Effect of flow change on brain injury during an experimental model of differential hypoxaemia in cardiogenic shock supported by extracorporeal membrane oxygenation.

Differential hypoxaemia (DH) is common in patients supported by femoral veno-arterial extracorporeal membrane oxygenation (V-A ECMO) and can cause cerebral hypoxaemia. To date, no models have studied the direct impact of flow on cerebral damage. We investigated the impact of V-A ECMO flow on brain injury in an ovine model of DH. After inducing severe cardiorespiratory failure and providing ECMO support, we randomised six sheep into two groups: low flow (LF) in which ECMO was set at 2.5 L min-1 ensuring that the brain was entirely perfused by the native heart and lungs, and high flow (HF) in which ECMO was set at 4.5 L min-1 ensuring that the brain was at least partially perfused by ECMO. We used invasive (oxygenation tension-PbTO2, and cerebral microdialysis) and non-invasive (near infrared spectroscopy-NIRS) neuromonitoring, and euthanised animals after five hours for histological analysis. Cerebral oxygenation was significantly improved in the HF group as shown by higher PbTO2 levels (+ 215% vs - 58%, p = 0.043) and NIRS (67 ± 5% vs 49 ± 4%, p = 0.003). The HF group showed significantly less severe brain injury than the LF group in terms of neuronal shrinkage, congestion and perivascular oedema (p < 0.0001). Cerebral microdialysis values in the LF group all reached the pathological thresholds, even though no statistical difference was found between the two groups. Differential hypoxaemia can lead to cerebral damage after only a few hours and mandates a thorough neuromonitoring of patients. An increase in ECMO flow was an effective strategy to reduce such damages.

Recovery of organ-specific tissue oxygen delivery at restrictive transfusion thresholds after fluid treatment in ovine haemorrhagic shock.

BACKGROUND: Fluid resuscitation is the standard treatment to restore circulating blood volume and pressure after massive haemorrhage and shock. Packed red blood cells (PRBC) are transfused to restore haemoglobin levels. Restoration of microcirculatory flow and tissue oxygen delivery is critical for organ and patient survival, but these parameters are infrequently measured. Patient Blood Management is a multidisciplinary approach to manage and conserve a patient's own blood, directing treatment options based on broad clinical assessment beyond haemoglobin alone, for which tissue perfusion and oxygenation could be useful. Our aim was to assess utility of non-invasive tissue-specific measures to compare PRBC transfusion with novel crystalloid treatments for haemorrhagic shock. METHODS: A model of severe haemorrhagic shock was developed in an intensive care setting, with controlled haemorrhage in sheep according to pressure (mean arterial pressure 30-40 mmHg) and oxygen debt (lactate > 4 mM) targets. We compared PRBC transfusion to fluid resuscitation with either PlasmaLyte or a novel crystalloid. Efficacy was assessed according to recovery of haemodynamic parameters and non-invasive measures of sublingual microcirculatory flow, regional tissue oxygen saturation, repayment of oxygen debt (arterial lactate), and a panel of inflammatory and organ function markers. Invasive measurements of tissue perfusion, oxygen tension and lactate levels were performed in brain, kidney, liver, and skeletal muscle. Outcomes were assessed during 4 h treatment and post-mortem, and analysed by one- and two-way ANOVA. RESULTS: Each treatment restored haemodynamic and tissue oxygen delivery parameters equivalently (p > 0.05), despite haemodilution after crystalloid infusion to haemoglobin concentrations below 70 g/L (p < 0.001). Recovery of vital organ-specific perfusion and oxygen tension commenced shortly before non-invasive measures improved. Lactate declined in all tissues and correlated with arterial lactate levels (p < 0.0001). The novel crystalloid supported rapid peripheral vasodilation (p = 0.014) and tended to achieve tissue oxygen delivery targets earlier. PRBC supported earlier renal oxygen delivery (p = 0.012) but delayed peripheral perfusion (p = 0.034). CONCLUSIONS: Crystalloids supported vital organ oxygen delivery after massive haemorrhage, despite haemodilution to < 70 g/L, confirming that restrictive transfusion thresholds are appropriate to support oxygen delivery. Non-invasive tissue perfusion and oximetry technologies merit further clinical appraisal to guide treatment for massive haemorrhage in the context of Patient Blood Management.

An innovative ovine model of severe cardiopulmonary failure supported by veno-arterial extracorporeal membrane oxygenation.

