Total DNA Levels Research Articles

Circulating tumor DNA dynamics using patient-customized assays are associated with outcome in neoadjuvantly treated breast cancer

Pathological complete response (pCR) is an accurate predictor of good outcome following neoadjuvant chemotherapy (NAC) for locally advanced breast cancer. The presence of circulating-tumor DNA (ctDNA) has recently been reported to be strongly predictive of poor outcome in similar patient groups. We monitored ctDNA levels from 10 women undergoing NAC for locally advanced breast cancer using a patient-specific, hybrid-capture sequencing technique sensitive to the level of one altered allele in 10,000. Plasma was collected prior to the start of NAC, prior to each infusion of NAC, and during follow-up for between 350 and 1150 d after the start of NAC. Prior to the start of NAC, ctDNA was detectable in 3/3 triple negative, 3/3 HER2+, and 2/4 HER2−, ER+ breast cancer patients. Total cell-free DNA levels were considerably higher when patients were on NAC than at other times. ctDNA dynamics during NAC showed that patients with pCR experienced rapid declines in ctDNA levels, whereas patients without pCR typically showed evidence of residual ctDNA after initiation of treatment. Intriguingly, two of three patients that showed marked increases in ctDNA while on NAC experienced rapid recurrences (<2 yr following start of NAC). The third patient that had increases in ctDNA levels while on NAC had low-grade ER+ disease and showed residual ctDNA after surgery, which became undetectable after local radiation. Taken together, these results demonstrate the ability of our approach to sensitively serially monitor ctDNA during NAC, and identifies a need to further investigate the possibility of stratifying patients who need additional treatment or identify therapies that are ineffective.

DNA Methylation in Yeast-Like Fungi of the Species Candida albicans Induced by Different Lengths of Exposure to Ozone

The objective of the study was to determine global DNA methylation induced by lengths of exposure to ozone in Candida albicans isolates of human and animal origin. The level of global DNA methylation was tested as the relative methylation level in the samples in relation to a methylated control. The analysis revealed differences in the methylation level in the human and animal samples. Ozone had a significant effect on the DNA methylation process, and longer exposure substantially increased the level of total methylated DNA. As expected, the degree of methylation depended on the length of exposure to ozone.

HIV controllers suppress viral replication and evolution and prevent disease progression following intersubtype HIV-1 superinfection.

The aim of this study was to investigate the impact of intersubtype HIV-1 superinfection on viremia, reservoir reseeding, viral evolution and disease progression in HIV controllers (HIC). A longitudinal analysis of two Brazilian HIC individuals (EEC09 and VC32) previously identified as dually infected with subtypes B and F1 viruses. Changes in plasma viremia, total HIV-1 DNA levels, CD4+ T-cell counts and HIV-1 quasispecies composition were measured over time. HIV-1 env diversity in peripheral blood mononuclear cell (PBMC) and plasma samples was accessed by single genome amplification and next-generation sequencing approaches, respectively. Viral evolution was evaluated by estimating nucleotide diversity and divergence. Individual EEC09 was probably initially infected with a CCR5-tropic subtype B strain and sequentially superinfected with a CXCR4-tropic subtype B strain and with a subtype F1 variant. Individual VC32 was infected with a subtype B strain and superinfected with a subtype F1 variant. The intersubtype superinfection events lead to a moderate increase in viremia and extensive turnover of viral population in plasma but exhibited divergent impact on the size and composition of cell-associated HIV DNA population. Both individuals maintained virologic control (<2000 copies/ml) and presented no evidence of viral evolution or immunologic progression for at least 2 years after the intersubtype superinfection event. These data revealed that some HIC are able to repeatedly limit replication and evolution of superinfecting viral strains of a different subtype with no signs of disease progression.

Reconstitution of HIV-1 reservoir following high-dose chemotherapy/autologous stem cell transplantation for lymphoma.

