo by bioinformatics analysis and quantitative real-time PCR (qPCR). In vitro cell crawling assay, transmigration assay and adhesion assay were subsequently applied to investigate the regulation of miR-223 on T cells. MiR-223 target gene was analyzed by western blot, luciferase analysis, and qPCR. Moreover, murine aGvHD model was established by infusing splenocytes and bone marrow nuclear cells from C57BL/6j mice (H-2Kb) into BALB/c recipient mice (H-2Kd). For therapeutic effect, MSCs or miR-223 Agomir were injected via tail vein. The general conditions of the mice in each group were monitored. Hematoxylin-eosin (H&E) staining was used to detect pathological changes of mice spleen, liver, and intestine. Mechanistically, immunofluorescence and flow cytometry were used to evaluate donor T cell migration, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of serum inflammatory cytokines IFN-γ, TNF-α, and IL-17. RESULTS: High-throughput sequencing revealed high expression of miR-223 in huc-MSCs-Exo and mb-MSCs-Exo. MiR-223 could restrain adhesion and migration of T cells by inhibiting ICAM-1 expression in mouse lymphatic endothelial cells. MiR-223Agomir infusion attenuated aGvHD clinical symptoms, reduced the donor T cell infiltration into the spleen, liver, and intestine, and decreased inflammatory cytokines IFN-γ, TNF-α, and IL-17. CONCLUSION: MSCs-Exo-derived miR-223 could attenuate aGvHD in mice through decreasing donor T cell migration. Our results unveil a new role of MSCs-Exo containing miR-223 in the treatment of aGvHD.

Incidence of and risk factors for surgical site infection after colorectal surgery: A multiple-center prospective study of 3,663 consecutive patients in China.

BACKGROUND: Surgical site infection (SSI) after colorectal surgery (CRS) remains a significant problem for its negative clinical outcomes. However, it is poorly understood in China. This study aims to investigate the incidence, risk factors and microbiology of SSI after CRS. METHODS: A nationwide prospective multicenter design was applied. Patients in 19 Chinese hospitals from 2015 to 2018 were prospectively monitored for SSI after CRS. Demographic data, hospital characteristics, and potential perioperative risk factors were collected and analyzed, using univariate and multivariate logistic regression models. RESULTS: Among 3,663 study participants, 134(3.66%) episodes of SSI were identified. The incidence rate of SSI decreased from 5.9 infections per 100 procedures in 2015 to 3.1 infections per 100 procedures in 2018 (incidence rate ratio, 0.52; 95% CI, 0.28-0.94). The SSI rates were 1.88, 4.15, 6.27 and 11.58 per 100 operations for the National Nosocomial Infections Surveillance system (NNIS) risk index categories of 0, 1, and 2 or 3, respectively. Escherichia coli (54/134, 40.3%) and Klebsiella pneumoniae (10/134, 7.5%) were the most frequently isolated microorganisms. A high prevalence of antibiotic resistance were observed in our study, with rates of extended spectrum beta-lactamase-producing or carbapenem-resistant Escherichia coli and Klebsiella pneumonia of 50.0%(27/54) and 30.0%(3/10) respectively. Preoperative hospital stay ≥ 48h (OR=2.28, 95% CI: 1.03-5.02, P=0.042) and contaminated or dirty wound (OR=3.38, 95% CI: 1.88-6.06, P=4.50×10-5) were significantly associated with increasing risk of SSI after CRS. CONCLUSION: A statistically significant but modest decrease in the incidence rate of CRS SSI over the 4-year study period was observed in this study. Noticeably, the relatively high rates of multidrug-resistant pathogens causing SSI after CRS should be alert, while more studies with large population are needed due to the small number of isolates identified in our survey.

Comparison of the health risks associated with different exposure pathways of multiple volatile chlorinated hydrocarbons in contaminated drinking groundwater.