Refractory cardiogenic shock (CS) often requires veno-arterial extracorporeal membrane oxygenation (VA-ECMO) to sustain end-organ perfusion. Current animal models result in heterogenous cardiac injury and frequent episodes of refractory ventricular fibrillation. Thus, we aimed to develop an innovative, clinically relevant, and titratable model of severe cardiopulmonary failure. Six sheep (60 ± 6 kg) were anaesthetized and mechanically ventilated. VA-ECMO was commenced and CS was induced through intramyocardial injections of ethanol. Then, hypoxemic/hypercapnic pulmonary failure was achieved, through substantial decrease in ventilatory support. Echocardiography was used to compute left ventricular fractional area change (LVFAC) and cardiac Troponin I (cTnI) was quantified. After 5 h, the animals were euthanised and the heart was retrieved for histological evaluations. Ethanol (58 ± 23 mL) successfully induced CS in all animals. cTnI levels increased near 5000-fold. CS was confirmed by a drop in systolic blood pressure to 67 ± 14 mmHg, while lactate increased to 4.7 ± 0.9 mmol/L and LVFAC decreased to 16 ± 7%. Myocardial samples corroborated extensive cellular necrosis and inflammatory infiltrates. In conclusion, we present an innovative ovine model of severe cardiopulmonary failure in animals on VA-ECMO. This model could be essential to further characterize CS and develop future treatments.

An Ovine Model of Hemorrhagic Shock and Resuscitation, to Assess Recovery of Tissue Oxygen Delivery and Oxygen Debt, and Inform Patient Blood Management.

BACKGROUND: Aggressive fluid or blood component transfusion for severe hemorrhagic shock may restore macrocirculatory parameters, but not always improve microcirculatory perfusion and tissue oxygen delivery. We established an ovine model of hemorrhagic shock to systematically assess tissue oxygen delivery and repayment of oxygen debt; appropriate outcomes to guide Patient Blood Management. METHODS: Female Dorset-cross sheep were anesthetized, intubated, and subjected to comprehensive macrohemodynamic, regional tissue oxygen saturation (StO2), sublingual capillary imaging, and arterial lactate monitoring confirmed by invasive organ-specific microvascular perfusion, oxygen pressure, and lactate/pyruvate levels in brain, kidney, liver, and skeletal muscle. Shock was induced by stepwise withdrawal of venous blood until MAP was 30 mm Hg, mixed venous oxygen saturation (SvO2) < 60%, and arterial lactate >4 mM. Resuscitation with PlasmaLyte® was dosed to achieve MAP > 65 mm Hg. RESULTS: Hemorrhage impacted primary outcomes between baseline and development of shock: MAP 89 ±â€Š5 to 31 ±â€Š5 mm Hg (P < 0.01), SvO2 70 ±â€Š7 to 23 ±â€Š8% (P < 0.05), cerebral regional tissue StO2 77 ±â€Š11 to 65 ±â€Š9% (P < 0.01), peripheral muscle StO2 66 ±â€Š8 to 16 ±â€Š9% (P < 0.01), arterial lactate 1.5 ±â€Š1.0 to 5.1 ±â€Š0.8 mM (P < 0.01), and base excess 1.1 ±â€Š2.2 to -3.6 ±â€Š1.7 mM (P < 0.05). Invasive organ-specific monitoring confirmed reduced tissue oxygen delivery; oxygen tension decreased and lactate increased in all tissues, but moderately in brain. Blood volume replacement with PlasmaLyte® improved primary outcome measures toward baseline, confirmed by organ-specific measures, despite hemoglobin reduced from baseline 10.8 ±â€Š1.2 to 5.9 ±â€Š1.1 g/dL post-resuscitation (P < 0.01). CONCLUSION: Non-invasive measures of tissue oxygen delivery and oxygen debt repayment are suitable outcomes to inform Patient Blood Management of hemorrhagic shock, translatable for pre-clinical assessment of novel resuscitation strategies.

Fibrinogen Early In Severe Trauma studY (FEISTY): results from an Australian multicentre randomised controlled pilot trial.

