A Practical Guide to 16S rRNA Microbiome Analysis in Musculoskeletal Disorders.

Microbial taxonomic assignment based on 16S marker gene amplification requires multiple data transformations, often encompassing the use of a variety of computational platforms. Bioinformatics analysis may represent a bottleneck for researchers as many tools require programmatic access in order to implement the software. Here we describe a step-by-step approach for taxonomic assignment using QIIME2 and highlight the utility of graphical-based microbiome tools for further analysis and identification of biological relevant taxa with reference to an outcome of interest.

Evidence review of physical distancing and partition screens to reduce healthcare acquired SARS-CoV-2.

We review the evidence base for two newly introduced Infection prevention and control strategies within UK hospitals. The new standard infection control precaution of 2 metres physical distancing and the use of partition screens as a means of source control of infection for SARS-CoV-2. Following review of Ovid-MEDLINE and governmental SAGE outputs there is limited evidence to support the use of 2 metres physical distancing and partition screens within healthcare.

Protecting the Microbiota.

We examine 3 different approaches to protecting the gut microbiome: highly targeted antibiotics, antibiotic destruction, and antibiotic binding. Each approach shows promise to prevent the off-target effects of antibiotics on the gut microbiome.

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden. OBJECTIVES: To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon. METHODS: Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing. RESULTS: CPE populations increased during inoculation, plateauing at ∼105 log10 cfu/mL in both models and persisting throughout the experiments (>65 days), with no evidence of CPE 'washout'. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742 bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (∼170 kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model). CONCLUSIONS: CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials.

Robust T cell responses to aspergillosis in chronic granulomatous disease: implications for immunotherapy.

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4(+) T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4-depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4(+) T cell-depleted mice, similar to wild-type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4(+) T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non-depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4(+) T cell response suggest that CD4(+) T cell-based immunotherapy for this disease is unlikely to be beneficial.

Combining T-cell immunotherapy and anti-androgen therapy for prostate cancer.

BACKGROUND: Prostate cancer remains a significant health problem for men in the Western world. Although treatment modalities are available, these do not confer long-term benefit and are accompanied by substantial side effects. Adoptive immunotherapy represents an attractive alternative to conventional treatments as a means to control tumor growth. METHODS: To selectively target the tumor-expressed form of Muc1 we constructed a retroviral vector encoding a chimeric antigen receptor (CAR) directed against the aberrantly-expressed extracellular portion of Muc1 called the 'variable number of tandem repeats'. RESULTS: We now demonstrate that T cells can be genetically engineered to express a CAR targeting the tumor-associated antigen Muc1. CAR-Muc1 T cells were able to selectively kill Muc1-expressing human prostate cancer cells. However, we noted that heterogeneous expression of the Muc1 antigen on tumor cells facilitated immune escape and the outgrowth of target-antigen loss variants of the tumor. Given the importance of androgen ablation therapy in the management of metastatic prostate cancer, we therefore also tested the value of combining conventional (anti-androgen) and experimental (CAR-Muc1 T cells) approaches. We show that CAR-Muc1 T cells were not adversely impacted by anti-androgen therapy and subsequently demonstrate the feasibility of combining the approaches to produce additive anti-tumor effects in vitro. CONCLUSIONS: Adoptive transfer of CAR-Muc1 T cells alone or in combination with other luteinizing hormone-releasing hormone analogs or antagonists should be tested in human clinical trials.

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety.

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19(+) T cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL-15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL-15). We found that compared with CAR.19(+) T cells, iC9/CAR.19/IL-15(+) T cells had: (1) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3- to 15-fold greater expansion in vivo) and reduced cell death rate (Annexin-V(+)/7-AAD(+) cells 10+/-6% for iC9/CAR.19/IL-15(+) T cells and 32+/-19% for CAR.19(+) T cells); (2) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1(+) cells <15% for iC9/CAR.19/IL-15(+) T cells versus >40% for CAR.19(+) T cells); and (3) improved antitumor effects in vivo (from 4.7- to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL-15(+) T cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies.

