In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.

Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.

Comprehensive mutational profiling identifies new driver events in cutaneous leiomyosarcoma.

BACKGROUND: Cutaneous leiomyosarcoma (cLMS) is a rare soft tissue neoplasm, showing smooth muscle differentiation, that arises from the mesenchymal cells of the dermis. To-date, genetic investigation of these tumours has involved studies with small sample sizes and limited analyses that identified recurrent somatic mutations in RB1 and TP53, copy number gain of MYCOD and IGF1R, and copy number loss of PTEN. OBJECTIVES: To better understand the molecular pathogenesis of cLMS, we comprehensively explored the mutational landscape of these rare tumours to identify candidate driver events. METHODS: In this retrospective, multi-institutional study, we performed whole-exome sequencing and RNA sequencing on 38 cases of cLMS. RESULTS: TP53 and RB1 were identified as significantly mutated, thus, represent validated driver genes of cLMS. COSMIC mutational signatures SBS7a/b and DBS1 were recurrent, thus, ultraviolet light exposure may be an aetiological factor driving cLMS. Analysis of significantly recurrent somatic copy number alterations, which represent candidate driver events, found focal (<10Mb) deletions encompassing TP53 and KDM6B, and amplifications encompassing ZMYM2, MYOCD, MAP2K4 and NCOR1. A larger (24 Mb) recurrent deletion encompassing CYLD was also identified as significant. Significantly recurrent broad copy number alterations, involving at least half of a chromosome arm, included deletions of 6p/q, 10p/q, 11q, 12q, 13q and 16p/q, and amplification of 15q. Notably PTEN is located on 10q, RB1 on 13q and IGFR1 on 15q. Fusion gene analysis identified recurrent CRTC1/3::MAML2 fusions, as well as many novel fusions in individual samples. CONCLUSIONS: Our analysis of the largest number of cLMS cases to-date highlights the importance of large cohort sizes and the exploration beyond small targeted gene panels when performing molecular analyses, as it allowed a comprehensive exploration of the mutational landscape of these tumours and identification of novel candidate driver events. It also uniquely afforded the opportunity to compare the molecular phenotype of cLMS with LMS of other tissue types, such as uterine and soft tissue LMS. Given that molecular profiling has resulted in the development of novel targeted treatment approaches for uterine and soft tissue LMS, our study now allows the same opportunities to become available for patients with cLMS.

Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

Seroprevalence of Pteropine orthoreovirus in humans remain similar after nearly two decades (2001-2002 vs. 2017) in Tioman Island, Malaysia.

Pteropine orthoreovirus (PRV) is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. In Malaysia, aside from PRV2P (Pulau virus) being isolated from Pteropus hypomelanus sampled in Tioman Island, PRV3M (Melaka virus), PRV4K (Kampar virus), and PRV7S (Sikamat virus) were all isolated from samples of patients who reported having a disease spectrum from acute respiratory distress to influenza-like illness and sometimes even with enteric symptoms such as diarrhea and abdominal pain. Screening of sera collected from human volunteers on Tioman Island in 2001-2002 demonstrated that 12.8% (14/109) were positive for PRV2P and PRV3M. Taking all these together, we aim to investigate the serological prevalence of PRV (including PRV4K and PRV7S) among Tioman Island inhabitants again with the assumption that the seroprevalence rate will remain nearly similar to the above reported if human exposure to bats is still happening in the island. Using sera collected from human volunteers on the same island in 2017, we demonstrated seroprevalence of 17.8% (28/157) against PRV2P and PRV3M, respectively. Seropositivity of 11.4% among Tioman Island inhabitants against PRV4K and PRV7S, respectively, was described in this study. In addition, the seroprevalence of 89.5% (17/19), 73.6% (14/19), 63.0% (12/19), and 73.6% (14/19) against PRV2P, PRV3M, PRV4K, and PRV7S, respectively, were observed among pteropid bats in the island. We revealed that the seroprevalence of PRV among island inhabitants remains nearly similar after nearly two decades, suggesting that potential spill-over events in bat-human interface areas in the Tioman Island. We are unclear whether such spillover was directly from bats to humans, as suspected for the PRV3M human cases, or from an intermediate host(s) yet to be identified. There is a high possibility of the viruses circulating among the bats as demonstrated by high seroprevalence against PRV in the bats.