Autologous stem cell transplantation following high-dose chemotherapy (HDC/ASCT) is the prime model to study the impact of HDC in HIV-1-infected participants. We analyzed the impact of HDC/ASCT on the resurgent reservoir composition and origin. We included retrospectively a homogenous group of HIV-1-infected patients treated for high-risk lymphoma in a reference center with similar chemotherapy regimens. Thirteen participants treated with HDC/ASCT from 2012 to 2015 were included. A median seven longitudinal blood samples per participant were available. Total HIV-1 DNA levels in peripheral blood mononuclear cells (PBMCs) were quantified by quantitative PCR. In six participants with sustained viral suppression, the highly variable C2V3 viral region was subjected to next-generation sequencing. Maximum-likelihood phylogeny trees were generated from the reconstructed viral haplotypes. Lymphocyte subsets were studied by flow cytometry. PBMC-associated HIV-1 DNA levels were stable over time. Viral diversity decreased along lymphoma treatment, but increased promptly back to prechemotherapy numbers after HDC/ASCT. Blood viral populations from all time-points were intermingled in phylogeny trees: the resurgent reservoir was similar to pre-HDC circulating proviruses. Memory subsets were the main contributor to the early restoration of the CD4+ T-cell pool, with a delayed increase in naïve cell counts. The characterization of HIV-1 reservoir in blood revealed a fast and consistent replenishment from memory CD4+ T cells after HDC/ASCT. As HDC/ASCT is increasingly involved in HIV cure trials with gene-modified hematopoietic stem cells, the management of infected T cells in HIV-positive autologous transplants will be critical.

HIV-1 DNA Is Maintained in Antigen-Specific CD4+ T Cell Subsets in Patients on Long-Term Antiretroviral Therapy Regardless of Recurrent Antigen Exposure

Memory CD4+ T cells (mCD4s) containing integrated HIV DNA are considered the main barrier to a cure for HIV infection. Here, we analyzed HIV DNA reservoirs in antigen-specific subsets of mCDs to delineate the mechanisms by which HIV reservoirs persist during antiretroviral therapy (ART). HIV Gag, cytomegalovirus (CMV), and tetanus toxoid (TT)-specific mCD4s were isolated from peripheral blood samples obtained from 11 individual subjects, 2-11 years after commencing ART. Antigen-specific mCD4s were identified by the sensitive OX40 assay and purified by cell sorting. Total HIV DNA levels were quantified by real-time PCR, and clonal viral sequences generated from mCD4 subsets and pre-ART plasma samples. Quantitative results and sequence analysis were restricted to five and three study participants, respectively, which was likely due to the low frequency of the antigen-specific mCD4s and relatively low HIV DNA proviral loads. Median HIV Gag-, CMV-, and TT-specific mCD4s were 0.61%, 2.46%, and 0.78% of total mCD4s, and they contained a median of 2.50, 2.38, and 2.55 log10 copies of HIV DNA per 106 cells, respectively. HIV DNA sequences were derived from antigen-specific mCD4s clustered with sequences derived from pre-ART plasma samples. There was a trend toward increased viral diversity in clonal viral sequences derived from CMV-specific mCD4s relative to TT-specific mCD4s. Despite limitations, this study provides direct evidence that HIV reservoirs persist in memory CD4+ T cell subsets maintained by homeostatic proliferation (TT) and adds to growing evidence against viral evolution during ART. Similar future studies require techniques that sample diverse HIV reservoirs and with improved sensitivity.

Galectin Secretion and Modulation in Sheep Blood

Galectins (GAL) are &amp;beta;-galactoside binding proteins that can modulate both pregnancy and the response to pathogens. Increased susceptibility to infection by pathogens is linked to periparturient immune suppression and a periparturient rise (PPR) in parasite eggs on pasture. The possible role of GAL in periparturient immune relaxation and PRR needs definition. &amp;nbsp;The objective of this study was to evaluate galectin secretion and its relationship to measures of the immune relaxation and PRR in periparturient sheep. Samples were collected from pregnant St. Croix sheep (n=6) weekly at days -21 to + 21 relative to lambing. Fecal samples were collected and evaluated for strongyle and coccidia parasite eggs. The concentration of IgA and IgE coproantibodies, total microbial DNA, Bifidobacteria and Lactobacillus levels in fecal samples were used as indicators of gut health.&amp;nbsp; Blood samples were collected by jugular venipuncture and assessed for Packed Cell Volume (PCV), total and differential white blood cell counts. Total protein concentrations and protein profile were evaluated in serum. Secretion of GAL 1, 3, 9 and 14 were evaluated using ELISAs. Data were analyzed by one-way ANOVA and statistical significance was declared at P &amp;lt;0.05. Galectins tested were secreted in sheep blood. Differential modulation of GAL secretion and correlation with periparturient immune suppression and parasite infection was observed. Galectin secretion was modulated by the periparturient period, type and status of parasite infection. This first insight into a possible role of secreted galectins in periparturient immune relaxation and PRR improves the understanding of the immune response, informs development of management programs and therapeutics and presents Galectin profiles as biomarkers with diagnostic potential.

Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA

BackgroundHepatitis B virus covalently closed circular DNA (HBV cccDNA) is an important biomarker of hepatitis B virus infection. However, the current methods are not specific and sensitive. The present study aimed to develop a specific and sensitive assay method for the quantification of HBV cccDNA. MethodsExonuclease I (Exo I) & Exonuclease III (Exo III) and specific primer probes are used in real-time PCR. The virus particles isolated from peripheral blood mononuclear cells were used as negative control and HBV1.3 recombinant plasmid 3.2 kb circular DNA fragment was used as positive control. The methods of cccDNA detection were evaluated in cell lines, plasmid, animal model, patient serum and liver biopsies. ResultsA linear range of 101–107 copies/assay using specific primers for HBV cccDNA was established. HBV cccDNA were only detected in cell lines, animal model and liver tissue. It cannot be detected in serum samples. Intrahepatic HBV cccDNA level had good correlation with intrahepatic total HBV DNA level (r = 0.765, P < 0.001). ConclusionsThe real-time quantitative PCR is an effective and feasible method for sensitive and specific detection of low copy number of cccDNA. The novel detection method is fast, provides high sensitivity and specificity and can be used in clinical practice.

Extracellular DNA in the Dynamics of Uncomplicated Pregnancy.

We analyzed the concentration of extracellular DNA and its fractions in the dynamics of uncomplicated pregnancy Thirty women with singleton pregnancy were examined. The concentration of total, maternal, and fetal cell-free DNA in maternal blood was measured at gestation weeks 11-14, 24-26, and 30-32. The level of total cell-free DNA was evaluated by measuring the concentration of RASSF1A gene using quantitative PCR analysis, the level of cell-free fetal DNA was assessed by determining the hypermethylated part of RASSF1A gene. The concentration of total cell-free DNA and cell-free maternal DNA did not change during the first half of pregnancy, but increased after 24-26 weeks. The level of cell-free fetal DNA increased from the first to the second and third trimester: 14.15 (2.32-36.25), 24.87 (6.29-129.32), and 32.62 (8.97-133.52) GE/ml (p<0.05), respectively. Our results characterize the dynamics of the content of cell-free DNA and its fractions during pregnancy, which should be taken into account when using cell-free DNA for prediction of placenta-associated complications.

Analytical antiretroviral therapy interruption does not irreversibly change preinterruption levels of cellular HIV.

The impact of short-term analytical treatment interruptions (ATI) on the levels of cellular HIV and of residual activation after subsequent antiretroviral therapy (ART)-mediated plasma HIV viral load re-suppression remains under active investigation. Peripheral blood mononuclear cells (PBMC) from 23 ART-suppressed, chronically HIV-1-infected patients were evaluated at the initiation of an ATI, during ATI, and following plasma re-suppression of HIV with ART. T-cell activation was measured by flow cytometry. Total cellular HIV DNA, and episomal 2-long terminal repeat (2-LTR) circles were measured by droplet digital PCR (ddPCR). Cellular HIV multiply spliced RNA (tat/rev), unspliced (gag), and poly(A) tailed transcripts [poly(A)] were measured by reverse transcriptase-ddPCR. Analyses were performed using R version 2.5.1 or JMP Pro 11. ATI (median ATI duration, 4 weeks) resulted in a rise of plasma HIV RNA (median = 72900 copies/ml), decrease in CD4+ T cells/μl (median = 511.5 cells/μl; P = 0.0001), increase in T-cell activation, and increase in cellular HIV DNA and RNA. Mean fluorescence intensity of CD38 on CD4+HLA-DR+ T cells at baseline was positively associated with total HIV DNA levels during ATI (pol: P = 0.03, Rho = 0.44). Upon ART resumption, plasma HIV re-suppression occurred after a median of 13 weeks and resulted in restoration of pre-ATI CD4+ T cells/μl, T-cell activation, and levels of cellular HIV DNA and RNA. Monitored viremia and immune activation during an ATI in ART-suppressed chronic HIV-infected patients does not change the amount of persistent cellular HIV RNA or total HIV DNA after ART-mediated re-suppression.