Background: Haemorrhage is a major cause of death in severe trauma. Fibrinogen plays a critical role in maintaining haemostasis in traumatic haemorrhage, and early replacement using fibrinogen concentrate (FC) or cryoprecipitate (Cryo) is recommended by several international trauma guidelines. Limited evidence supports one product over the other, with widespread geographic and institutional variation in practice. Two previous trials have investigated the feasibility of rapid FC administration in severely injured trauma patients, with conflicting results. Objective: To compare the time to fibrinogen replacement using FC or Cryo in severely injured trauma patients with major haemorrhage and hypofibrinogenaemia. Design, setting, patients and interventions: A multicentre controlled pilot trial in which adult trauma patients with haemorrhage were randomly assigned (1:1) to receive FC or Cryo for fibrinogen replacement, guided by FIBTEM A5 (functional fibrinogen assessment at 5 minutes after clot formation, using rotational thromboelastometry). Main outcome measures: The primary outcome was time to commencement of fibrinogen replacement. Secondary outcomes included effects of the intervention on plasma fibrinogen levels and clinical outcomes including transfusion requirements and mortality. Results: Of the 100 randomly assigned patients, 62 were hypofibrinogenaemic and received the intervention (n = 37) or Cryo (n = 25). Median (interquartile range [IQR]) time to delivery of FC was 29 min (23-40 min) compared with 60 min (40-80 min) for Cryo (P = 0.0001). All 62 patients were hypofibrinogenaemic before receiving FC or Cryo (FC: median FIBTEM A5, 8 mm [IQR, 7-9 mm]; Cryo: median FIBTEM A5, 9 mm [IQR, 5-10 mm]). In the FC arm patients received a median of 3 g FC (IQR, 2-4 g), and in the Cryo arm patients received a median of 8 units of Cryo (IQR, 8-14 units). Restoration of fibrinogen levels was achieved in both arms after the intervention. Blood product transfusion, fluid resuscitation and thromboembolic complications were similar in both arms. Overall mortality was 15.3%, with more deaths in the FC arm. Conclusion: Fibrinogen replacement in severely injured trauma patients with major haemorrhage and hypofibrinogenaemia was achieved substantially faster using FC compared with Cryo. Fibrinogen levels increased appropriately using either product. The optimal method for replacing fibrinogen in traumatic haemorrhage is controversial. Our results will inform the design of a larger trial powered to assess patient-centred outcomes.

Essential Role of Patient Blood Management in a Pandemic: A Call for Action.

The World Health Organization (WHO) has declared coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a pandemic. Global health care now faces unprecedented challenges with widespread and rapid human-to-human transmission of SARS-CoV-2 and high morbidity and mortality with COVID-19 worldwide. Across the world, medical care is hampered by a critical shortage of not only hand sanitizers, personal protective equipment, ventilators, and hospital beds, but also impediments to the blood supply. Blood donation centers in many areas around the globe have mostly closed. Donors, practicing social distancing, some either with illness or undergoing self-quarantine, are quickly diminishing. Drastic public health initiatives have focused on containment and "flattening the curve" while invaluable resources are being depleted. In some countries, the point has been reached at which the demand for such resources, including donor blood, outstrips the supply. Questions as to the safety of blood persist. Although it does not appear very likely that the virus can be transmitted through allogeneic blood transfusion, this still remains to be fully determined. As options dwindle, we must enact regional and national shortage plans worldwide and more vitally disseminate the knowledge of and immediately implement patient blood management (PBM). PBM is an evidence-based bundle of care to optimize medical and surgical patient outcomes by clinically managing and preserving a patient's own blood. This multinational and diverse group of authors issue this "Call to Action" underscoring "The Essential Role of Patient Blood Management in the Management of Pandemics" and urging all stakeholders and providers to implement the practical and commonsense principles of PBM and its multiprofessional and multimodality approaches.

Mapping the extent of heterogeneity of human CCR5+ CD4+ T cells in peripheral blood and lymph nodes.

BACKGROUND: CD4 T cells that express the chemokine receptor, CCR5, are the most important target of HIV-1 infection, but their functions, phenotypes and anatomical locations are poorly understood. We aimed to use multiparameter flow cytometry to better define the full breadth of these cells. METHODS: High-parameter fluorescence flow and mass cytometry were optimized to analyse subsets of CCR5 memory CD4 T cells, including CD25CD127 Tregs, CXCR3CCR6- Th1-like, CCR6CD161CXCR3- Th17-like, integrins α4ß7 gut-homing, CCR4 skin-homing, CD62L lymph node-homing, CD38HLA-DR activated cells, and CD27-CD28- cytotoxic T lymphocytes, in a total of 22 samples of peripheral blood, ultrasound-guided fine needle biopsies of lymph nodes and excised tonsils. CCR5 antigen-specific CD4 T cells were studied using the OX40 flow-based assay. RESULTS: 10-20% of CCR5 memory CD4 T cells were Tregs, 10-30% were gut-homing, 10-30% were skin-homing, 20-40% were lymph node-homing, 20-50% were Th1-like and 20-40% were Th17-like cells. Up to 30% were cytotoxic T lymphocytes in CMV-seropositive donors, including cells that were either CCR5Granzyme K or CCR5Granzyme B. When all possible phenotypes were exhaustively analysed, more than 150 different functional and trafficking subsets of CCR5 CD4 T cells were seen. Moreover, a small population of resident CD69Granzyme KCCR5 CD4 T cells was found in lymphoid tissues. CMV- and Mycobacterium tuberculosis-specific CD4 T cells were predominantly CCR5. CONCLUSION: These results reveal for the first time the prodigious heterogeneity of function and trafficking of CCR5 CD4 T cells in blood and in lymphoid tissue, with significant implications for rational approaches to prophylaxis for HIV-1 infection and for purging of the HIV-1 reservoir in those participants already infected.

Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption.

Anti-HIV envelope (Env) antibodies elicit important Fc receptor functions, including FcγRIIIa-mediated natural killer cell killing of opsonized infected targets. How these antibodies evolve during HIV infection and treatment remains poorly understood. We describe changes in anti-HIV Env IgG using longitudinal samples from seroconverter subjects treated soon after infection and later during periods of structured treatment interruption (STI). Our well-validated dimeric rsFcγR binding assays combine effects of opsonizing antibody subclasses, epitopes, and geometries to provide a measure of FcγR (Fcγ receptor)-mediated functionality. IgG1 anti-Env titers diminished rapidly during antiretroviral therapy (ART; t1/2 3.0 ± 0.8 months), while the dimeric rsFcγRIIIa activity persisted longer (t1/2 33 ± 11 months), suggesting that there is maintenance of functional antibody specificities within the diminished pool of anti-HIV Env Abs. The initial antibody response to infection in two subjects was characterized by approximately fivefold higher FcγRIIIa compared with FcγRIIa binding activity. Uncoupling of FcγRIIa and FcγRIIIa activities may be a distinct feature of the early antibody response that preferentially engages FcγRIIIa-mediated effector functions. Two to three STI cycles, even with low viremia, were sufficient to boost dimeric FcγR activity in these seroconverter subjects. We hypothesize that increased humoral immunity induced by STI is a desirable functional outcome potentially achievable by therapeutic immunization during ART. We conclude that controlled viral antigen exposure under the protection of suppressive ART may be effective in eliciting FcγR-dependent function in support of viral reactivation and kill strategies.

Possible clearance of transfusion-acquired nef/LTR-deleted attenuated HIV-1 infection by an elite controller with CCR5 Δ32 heterozygous and HLA-B57 genotype.

BACKGROUND: Subject C135 is one of the members of the Sydney Blood Bank Cohort, infected in 1981 through transfusion with attenuated nef/3' long terminal repeat (LTR)-deleted HIV-1, and has maintained undetectable plasma viral load and steady CD4 cell count, in the absence of therapy. Uniquely, C135 combines five factors separately associated with control of viraemia: nef/LTR-deleted HIV-1, HLA-B57, HLA-DR13, heterozygous CCR5 Δ32 genotype and vigorous p24-stimulated peripheral blood mononuclear cell (PBMC) proliferation. Therefore, we studied in detail viral burden and immunological responses in this individual. METHODS: PBMC and gut and lymph node biopsy samples were analysed for proviral HIV-1 DNA by real-time and nested PCRs, and nef/LTR alleles by nested PCR. HIV-specific antibodies were studied by Western blotting, and CD4+ and CD8+ T lymphocyte responses were measured by proliferation and cytokine production in vitro. RESULTS: PBMC samples from 1996, but not since, showed amplification of nef alleles with gross deletions. Infectious HIV-1 was never recovered. Proviral HIV-1 DNA was not detected in recent PBMC or gut or lymph node biopsy samples. C135 has a consistently weak antibody response and a substantial CD4+ T cell proliferative response to a previously described HLA-DR13-restricted epitope of HIV-1 p24 in vitro, which augmented a CD8+ T cell response to an immunodominant HLA-B57-restricted epitope of p24, while his T cells show reduced levels of CCR5. CONCLUSIONS: Subject C135's early PCR and weak antibody results are consistent with limited infection with a poorly replicating nef/LTR-deleted strain of HIV-1. With his HLA-B57-restricted gag-specific CD8 and helper HLA-DR13-restricted CD4 T cell proliferative responses, C135 appears to have cleared his HIV-1 infection 37 years after transfusion.