Adoptive cellular immunotherapy for viral diseases.

Viral infections remain a major cause of morbidity and mortality after pediatric hematopoietic stem cell transplantation. Adoptive transfer of donor-derived virus-specific T cells can reconstitute antiviral immunity in recipients and be effective both in preventing and treating cytomegalovirus, Epstein-Barr virus and adenovirus infection. Current efforts are focused on providing protection toward a broader range of viruses safely, rapidly and effectively.

Reconstitution of adenovirus-specific cell-mediated immunity in pediatric patients after hematopoietic stem cell transplantation.

Adenovirus (adv) is a significant cause of morbidity and mortality in pediatric hematopoietic stem cell transplant recipients, and control of infection seems to require antigen-specific T cells. We evaluated the recovery of adv-specific cellular immunity in this patient population related to degree of T-cell immunosuppressive therapy and compared this to adv cellular immunity of normal donors. Over 12 months, we monitored for adv DNA in stool and blood of patients and in the blood of a normal donor group. Twenty-two pediatric hematopoietic stem cell transplant (HSCT) patients (14 months-20 years) who received matched-related (MRD n=6), mismatched related (Haplo n=6) or matched unrelated donor (MUD n=10) grafts, were followed and results compared to healthy controls (n=8). Adv was detected by polymerase chain reaction in blood and/or stool from 81.8% of patients on at least one occasion post-HSCT, but only 68% of patients developed symptomatic adv infections. Recovery of adv-specific T cells was significantly delayed in the MUD and Haplo recipients, whereas recovery in the MRD group was similar to levels detected in healthy donors within 30 days post-transplant. In conclusion, recipients of alternative donor transplants at our institution have significantly delayed adv-specific cellular immune recovery, which correlates to an increased risk of adv-associated morbidity and mortality.

Slc11a1 (formerly Nramp1) polymorphisms and susceptibility to post-transplant lymphoproliferative disease following pediatric liver transplantation.

BACKGROUND: Polymorphisms of the solute carrier family 11 member 1 (Slc11a1) gene have previously been associated with susceptibility to infectious disease, anti-tumor defenses, and autoimmune diseases. We postulated that polymorphisms of the gene may also be associated with susceptibility to post-transplant lymphoproliferative disease (PTLD), a disease thought to be related to an impaired immune response to Epstein-Barr virus (EBV) in immunosuppressed patients. METHODS: Whole blood samples were obtained from 45 pediatric patients who underwent liver transplantation. Polymerase chain reaction (PCR) was used to amplify a 3' region of the gene that includes an exon 15 single-nucleotide substitution (referred to as D543N) and a 4-bp deletion polymorphism (referred to as 3'-UTR). PCR products were digested using AvaII and FokI restriction enzymes for the D543N and 3'-UTR polymorphisms, respectively. PTLD disease status and EBV virus serum titers of all patients were obtained from hospital records. RESULTS: Six of the 45 pediatric transplant recipients developed PTLD. An association was found between 3'-UTR polymorphisms of Slc11a1 and incidence of PTLD after liver transplantation (P = 0.005). In addition, post-transplant serum EBV titers were higher (P = 0.009) for recipients with certain Slc11a1 polymorphisms. No association was found between the D543N polymorphism and incidence of PTLD. CONCLUSION: 3'-UTR polymorphisms of the Slc11a1 gene appear to be associated with susceptibility to PTLD and the immune response to EBV in pediatric liver transplant recipients. Genotyping of pediatric patients undergoing liver transplantation may enable early identification of patients at high risk for developing high EBV titers and/or PTLD.

T cell therapies.