Urinary measurement of circulating tumor DNA for treatment monitoring and prognosis of metastatic colorectal cancer patients.

Background Solid tumor tissue testing is the gold standard for molecular-based assays for metastatic colorectal cancer (mCRC). This poses challenges during treatment monitoring. Total DNA derived from urine specimens offers clear advantages to track the disease dynamics. Our study aims to evaluate the sensitivity for total DNA recovered from urine and its clinical relevance to mCRC. Methods KRAS mutations in urine specimens were examined in 150 mCRC patients. Baseline concordance was established to determined clinical relevance. The total DNA quantities were also prospectively examined in serial samplings during treatment. Results Analysis of the genetic mutations showed good agreement for baseline samples. Matched tumor and urine specimens' molecular profiles were observed to have 90% concordance. Comparing with healthy volunteers, we established a cutoff of 8.15 ng that demonstrated elevated total DNA levels was associated with mCRC patients (sensitivity: 90.7%; specificity: 82.0%). For patients treated with chemotherapy or anti-epidermal growth factor receptor inhibitors, DNA quantity mirrored early treatment response. Survival analysis showed that patients with sustained elevated quantities of KRAS mutations had poorer outcome. Conclusions Total urine DNA offers a viable complement for mutation profiling in mCRC patients, given the good agreement with matched tumor samples. Our study also established that this is specific based on the results from healthy individuals. Serial monitoring of total DNA levels allowed early prediction to treatment response and was effective to identify high risk patients. This is potentially useful to complement current disease management.

Total HIV-1 DNA Dynamics and Influencing Factors in Long-Term ART-Treated Japanese Adults: A Retrospective Longitudinal Analysis.

Understanding HIV persistence in treated patients is an important milestone toward drug-free control. We aimed at analyzing total HIV DNA dynamics and influencing factors in Japanese patients who received more than a decade of suppressive antiretroviral treatment (ART). A retrospective study including clinical records and 840 peripheral blood mononuclear cells samples (mean 14 samples/patient) for 59 patients (92% male) was performed. Subjects were divided into 2 groups: with and without hematological comorbidity (mainly hemophilia) plus hepatitis C virus coinfection. Total HIV DNA was measured in peripheral blood mononuclear cells by quantitative polymerase chain reaction. The dynamics, regression over time, and influence of antiretrovirals by group were estimated using a novel regression model (R software v 3.2.3). Total HIV DNA decreased on ART initiation, and subsequently, its dynamics varied between groups with previously undescribed fluctuations. If calculated by on-treatment, the mean total HIV DNA levels were similar. The comorbidity group had unstable levels showing different regression over time (P = 0.088/0.094 in year 1/after year 8 of ART) and significantly different treatment responses as shown by antiretroviral group switching estimates. Furthermore, curing hepatitis C virus in hemophiliacs did not significantly alter total HIV DNA levels or regression. Our data identified some effects of the long-term treatment on total HIV DNA levels and highlighted the partial influence of comorbidities and coinfections. Total HIV DNA monitoring contributed to therapy response estimates and HIV reservoir quantification. The results suggest that HIV DNA monitoring during ART might be useful as a persistence marker in both HIV-monoinfected patients and those with comorbidities and coinfections.

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism (MSAP) Technique

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism (MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15°C, 22°C and 26°C, respectively. Compared with the control group (22°C), the fully methylated and totally methylated ratios were increased in both experiment groups (15°C and 26°C). All of the cytosine methylation/demethylation transform (CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Frontline Science: Plasma and immunoglobulin G galactosylation associate with HIV persistence during antiretroviral therapy.