D-immunized blood donors who are female and who possess at least one HLA-DRB1*15 allele show a propensity for high serum RhIG production.

BACKGROUND: D- individuals with previous D-incompatible pregnancies and/or blood transfusions, as well as those who are actively immunized with small-volume D+ red blood cells (RBCs), are stimulated to produce RhIG. Many factors could influence the stimulation of immunoglobulin production in response to foreign antigen (such as antigen immunogenicity and genetic factors), and it is unknown whether genetic markers could potentially identify responder anti-D donors. STUDY DESIGN AND METHODS: Anti-D donors were assigned a responder profile based on their serum RhIG levels (n = 431). A subset of donors (n = 272) had DNA extracted for polymerase chain reaction genotyping assays for target genes in antigen presentation and pathogen recognition receptors (TLR2, TLR4, CD14, FcγRIIA, and the MHC Class II locus HLA-DRB1). Statistical tests for associations between anti-D donor responder profiles and genetic factors were performed. RESULTS: A large proportion of our donors (38.7%) were classified as nonresponder donors, despite receiving multiple D+ RBC immunizations, whereas female sex was significantly associated with an all-responder profile (p < 0.001). The presence of the DRB1*15 allele and absence of the DRB1*04 allele were more likely to be associated with a responder anti-D donor, although not significantly after Bonferroni correction. A combination of the DRB1*15 allele and female sex was significantly associated with an anti-D donor responder profile. CONCLUSION: This study has identified female sex and the HLA-DRB1*15 allele as potentially useful markers that could be used to screen donors before entry into D immunization programs.

Fibrinogen Early In Severe Trauma studY (FEISTY): study protocol for a randomised controlled trial.

BACKGROUND: Haemorrhage is a leading cause of death in severe trauma. Fibrinogen plays a critical role in maintaining haemostasis in traumatic haemorrhage. Early fibrinogen replacement is recommended by several international trauma guidelines using either fibrinogen concentrate (FC) or cryoprecipitate (Cryo). There is limited evidence to support one product over the other with widespread geographic and institutional variation in practice. This pilot trial is the first randomised controlled trial comparing FC to Cryo in traumatic haemorrhage. METHODS/DESIGN: The Fibrinogen Early In Severe Trauma studY (FEISTY) is an exploratory, multicentre, randomised controlled trial comparing FC to Cryo for fibrinogen supplementation in traumatic haemorrhage. This trial will utilise thromboelastometry (ROTEM®) to guide and dose fibrinogen supplementation. The trial will recruit 100 trauma patients at four major trauma centres in Australia. Adult trauma patients with evidence of haemorrhage will be enrolled on arrival in the trauma unit and randomised to receiving fibrinogen supplementation with either FC or Cryo. The primary outcome is the differential time to fibrinogen supplementation. There are a number of predetermined secondary outcomes including: effects of the intervention on plasma fibrinogen levels, feasibility assessments and clinical outcomes including transfusion requirements and mortality. DISCUSSION: The optimal method for replacing fibrinogen in traumatic haemorrhage is fiercely debated. In this trial the feasibility and efficacy of fibrinogen supplementation using FC will be compared to Cryo. The results of this pilot study will facilitate the design of a larger trial with sufficient power to address patient-centred outcomes. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02745041 . Registered 4 May 2016.

Fibrinogen in traumatic haemorrhage: A narrative review.

Haemorrhage in the setting of severe trauma is associated with significant morbidity and mortality. There is increasing awareness of the important role fibrinogen plays in traumatic haemorrhage. Fibrinogen levels fall precipitously in severe trauma and the resultant hypofibrinogenaemia is associated with poor outcomes. Hence, it has been postulated that early fibrinogen replacement in severe traumatic haemorrhage may improve outcomes, although, to date there is a paucity of high quality evidence to support this hypothesis. In addition there is controversy regarding the optimal method for fibrinogen supplementation. We review the current evidence regarding the role of fibrinogen in trauma, the rationale behind fibrinogen supplementation and discuss current research.

Immunomodulation of inflammatory leukocyte markers during intravenous immunoglobulin treatment associated with clinical efficacy in chronic inflammatory demyelinating polyradiculoneuropathy.