A total of 152 groundwater samples were collected around a contaminated site to evaluate the carcinogenic and noncarcinogenic risks of exposure to twelve volatile chlorinated hydrocarbons (VCHs) by oral ingestion, dermal contact and inhalation absorption during showering. Although toxicity data of some VCHs are fragmentary, the results showed that the carcinogenic and noncarcinogenic risks of exposure to VCHs in contaminated drinking groundwater for local residents needs immediate attention. The main risk contributors for the carcinogenic and noncarcinogenic risks are carbon tetrachloride and 1,1,2-trichloroethane through inhalation exposure pathway, respectively. The health risk contribution rates associated with three exposure pathways for a specific VCH were intrinsic to the compound, and the dermal contact corresponded to a negligible contribution for almost every VCH species. Although most of the evaluated VCHs had a higher risk contribution by inhalation than by oral ingestion pathway, the integrated multi-VCH health risk contributions of the three exposure pathways were mainly dependent on the VCH compositions. Drinking boiled water not only decreased the exposure risk but also affected the risk contribution rates of three exposure pathways, which indicates that it is feasible to reduce the risk of VCH exposure during daily activities based on the risk contribution of the exposure pathways. In addition to the VCHs included in the drinking water standards, species such as 1,1,2,2-tetrachloroethane and hexachloroethane also showed a remarkable exposure health risk according to the standardized health risk calculation, which implied that improved drinking water standards are urgently required.

The Therapeutic Effect of ICAM-1-Overexpressing Mesenchymal Stem Cells on Acute Graft-Versus-Host Disease.

BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) do not readily migrate to appropriate sites, and this creates a major obstacle for their use in the treatment of graft-versus-host disease (GVHD). Intercellular adhesion molecule-1 (ICAM-1) can guide the homing of various immune cells to the proper anatomical location within secondary lymphoid organs (SLOs), which are the major niches for generating immune responses or tolerance. MSCs rarely migrate to SLOs after intravenous infusion, and are constitutively low expression of ICAM-1. So in our previous work, ICAM-1 was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (ICAM-1MSCs). Here, we hypothesized that ICAM-1highMSCs may significantly improve their immunomodulatory effect. METHODS: We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate ICAM-1highMSCs immunomodulatory effect on dendritic cells (DCs) and T cells in vitro and in vivo. MSCs were labeled with carboxyfluorescein diacetate succinimidylester (CFSE) to detect its distribution in mouse model. RESULTS: Our in vitro analyses revealed ICAM-1 MSCs could suppress DCs maturation according to co-culture methods and suppress the T cell immune response according to the mixed lymphocyte response (MLR) and lymphoblast transformation test (LTT) tests. We found that infusion of ICAM-1highMSCs potently prolonged the survival of GVHD mouse model. The infused ICAM-1highMSCs migrate to SLOs in vivo, and suppressed DCs maturation, suppressed CD4+ T cell differentiation to Th1 cells, and increased the ratios of Treg cells. CONCLUSIONS: Taken together, these data demonstrate that ICAM-1highMSCs had an enhanced immunosuppressive effect on DCs and T cells, which may help explain the protective effect in a GVHD model. This exciting therapeutic strategy may improve the clinical efficacy of MSC-based therapy for GVHD.

Performance and mechanism of Cr(VI) removal by zero-valent iron loaded onto expanded graphite.