The mutational landscape of melanoma brain metastases presenting as the first visceral site of recurrence.

Brain metastases are a major cause of melanoma-related mortality and morbidity. We undertook whole-exome sequencing of 50 tumours from patients undergoing surgical resection of brain metastases presenting as the first site of visceral disease spread and validated our findings in an independent dataset of 18 patients. Brain metastases had a similar driver mutational landscape to cutaneous melanomas in TCGA. However, KRAS was the most significantly enriched driver gene, with 4/50 (8%) of brain metastases harbouring non-synonymous mutations. Hotspot KRAS mutations were mutually exclusive from BRAFV600, NRAS and HRAS mutations and were associated with a reduced overall survival from the resection of brain metastases (HR 10.01, p = 0.001). Mutations in KRAS were clonal and concordant with extracranial disease, suggesting that these mutations are likely present within the primary. Our analyses suggest that KRAS mutations could help identify patients with primary melanoma at higher risk of brain metastases who may benefit from more intensive, protracted surveillance.

The clinicopathologic spectrum and genomic landscape of de-/trans-differentiated melanoma.

Dedifferentiation and transdifferentiation are rare and only poorly understood phenomena in cutaneous melanoma. To study this disease more comprehensively we have retrieved 11 primary cutaneous melanomas from our pathology archives showing biphasic features characterized by a conventional melanoma and additional areas of de-/trans-differentiation as defined by a lack of immunohistochemical expression of all conventional melanocytic markers (S-100 protein, SOX10, Melan-A, and HMB-45). The clinical, histologic, and immunohistochemical findings were recorded and follow-up was obtained. The patients were mostly elderly (median: 81 years; range: 42-86 years) without significant gender predilection, and the sun-exposed skin of the head and neck area was most commonly affected. The tumors were deeply invasive with a mean depth of 7 mm (range: 4-80 mm). The dedifferentiated component showed atypical fibroxanthoma-like features in the majority of cases (7), while additional rhabdomyosarcomatous and epithelial transdifferentiation was noted histologically and/or immunohistochemically in two tumors each. The background conventional melanoma component was of desmoplastic (4), superficial spreading (3), nodular (2), lentigo maligna (1), or spindle cell (1) types. For the seven patients with available follow-up data (median follow-up period of 25 months; range: 8-36 months), two died from their disease, and three developed metastases. Next-generation sequencing of the cohort revealed somatic mutations of established melanoma drivers including mainly NF1 mutations (5) in the conventional component, which was also detected in the corresponding de-/trans-differentiated component. In summary, the diagnosis of primary cutaneous de-/trans-differentiated melanoma is challenging and depends on the morphologic identification of conventional melanoma. Molecular analysis is diagnostically helpful as the mutated gene profile is shared between the conventional and de-/trans-differentiated components. Importantly, de-/trans-differentiation does not appear to confer a more aggressive behavior.

Home before Hospital: a whole of system re-design project to improve rates of home-based dialysis therapy: Experience and outcomes over 8 years.