T cell therapies are increasingly used for the treatment of malignancies and viral-associated diseases. Initial studies focused on the use of unmanipulated T cell populations after allogeneic stem cell transplantation. More recently, the use of antigen-specific T cells has been explored. This chapter reviews the clinical experience with polyclonal Epstein-Barr virus (EBV)-specific cytotoxic T cells (CTL) for the treatment of EBV-associated malignancies. Strategies on how to improve the antitumor activity of EBV-specific CTL are being discussed. If effective, these strategies will have broad implications for T cell therapies for a range of human tumors with defined antigens.

T-cell immunotherapy for adenoviral infections of stem-cell transplant recipients.

Human adenoviruses are ubiquitous lytic DNA viruses that can be divided into 51 different serotypes, grouped from A to F on the basis of genome size, composition, homology, and organization. Adenovirus infections, although frequent, are rarely fatal in immunocompetent individuals, due to potent innate and adaptive immune responses. By contrast, adenoviruses are a significant cause of morbidity and mortality in immunosuppressed individuals, for whom there are limited treatment options. Since antiviral drugs have variable efficacy in the treatment of severe adenovirus disease, iatrogenic reconstitution with in vitro expanded virus-specific cytotoxic T lymphocytes (CTLs) is an attractive option for prophylaxis and treatment, particularly because the endogenous recovery of adenovirus-specific T cells has proved important in controlling infection in vivo. Thus, we have characterized human T-cell responses to adenovirus in vitro and explored the potential of adoptive T-cell immunotherapy as a prophylactic or therapeutic strategy for adenovirus infections posttransplant.

Suicide genes as safety switches in T lymphocytes.

Broader application of adoptive transfer of tumor-specific T-lymphocytes is accompanied by the need for effective suicide genes to ensure the safety of this cell-based therapy. In vivo elimination of T-lymphocytes expressing the herpes simplex virus-derived thymidine kinase gene has demonstrated the feasibility of this suicide gene as safety switch. However, improvements are required to overcome initial problems, such as immunogenicity. Here, newly developed suicide genes, including inducible Fas, inducible caspase and CD20 are discussed. In addition, problems of clinical application of marker genes and gene transfer techniques, which are prerequisites for suicide gene therapy, are addressed.

Expansion of EBV latent membrane protein 2a specific cytotoxic T cells for the adoptive immunotherapy of EBV latency type 2 malignancies: influence of recombinant IL12 and IL15.

BACKGROUND: EBV-associated malignancies with a Type II latency gene expression pattern, such as EBV-positive HD, or nasopharyngeal carcinoma, frequently express the EBV latency Ag LMP2a. Hence, they provide a potential target for adoptive immunotherapy using in vitro-generated LMP2a-specific cytotoxic T lymphocytes (CTL). In this study, LMP2a-specific CTL were specifically amplified and the influence of rIL12 and rIL15 on the culture outcome was tested. METHODS: PBMC from donors were stimulated twice with autologous DC transduced with an adenovirus vector expressing LMP2a. This led to a significant expansion of LMP2a-tetramer-specific CTL, which were subsequently further expanded with autologous EBV-transformed B-lymphoblastoid cells (LCL). The addition of rIL12 and rIL15 to the standard IL2-containing culture medium enhanced the proliferation of LMP2a-specific CTL. RESULTS: While rIL15 did not change the pattern of cytokines secreted by LMP2a-CTL, rIL12 enhanced the production of Th1/Tc1 cytokines, such as IFN-n, while suppressing the production of the Th2/Tc2 cytokine IL5. DISCUSSION: Stimulation of CTL cultures with rIL12 or rIL15 will generate CTL more rapidly, facilitating the application of this approach for patients with these EBV-associated disorders.

Targeting CD19 with genetically modified EBV-specific human T lymphocytes.