Zero-valent iron (ZVI) was loaded on expanded graphite (EG) to produce a composite material (EG-ZVI) for efficient removal of hexavalent chromium (Cr(VI)). EG and EG-ZVI were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy and Brunauer-Emmett-Teller (BET) analysis. EG-ZVI had a high specific surface area and contained sub-micron sized particles of zero-valent iron. Batch experiments were employed to evaluate the Cr(VI) removal performance. The results showed that the Cr(VI) removal rate was 98.80% for EG-ZVI, which was higher than that for both EG (10.00%) and ZVI (29.80%). Furthermore, the removal rate of Cr(VI) by EG-ZVI showed little dependence on solution pH within a pH range of 1-9. Even at pH11, a Cr(VI) removal rate of 62.44% was obtained after reaction for 1hr. EG-ZVI could enhance the removal of Cr(VI) via chemical reduction and physical adsorption, respectively. X-ray photoelectron spectroscopy (XPS) was used to analyze the mechanisms of Cr(VI) removal, which indicated that the ZVI loaded on the surface was oxidized, and the removed Cr(VI) was immobilized via the formation of Cr(III) hydroxide and Cr(III)-Fe(III) hydroxide/oxyhydroxide on the surface of EG-ZVI.

[MicroRNA-3963 Promotes Adipogenic Differentiation of Mouse-Derived Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of MicroRNA-3963(miR-3963) on the adipogenic differentiation of mouse bone-derived mesenchymal stem cells(MSC). METHODS: MSCs were isolated from C57BL/6 mice bone fragment and transfected with miR-3963 mimic, miR-3963 inhibitor and negative control. The expression of miR-3963 and transfection efficiency were detected by q-PCR. These transfected cells were induced to adipocytes and stained with oil red O after 14 days culture. q-PCR and Western blot were used to detect the expression of adipogenic differentiation marker genes C/EBPα and PPARγ at transcriptional level and protein level. RESULTS: The results of q-PCR revealed that miR-3963 expression level was up-regulated after transfection with miR-3963 mimic (P<0.0001), and down-regulated after transfection with miR-3963 inhibitor (P<0.0001). After oil red staining, overexpression of miR-3963 in MSCs could promote the formation of lipid droplet. The q-PCR and Western blot analyses showed the significant increase of expression of adipogenic marker genes C/EBPα and PPARγ in MSC transfected with miR-3963 mimic. Additionally, compared with the control group, miR-3963 inhibitor could decrease adipogenic differentiation of MSC. CONCLUSION: miR-3963 can regulate and promote adipogenic differentiation of mouse bone-derived MSC.

[Effect of MicroRNA-146b-5p on Adipogenesis of Mouse Bone Essence Derived Mesenchymal Stem Cells].

OBJECTIVE: To explore the effect of MicroRNA-146b-5p (miR-146b-5p) on the mouse bone essence derived MSC adipogenic differentiation. METHODS: MSC were isolated from bone essence of C57BL/6 mice. The expression level of miR-146b-5p in the process of adipogenic differentiation of MSC was detected by q-PCR; the role of miR-146b-5p mimics or inhibitors in the process of mouse bone essence derived MSC adipogenic differentiation was analyzed through oil red staining the expression of C/EBPα and PPARγ after cultured for 14 days was detected by q-PCR; the protein level of PPARγ after miR-146b-5p transfection was detected by Western blot. RESULTS: The MSC were successfully isolated from bone essence of mice, the q-PCR results showed an increasing expression level of miR-146-5p in the process of MSC adipogenic differentiation. Compared with the control group, MSC transfected with miR-146b-5p mimic could up-regulate the expression of miR-146b-5p (P<0.001), while miR-146b-5p inhibitor transfection could down-regulate the endogenous miR-146b-5p expression (P<0.01). After culture for 14 d, the result of Oil red staining showed that the miR-146b-5p inhibitor could inhibit adipogenic differentiation, while the miR-146b-5p mimic could promote the adipogenic differentiation of MSC. After induction for 14 d, compared with control, the PPARγ and C/EBPα in mimic group were higher expressed PPARγ and C/EBPα (P<0.01). Compared with induced group, the PPAPγ and C/EBPα were lower expressed in inhibitor group (P<0.05). The results of Western blot showed that the expression level of PPARγ was high in minic group, and it was low in inhibitor group. CONCLUSION: miR-146b-5p is up-regulated in the process of MSC adipogenic differentiation, and it promotes the adipogenesis of MSC originated from mouse bone essence.