BACKGROUND: Despite evidence that clinical outcomes for patients treated with peritoneal dialysis (PD) or home haemodialysis are better than for patients treated with conventional satellite or hospital-based haemodialysis, rates of home-based dialysis therapies world-wide remain low. Home-based dialysis care is also cost-effective and indeed the favoured dialysis option for many patients. METHODS & OBJECTIVES: Using a lean-thinking framework and established change management methodology, a project embracing a system-wide approach at making a change where a 'Home before Hospital' philosophy underpinned all approaches to dialysis care was undertaken. Three multidisciplinary working groups (pathway, outreach and hybrid) were established for re-design and implementation. The primary aim was to improve home-based dialysis therapy prevalence rates from a baseline of 14.8% by ≥2.5%/year to meet a target of 35%, whilst not only maintaining but improving the quality of care provided to patients requiring maintenance dialysis. A 'future' state pathway was developed after review of the 'current' state (Pathway Working Group) and formed the basis on which a nurse-led outreach service (Outreach Working Group) was established. With the support of the multidisciplinary team, the outreach service model focussed on early, consistent, and frequent education, patient support in decision-making, and clinician engagement. RESULTS: A target prevalence of >30% for home-based therapies (mainly achieved with PD) was achieved within 2 years. This prevalence rate reached 35% within 3 years and was maintained at 8 years. In addition, selected patients already on maintenance satellite-based haemodialysis (Hybrid Working Group) were educated to achieve high levels of proficiencies in self-care. CONCLUSION: Having the system-wide approach to a Quality Improvement Process and using established principles and change management processes, the successful implementation of a new sustainable model of care focussed on home-based dialysis therapy was achieved. A key feature of the model (through outreach) was early nurse-led education and support of patients in decision-making and ongoing support through multidisciplinary care.

Knowing More of the Iceberg: How Detecting a Greater Proportion of Carbapenem-Resistant Enterobacteriaceae Carriers Influences Transmission.

BACKGROUND: Clinical testing detects a fraction of carbapenem-resistant Enterobacteriaceae (CRE) carriers. Detecting a greater proportion could lead to increased use of infection prevention and control measures but requires resources. Therefore, it is important to understand the impact of detecting increasing proportions of CRE carriers. METHODS: We used our Regional Healthcare Ecosystem Analyst-generated agent-based model of adult inpatient healthcare facilities in Orange County, California, to explore the impact that detecting greater proportions of carriers has on the spread of CRE. RESULTS: Detecting and placing 1 in 9 carriers on contact precautions increased the prevalence of CRE from 0% to 8.0% countywide over 10 years. Increasing the proportion of detected carriers from 1 in 9 up to 1 in 5 yielded linear reductions in transmission; at proportions >1 in 5, reductions were greater than linear. Transmission reductions did not occur for 1, 4, or 5 years, varying by facility type. With a contact precautions effectiveness of ≤70%, the detection level yielding nonlinear reductions remained unchanged; with an effectiveness of >80%, detecting only 1 in 5 carriers garnered large reductions in the number of new CRE carriers. Trends held when CRE was already present in the region. CONCLUSION: Although detection of all carriers provided the most benefits for preventing new CRE carriers, if this is not feasible, it may be worthwhile to aim for detecting >1 in 5 carriers.

ST Spot Detector: a web-based application for automatic spot and tissue detection for spatial Transcriptomics image datasets.

Motiviation: Spatial Transcriptomics (ST) is a method which combines high resolution tissue imaging with high troughput transcriptome sequencing data. This data must be aligned with the images for correct visualization, a process that involves several manual steps. Results: Here we present ST Spot Detector, a web tool that automates and facilitates this alignment through a user friendly interface. Contact: jose.fernandez.navarro@scilifelab.se. Supplementary information: Supplementary data are available at Bioinformatics online.

Increased HIV-1 vaccine efficacy against viruses with genetic signatures in Env V2.

The RV144 trial demonstrated 31% vaccine efficacy at preventing human immunodeficiency virus (HIV)-1 infection. Antibodies against the HIV-1 envelope variable loops 1 and 2 (Env V1 and V2) correlated inversely with infection risk. We proposed that vaccine-induced immune responses against V1/V2 would have a selective effect against, or sieve, HIV-1 breakthrough viruses. A total of 936 HIV-1 genome sequences from 44 vaccine and 66 placebo recipients were examined. We show that vaccine-induced immune responses were associated with two signatures in V2 at amino acid positions 169 and 181. Vaccine efficacy against viruses matching the vaccine at position 169 was 48% (confidence interval 18% to 66%; P = 0.0036), whereas vaccine efficacy against viruses mismatching the vaccine at position 181 was 78% (confidence interval 35% to 93%; P = 0.0028). Residue 169 is in a cationic glycosylated region recognized by broadly neutralizing and RV144-derived antibodies. The predicted distance between the two signature sites (21 ± 7 Å) and their match/mismatch dichotomy indicate that multiple factors may be involved in the protection observed in RV144. Genetic signatures of RV144 vaccination in V2 complement the finding of an association between high V1/V2-binding antibodies and reduced risk of HIV-1 acquisition, and provide evidence that vaccine-induced V2 responses plausibly had a role in the partial protection conferred by the RV144 regimen.