Global antibody glycosylation is dynamic and plays critical roles in shaping different immunological outcomes and direct antibody functionality during HIV infection. However, the relevance of global antibody or plasma glycosylation patterns to HIV persistence after antiretroviral therapy (ART) has not been characterized. First, we compared glycomes of total plasma and isolated immunoglobulin G (IgG) from HIV+ ART-suppressed, HIV+ viremic, and HIV-negative individuals. Second, in ART-suppressed individuals, we examined the associations between glycomes and (1) levels of cell-associated HIV DNA and RNA in PBMCs and isolated CD4+ T cells, (2) CD4 count and CD4%, and (3) expression of CD4+ T-cell activation markers. HIV infection is associated with persistent alterations in the IgG glycome including decreased levels of disialylated glycans, which is associated with a lower anti-inflammatory activity, and increased levels of fucosylated glycans, which is associated with lower antibody-dependent cell-mediated cytotoxicity (ADCC). We also show that levels of certain mono- and digalactosylated nonfucosylated glycomic traits (A2G1, A2G2, and A2BG2), which have been reported to be associated with higher ADCC and higher anti-inflammatory activities, exhibit significant negative correlations with levels of cell-associated total HIV DNA and HIV RNA in ART-suppressed individuals. Finally, levels of certain circulating anti-inflammatory glycans are associated with higher levels of CD4 T cells and lower levels of T-cell activation. Our findings represent the first proof-of-concept evidence that glycomic alterations, known to be associated with differential states of inflammation and ADCC activities, are also associated with levels of HIV persistence in the setting of ART suppression.

HIV-1 non-R5 tropism correlates with a larger size of the cellular viral reservoir and a detectable residual viremia in patients under suppressive ART

BackgroundThe influence of HIV-1 co-receptor usage on the course of therapy in subjects fully responding to ART has been poorly investigated. ObjectivesTo explore the relationship between co-receptor tropism and cellular reservoir size, residual viremia and subsequent virological outcome in ART-treated patients with HIV-1 RNA stable <50 copies/mL. Study designViral co-receptor usage was predicted by viral env DNA sequencing with geno2pheno interpretation (FPR20%) and classified as R5 and non-R5. Total blood-associated HIV-1 DNA levels (log10 copies/106 leukocytes) were measured by qRT-PCR (5′LTR). Residual plasma viremia was categorized as detectable (1–49 cps/mL) or undetectable (<1 copy/mL). Virological rebounds (any HIV-1 RNA >50 copies/mL) were evaluated over 96 weeks. ResultsThe study included 116 subjects. Patients with R5 virus (n = 59) and non-R5 virus (n = 57) were homogeneous for the main characteristics except for the lower nadir CD4 cell count in the non-R5 group. Patients with non-R5 variants showed higher levels of HIV-1 DNA as compared to patients with R5 virus: mean 2.47 (95% CI 2.37–2.56) vs 2.17 (2.08–2.26) (p < 0.001). Moreover, a higher proportion of patients in the non-R5 group displayed detectable residual viremia with respect to the R5-group (54.4% vs 32.2%, p = .016). Detectable residual viremia was found to be significantly associated with viral rebounds. ConclusionThe presence of non-R5 viral DNA variants is related to a higher probability of residual viremia and to a larger size of the cellular viral reservoir in this setting. These data highlight a potential role of viral tropism in the monitoring of HIV-1 infection in virologically controlled subject.

Complex Analysis of Total and Fetal DNA and Cytokines in Blood Plasma of Pregnant Women with Preeclampsia.

We performed a complex analysis of total and fetal extracellular DNA, 8 cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage CSF, IFNγ, and TNFα) in blood plasma obtained from women with preeclampsia prior to labor onset. Total (sensitivity 89.47%, specificity 93.75%) and fetal extracellular DNA (sensitivity 73.68%, specificity 87.5%) were the most accurate parameters determining preeclampsia. We revealed a high correlation (p=3×10-6) between total and fetal extracellular DNA levels in the group of preeclampsia. Preeclampsia significantly increased the levels of macrophage factors IL-10 and IL-6. These cytokines significantly correlated with the levels of total and fetal extracellular DNA in the preeclampsia group. In the control group, such correlations were not observed. These findings obtained suggest that preeclampsia develops upon increased macrophage activity, leading to destruction of the placenta trophoblast cells.