Human Epstein-Barr virus-specific T cells were genetically modified to express chimeric receptors specific for human CD19, which is expressed on the cell surface of most B cell malignancies. The receptor-modified EBV-specific T cells can be expanded and maintained long term in the presence of EBV-infected B cells. They recognize autologous EBV-infected targets through their conventional T cell receptor, and allogeneic EBV-infected targets and tumor targets through their chimeric receptor. They efficiently lyse both EBV and CD19-positive tumor targets in the absence of background cytotoxicity against CD19-negative targets. Donor-derived EBV-specific T cells expressing chimeric anti-tumor receptors may represent a source of effector cells that could be safely administered to leukemia patients to eradicate minimal residual disease after allogeneic bone marrow transplantation.

Immunotherapy for Hodgkin's disease.

EBV proteins present in the malignant Hodgkin Reed-Sternberg (HR-S) cells of about 40% of patients with Hodgkin's Disease (HD) provide targets for immunotherapy with virus-specific cytotoxic T lymphocytes (CTL). However, Hodgkin tumors use multiple strategies to avoid CTL, including down-regulation of immunodominant EBV antigens, and secretion of cytokines and chemokines such as TGF-beta, that inhibit the activation of CTL and professional antigen-presenting cells (APC). To be effective against this tumor, CTL must resist some or all of these strategies. Thirteen patients with multiply-relapsed HD received EBV-specific CTL, generated ex vivo using the autologous EBV-transformed B cells (LCL) as stimulator cells. After CTL infusion, EBV-specific immunity increased, virus load decreased, CTL homed to sites of malignancy and persisted for up to ten months. Clinically, CTL produced resolution of B symptoms and mixed tumor responses including one complete remission of residual disease remaining after autologous bone marrow transplant. However, no complete remission of bulky disease was achieved. Although LMP2-specific CTL activity could be detected in some of the infused CTL lines, they were present in low frequency. In pre-clinical studies, LMP1 and LMP2-specific CTL could be produced by stimulating PBMC from patients and normal donors with autologous dendritic cells expressing LMP1 or LMP2 from adenoviral vectors. Further, CTL could be rendered resistant to the devastating effects of TGF-beta by transduction with a retrovirus vector expressing a dominant-negative TGF-beta receptor, while transgenic IL-12 could increase the expression of Th1 and decrease that of Th2 cytokines. Future clinical studies will test the efficacy of CTL with improved antigen-specificity and resistance to Hodgkin immune evasion strategies.

Generation of autologous Epstein-Barr virus-specific cytotoxic T cells for adoptive immunotherapy in solid organ transplant recipients.

BACKGROUND: Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorders (PTLD) affect 2%-27% of solid organ transplant (SOT) recipients. Adoptive immunotherapy may have therapeutic potential in this setting, but there is little experience in generating autologous EBV-specific cytotoxic T-cell lymphocytes (EBV-CTLs) from SOT recipients, and their efficacy and persistence in an immunosuppressed environment is unknown. METHODS: EBV-CTLs were generated from eight SOT recipients, using weekly stimulations with autologous lymphoblastoid cell lines (LCLs) and interleukin-2. CTL phenotype and function were evaluated in the presence of therapeutic concentration of cyclosporin A or FK506. RESULTS: In all cases, CTLs expanded with normal kinetics. The majority was CD3+CD8+ (mean, 76%), with less than 3% of natural killer cells. All ex vivo-generated CTLs produced significantly higher killing of autologous LCLs than of HLA-mismatched LCLs (mean, 56% vs. 14% at 20:1 ratio). No lysis of autologous or allogeneic PHA blasts was observed. The CTL expansion rate was reduced in a concentration-dependent manner in the presence of immunosuppressive drugs; however, neither lytic activity nor phenotype was affected. CONCLUSIONS: Using methods that are approved for clinical application, EBV-CTLs can be generated from SOT recipients, even those with frank lymphoma, or who are receiving immunosuppressive drugs. These CTLs retain their function in the presence of immunosuppressive agents. Although in vivo efficacy, safety, and persistence can be assessed only in clinical trials, our results suggest that CTLs can be effective for the treatment of PTLD, even when immunosuppression cannot be reduced because of the high risk of graft rejection.