Accuracy and Utility of Estimating Lean Body Mass and Nutritional Status in Patients with Chronic Kidney Disease on Long-Term Hemodialysis Using Anthropometric Skinfold Thickness Measurements.

Malnutrition is common in patients on hemodialysis (prevalence of 30% to 50%) and is associated with higher mortality. Lean body mass (LBM) assessment is an accurate way of assessing nutritional status. The dual-energy X-ray absorptiometry (DEXA) scan is a reliable method in assessing body compositions and LBM; however, it is expensive and largely inaccessible. Anthropometric skinfold thickness measurement (ASFM) is useful in assessing LBM. It is cheaper and accessible, but underutilized clinically. The subjective global score (SGA) is a well-established method of assessing nutritional status. All three methods of assessing nutritional status were compared. In this pilot observational study, there was a significant correlation between LBM% estimated by DEXA and ASFM (mean difference -1.46% [95% CI -4.09 to 1.18]; LOA -14.0 to 11.1). Nutritional status by SGA could only detect those severely malnourished when using LBM% by ASFM as comparison. Our study demonstrated that ASFM is a useful method of assessing LBM and nutritional status, which can be easily utilized clinically.

Sequence-based characterization of structural variation in the mouse genome.

Structural variation is widespread in mammalian genomes and is an important cause of disease, but just how abundant and important structural variants (SVs) are in shaping phenotypic variation remains unclear. Without knowing how many SVs there are, and how they arise, it is difficult to discover what they do. Combining experimental with automated analyses, we identified 711,920 SVs at 281,243 sites in the genomes of thirteen classical and four wild-derived inbred mouse strains. The majority of SVs are less than 1 kilobase in size and 98% are deletions or insertions. The breakpoints of 160,000 SVs were mapped to base pair resolution, allowing us to infer that insertion of retrotransposons causes more than half of SVs. Yet, despite their prevalence, SVs are less likely than other sequence variants to cause gene expression or quantitative phenotypic variation. We identified 24 SVs that disrupt coding exons, acting as rare variants of large effect on gene function. One-third of the genes so affected have immunological functions.

Mouse genomic variation and its effect on phenotypes and gene regulation.

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.

Beyond the Intensive Care Unit (ICU): Countywide Impact of Universal ICU Staphylococcus aureus Decolonization.

A recent trial showed that universal decolonization in adult intensive care units (ICUs) resulted in greater reductions in all bloodstream infections and clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) than either targeted decolonization or screening and isolation. Since regional health-care facilities are highly interconnected through patient-sharing, focusing on individual ICUs may miss the broader impact of decolonization. Using our Regional Healthcare Ecosystem Analyst simulation model of all health-care facilities in Orange County, California, we evaluated the impact of chlorhexidine baths and mupirocin on all ICU admissions when universal decolonization was implemented for 25%, 50%, 75%, and 100% of ICU beds countywide (compared with screening and contact precautions). Direct benefits were substantial in ICUs implementing decolonization (a median 60% relative reduction in MRSA prevalence). When 100% of countywide ICU beds were decolonized, there were spillover effects in general wards, long-term acute-care facilities, and nursing homes resulting in median 8.0%, 3.0%, and 1.9% relative MRSA reductions at 1 year, respectively. MRSA prevalence decreased by a relative 3.2% countywide, with similar effects for methicillin-susceptible S. aureus. We showed that a large proportion of decolonization's benefits are missed when accounting only for ICU impact. Approximately 70% of the countywide cases of MRSA carriage averted after 1 year of universal ICU decolonization were outside the ICU.

The Potential Trajectory of Carbapenem-Resistant Enterobacteriaceae, an Emerging Threat to Health-Care Facilities, and the Impact of the Centers for Disease Control and Prevention Toolkit.