Rapid decline of HIV-1 DNA and RNA in infants starting very early antiretroviral therapy may pose a diagnostic challenge.

Birth diagnosis of HIV-1 infection offers an ideal opportunity for early antiretroviral therapy (ART) to limit HIV-1 reservoir size and limit disease progression. Although data on cellular HIV-1 DNA decay exist for children commencing treatment from 2 to 3 months of age, data are lacking for starting shortly after birth. We studied infants who initiated ART within 8 days after birth to assess HIV-1 DNA levels longitudinally. Children were recruited from public health clinics in Cape Town where birth diagnosis of HIV-1 coupled with early ART initiation occurred. Total cellular HIV-1 DNA levels were determined using a sensitive quantitative PCR targeting a conserved region in integrase. Of 11 infants diagnosed and beginning ART within 8 days of birth with detectable pre-ART HIV-1 DNA, three subsequently had undetectable HIV-1 DNA after 6 days, 3 months and 4 months on treatment, respectively. In seven who had virologic suppression (defined as a continuous downward trend in plasma HIV-1 RNA, and <100 copies/ml after 6 months) total HIV-1 DNA continued to decay over 12 months [mean half-life of 64.8 days (95% confidence interval: 47.9-105.7)]. In infants initiated on ART within 8 days of life the combination of maternal ART, and early ART for prophylaxis and treatment contribute to rapid decline of HIV-1 infected cells to low or undetectable levels. However, rapid decline of HIV-1 RNA and DNA may complicate definitive diagnosis when confirmatory testing is delayed.

No Effect of Pegylated Interferon-α on Total HIV-1 DNA Load in HIV-1/HCV Coinfected Patients.

Pegylated interferon-alpha (pIFN-α) is suggested to lower human immunodeficiency virus type-1 (HIV-1) DNA load in antiretroviral therapy (ART)-treated patients. We studied kinetics of HIV-1 DNA levels in 40 HIV-1/hepatitis C virus (HCV) coinfected patients, treated with pIFN-α for HCV and categorized into 3 groups according to start of ART: chronic HIV-1 infection (n = 22), acute HIV-1 infection (n = 8), no-ART (n = 10). Total HIV-1 DNA levels in 247 peripheral blood mononuclear cell samples were stable before, during, and after pIFN-α treatment in all groups. Our results question the benefit of pIFN-α as an immunotherapeutic agent for reducing the HIV-1 reservoir.

A higher CD4/CD8 ratio correlates with an ultralow cell-associated HIV-1 DNA level in chronically infected patients on antiretroviral therapy: a case control study

BackgroundThe HIV-1 DNA reservoir is an important marker that reflects viro-immunological status and can be affected by multiple viral or cellular factors. Determining the potential factors associated with the size of the HIV-1 DNA reservoir benefits the surveillance of disease progression and antiretroviral treatments.MethodsWe conducted a case control study to explore the factors that may affect the level of HIV-1 DNA. The level of HIV-1 total DNA in peripheral blood at 5 time points was quantified by quantitative PCR. Chronically HIV-1-infected patients whose cell-associated HIV-1 DNA levels were below the detection limit after receiving antiretroviral therapy (ART) for 96 weeks were identified (group 1), and patients who still had detectable levels of cell-associated HIV-1 DNA after ATR treatment were used as the control (group 2).ResultsTwenty-one patients with ultralow levels of cell-associated HIV-1 DNA [<20 copies/106 peripheral blood mononuclear cells (PBMCs)] presented with a lower CD8+ T-cell count (average: 511 ± 191 versus 715 ± 256 cells/μL, p = 0.013) and a higher CD4/CD8 ratio (average: 1.04 ± 0.37 versus 0.72 ± 0.32, respectively, p = 0.002) at week 96. In the multivariate analysis, patients with a higher CD4/CD8 ratio at week 96 were more likely to have levels of HIV-1 DNA below the detection limit (per 0.1 increase, OR = 1.29, 95% CI, 1.05–1.59, p = 0.017).ConclusionAfter matching baseline HIV-1 DNA levels, a higher CD4/CD8 ratio at week 96 was the only factor associated with an ultralow level of HIV-1 DNA. The CD4/CD8 ratio can be used as an easy biomarker to help monitor patients on ART who will be selected to participate in eradication studies.