Carbapenem-resistant Enterobacteriaceae (CRE), a group of pathogens resistant to most antibiotics and associated with high mortality, are a rising emerging public health threat. Current approaches to infection control and prevention have not been adequate to prevent spread. An important but unproven approach is to have hospitals in a region coordinate surveillance and infection control measures. Using our Regional Healthcare Ecosystem Analyst (RHEA) simulation model and detailed Orange County, California, patient-level data on adult inpatient hospital and nursing home admissions (2011-2012), we simulated the spread of CRE throughout Orange County health-care facilities under 3 scenarios: no specific control measures, facility-level infection control efforts (uncoordinated control measures), and a coordinated regional effort. Aggressive uncoordinated and coordinated approaches were highly similar, averting 2,976 and 2,789 CRE transmission events, respectively (72.2% and 77.0% of transmission events), by year 5. With moderate control measures, coordinated regional control resulted in 21.3% more averted cases (n = 408) than did uncoordinated control at year 5. Our model suggests that without increased infection control approaches, CRE would become endemic in nearly all Orange County health-care facilities within 10 years. While implementing the interventions in the Centers for Disease Control and Prevention's CRE toolkit would not completely stop the spread of CRE, it would cut its spread substantially, by half.

Impact of Delays between Clinical and Laboratory Standards Institute and Food and Drug Administration Revisions of Interpretive Criteria for Carbapenem-Resistant Enterobacteriaceae.

Delays often occur between CLSI and FDA revisions of antimicrobial interpretive criteria. Using our Regional Healthcare Ecosystem Analyst (RHEA) simulation model, we found that the 32-month delay in changing carbapenem-resistant Enterobacteriaceae (CRE) breakpoints might have resulted in 1,821 additional carriers in Orange County, CA, an outcome that could have been avoided by identifying CRE and initiating contact precautions. Policy makers should aim to minimize the delay in the adoption of new breakpoints for antimicrobials against emerging pathogens when containment of spread is paramount; delays of <1.5 years are ideal.

Interplay of metabolizing enzymes and transporter of xenobiotics.

1. Xenobiotics are metabolized and eliminated through the coordinated interplay of their metabolizing enzymes and transporters. However, these two activities in vitro are measured separately, with the addition of ATP as a pre-requisite. 2. We propose a human renal cell-line model which integrates the sulfate and glutathione conjugation of xenobiotics with the efflux of their respective conjugates. Sulfation and glutathionylation represent two major Phase II detoxification of xenobiotics in man. The reactions are catalyzed, respectively, by phenolsulfotransferase and glutathione-S-transferase followed by extrusion of their respective conjugates. 3. Using Ko-143, a specific inhibitor of breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) transporter, we identified this transporter to be responsible for the efflux of p-cresol sulfate, harmol sulfate and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene. 4. The conjugation-cum-efflux was inhibited by oligomycin and uncouplers, which highlights the role of cellular mitochondria in providing ATP for the biosynthesis of their conjugating agents, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and reduced glutathione as well as for the transport function of BCRP. 5. The human 786-O renal cell-line provides a "3-in-1" system linking ATP biosynthesis to metabolism of xenobiotics and their ultimate transport and elimination by BCRP; this integrated system was not apparent in other human cell-lines examined.

Neurocognitive Functioning and Treatment Outcome Following Detoxification Among Asian Alcohol-Dependent Inpatients.

BACKGROUND: The prevalence of alcohol use disorders in Asia is increasing and relapse among treated populations remains the norm, not the exception. The extent to which cognitive impairment influences clinical outcome remains unclear, with research dominated by studies of Caucasian populations. OBJECTIVES: This study examines behavioral and self-reported cognitive functioning in detoxified alcohol-dependent (AD) patients in Singapore and its association with outcome. METHODS: The cognitive performance of 30 recently-detoxified AD inpatients and 30 demographically-matched controls was compared using visuospatial memory, working memory, set-shifting, planning and reflection impulsivity tests of the CANTAB®, and self-reported dysexecutive symptoms and everyday cognitive difficulties. Patients' alcohol use and self-reported cognitive functioning were reassessed 3-months post-discharge. RESULTS: Compared to matched controls, AD inpatients exhibited significantly poorer fluid intelligence, visuospatial memory, working memory, set-shifting flexibility and planning/organization, but not reflection impulsivity. In support of Western studies, a significant proportion (three-quarters) were "clinically impaired" on subtests. Significant reductions were observed in alcohol units, frequency and dependency scores at follow-up, though improvements in self-reported cognitive functioning were limited to abstainers. Baseline cognitive performance did not differentiate those who had abstained from alcohol and relapsed at follow-up. CONCLUSIONS/IMPORTANCE: Memory and executive functioning impairments were evident among Asian AD patients alongside self-reported cognitive difficulties, thus cognitively demanding psychological interventions may have limited impact during early detoxification. Future studies can build on these findings, with larger samples and measurement of moderating and mediating factors to extend our understanding of how cognitive impairment influences outcome.

Metabolic signatures of renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by the constitutive up-regulation of the hypoxia inducible factor-1. One of its target enzymes, pyruvate dehydrogenase (PDH) kinase 1 (PDHK1) showed increased protein expression in tumor as compared to patient-matched normal tissues. PDHK1 phosphorylated and inhibited PDH whose enzymatic activity was severely diminished, depriving the TCA cycle of acetylCoA. We and others have shown a decrease in the protein expressions of all respiratory complexes alluding to a compromise in oxidative phosphorylation (OXPHOS). On the contrary, we found that key parameters of OXPHOS, namely ATP biosynthesis and membrane potential were consistently measurable in mitochondria isolated from ccRCC tumor tissues. Interestingly, an endogenous mitochondrial membrane potential (MMP) was evident when ADP was added to mitochondria isolated from ccRCC but not in normal tissues. In addition, the MMP elicited in the presence of ADP by respiratory substrates namely malate/glutamate, succinate, α-ketoglutarate and isocitrate was invariably higher in ccRCC. Two additional hallmarks of ccRCC include a loss of uncoupling protein (UCP)-2 and an increase in UCP-3. Based on our data, we proposed that inhibition of UCP3 by ADP could contribute to the endogenous MMP observed in ccRCC and other cancer cells.

Dendritic cells restore CD8+ T cell reactivity to autologous HIV-1.

UNLABELLED: Recall T cell responses to HIV-1 antigens are used as a surrogate for endogenous cellular immune responses generated during infection. Current methods of identifying antigen-specific T cell reactivity in HIV-1 infection use bulk peripheral blood mononuclear cells (PBMC) yet ignore professional antigen-presenting cells (APC) that could reveal otherwise hidden responses. In the present study, peptides representing autologous variants of major histocompatibility complex (MHC) class I-restricted epitopes from HIV-1 Gag and Env were used as antigens in gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and polyfunctional cytokine assays. Here we show that dendritic cells (DC) enhanced T cell reactivity at all stages of disease progression but specifically restored T cell reactivity after combination antiretroviral therapy (cART) to early infection levels. Type 1 cytokine secretion was also enhanced by DC and was most apparent late post-cART. We additionally show that DC reveal polyfunctional T cell responses after many years of treatment, when potential immunotherapies would be implemented. These data underscore the potential efficacy of DC immunotherapy that aims to awaken a dormant, autologous, HIV-1-specific CD8+ T cell response. IMPORTANCE: Assessment of endogenous HIV-1-specific T cell responses is critical for generating immunotherapies for subjects on cART. Current assays ignore the ability of dendritic cells to reveal these responses and may therefore underestimate the breadth and magnitude of T cell reactivity. As DC do not prime new responses in these assays, it can be assumed that the observed responses are not detected without appropriate stimulation. This is important because dogma states that HIV-1 mutates to evade host recognition and that CD8+ cytotoxic T lymphocyte (CTL) failure is due to the inability of T cells to recognize the autologous virus. The results presented here indicate that responses to autologous virus are generated during infection but may need additional stimulation to be effective. Detecting the breadth and magnitude of HIV-1-specific T cell reactivity generated in vivo is of the utmost importance for generating effective DC immunotherapies.