Control of the HIV-1 DNA Reservoir Is Associated In Vivo and In Vitro with NKp46/NKp30 (CD335 CD337) Inducibility and Interferon Gamma Production by Transcriptionally Unique NK Cells.

The size of lentiviral DNA reservoirs reflects the effectiveness of immune responses against lentiviruses. So far, abundant information has been gathered on the control of HIV-1 replication. Understanding the innate mechanisms contributing to containment of the HIV DNA reservoir, however, are only partly clarified and are relevant to guiding interventions for reservoir containment or eradication. We studied the contribution of natural killer (NK) cell functional features in HIV patients controlling replication either spontaneously (HIV controllers [HIC]) or after progression and antiretroviral treatment (progressor patients [PP]). An inverse correlation between HIV DNA copy numbers (either total or integrated) in circulating CD4+ cells and NK cell function was observed. Induced interferon gamma (IFN-γ) production and NKp46/NKp30 activating receptor-induced expression correlated inversely with reservoir size. The correlation was present not only for a homogeneous cohort of HIC patients but also when PP were included in the analysis. Adaptive (NKG2C+ CD57+) NK cell features were not associated with reservoir size. However, a distinct set of 370 differentially expressed transcripts was found to underlie functional differences in NK cells controlling HIV DNA reservoir size. In proof-of-principle in vitro experiments of CD4+ cell infection with HIV-1, purified NK cells with the above-mentioned functional/transcriptional features displayed 10- and 30-fold higher abilities to control HIV replication and DNA burdens in vitro, respectively, than those of other NK cells. Thus, NK cells with a specific functional and transcriptional signature contribute to control of the HIV reservoir in CD4+ cells. Their selection, expansion, and/or adoptive transfer may support strategies to eradicate HIV-1 infection or to safely deescalate antiretroviral treatment.IMPORTANCE The most relevant feature of HIV-1 infection is represented by its DNA reservoir size in the body, which guarantees lifelong infection and resumption of virus replication after antiretroviral treatment interruption. So far, there has been little success in the identification of factors contributing to HIV-1 reservoir containment. In this study, by studying quantitative total and integrated HIV-1 DNA levels and NK cells in HIV-1 patients with either progressive or nonprogressive disease, we observed that inducible IFN-γ and natural cytotoxicity receptor (NCR) expression in a specific subset of NK cells with a characteristic transcriptional signature represents a correlate for HIV-1 reservoir control. This represents an advance in our understanding of the mechanism(s) that controls the lentivirus reservoir. Monitoring, selection, expansion, and adoptive transfer of these NK cells may allow monitoring of treatment efficacy and the likelihood of reservoir control and may support protocols for HIV-1 eradication.

Cell-free DNA levels and correlation to stage and outcome following treatment of locally advanced rectal cancer.

Accurate staging of rectal cancer remains essential for optimal patient selection for combined modality treatment, including radiotherapy, chemotherapy and surgery. We aimed at examining the correlation of cell free DNA with the pathologic stage and subsequent risk of recurrence for patients with locally advanced rectal cancer undergoing preoperative chemoradiation. We examined 75 patients with locally advanced rectal cancer receiving preoperative chemoradiation. Blood samples for translational use were drawn prior to rectal surgery. The level of cell free DNA was quantified by digital droplet PCR and expressed as copy number of beta 2 microglobulin. We found a median level of cell free DNA in the AJCC stages I-III of 3100, 8300, and 10,700 copies/mL respectively. For patients with 12 sampled lymph nodes or above, the median level of cell free DNA were 2400 copies/mL and 4400 copies/mL (p = 0.04) for node negative and node positive disease respectively. The median follow-up was 39 months and 11 recurrences were detected (15%). The median level for patients with recurrent disease was 13,000 copies/mL compared to 5200 copies/mL for non-recurrent patients (p = 0.08). We have demonstrated a correlation between the level of total cell free DNA and the pathologic stage and nodal involvement. Furthermore, we have found a trend towards a correlation with the risk of recurrence following resection of localized rectal cancer.