OBJECTIVE: The objective of the study was to profile leukocyte markers modulated during intravenous immunoglobulin (IVIg) treatment, and to identify markers and immune pathways associated with clinical efficacy of IVIg for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with potential for monitoring treatment efficacy. METHODS: Response to IVIg treatment in newly diagnosed IVIg-naïve and established IVIg-experienced patients was assessed by changes in expression of inflammatory leukocyte markers by flow cytometry. The adjusted INCAT disability and Medical Research Council sum scores defined clinical response. RESULTS: Intravenous immunoglobulin modulated immunopathogenic pathways associated with inflammatory disease in CIDP. Leukocyte markers of clinical efficacy included reduced CD185+ follicular helper T cells, increased regulatory markers (CD23 and CD72) on B cells, and reduction in the circulating inflammatory CD16+ myeloid dendritic cell (mDC) population and concomitant increase in CD62L and CD195 defining a less inflammatory lymphoid homing mDC phenotype. A decline in inflammatory CD16+ dendritic cells was associated with clinical improvement or stability, and correlated with magnitude of improvement in neurological assessment scores, but did not predict relapse. IVIg also induced a nonspecific improvement in regulatory and reduced inflammatory markers not associated with clinical response. CONCLUSIONS: Clinically effective IVIg modulated inflammatory and regulatory pathways associated with ongoing control or resolution of CIDP disease. Some of these markers have potential for monitoring outcome.

Retrospective Proteomic Analysis of Cellular Immune Responses and Protective Correlates of p24 Vaccination in an HIV Elite Controller Using Antibody Arrays.

BACKGROUND: HIV p24 is an extracellular HIV antigen involved in viral replication. Falling p24 antibody responses are associated with clinical disease progression and their preservation with non-progressive disease. Stimulation of p24 antibody production by immunization to delay progression was the basis of discontinued p24 vaccine. We studied a therapy-naive HIV+ man from Sydney, Australia, infected in 1988. He received the HIV-p24-virus like particle (VLP) vaccine in 1993, and continues to show vigorous p24 antigen responses (>4% p24-specific CD4⁺ T cells), coupled with undetectable plasma viremia. We defined immune-protective correlates of p24 vaccination at the proteomic level through parallel retrospective analysis of cellular immune responses to p24 antigen in CD4⁺ and CD8⁺ T cells and CD14⁺ monocytes at viremic and aviremic phases using antibody-array. We found statistically significant coordinated up-regulation by all three cell-types with high fold-changes in fractalkine, ITAC, IGFBP-2, and MIP-1α in the aviremic phase. TECK and TRAIL-R4 were down-regulated in the viremic phase and up-regulated in the aviremic phase. The up-regulation of fractalkine in all three cell-types coincided with protective effect, whereas the dysfunction in anti-apoptotic chemokines with the loss of immune function. This study highlights the fact that induction of HIV-1-specific helper cells together with coordinated cellular immune response (p < 0.001) might be important in immunotherapeutic interventions and HIV vaccine development.

First Evidence for the Disease-Stage, Cell-Type, and Virus Specificity of microRNAs during Human Immunodeficiency Virus Type-1 Infection.

The potential involvement of host microRNAs (miRNAs) in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+) individuals (viremic and aviremic individuals while receiving highly active antiretroviral therapy (HAART), therapy-naïve long-term non-progressors (LTNP), and HIV-negative (HIV-) healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV- samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted p-value < 0.05. The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV- controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs.

Identification of genetic polymorphisms that predict responder/non-responder profiles to the RhD antigen.

BACKGROUND: Regular plasma donors who produce high titre anti-D immunoglobulin (Ig) are overseen by the Australian Red Cross Blood Service RhD Program. New donors to the program are immunised with small amounts of RhD-positive RBCs, whilst donors who have developed anti-D due to previous RhD-incompatible blood transfusion or pregnancy are boosted with RhD-positive RBCs to maintain a high level of serum anti-D Ig. A significant proportion of primarily immunised individuals do not respond to RhD immunisation and are therefore unnecessarily exposed to the risks involved in RBC sensitisation. STUDY DESIGN AND METHODS: We genotyped 184 anti-D donors for ∼9000 immunological and inflammatory genetic polymorphisms on an Affymetrix GeneChip, and validated the results with a High-Resolution Melt analysis assay. We built and validated a predictive logistic regression model using High Responder and Non-Responder anti-D donors that incorporated highly-associated polymorphisms and gender. RESULTS: High Responder and Non-Responder profiles in anti-D donors were significantly associated with a shortlist of 13 genetic polymorphisms and sex of the donor. The derivation of a logistic regression model showed an accuracy rate of 92.6% that was subsequently validated as 60.0% with an independent set of donor samples. CONCLUSION: This study has developed a logistic regression model and a genotyping assay that can predict the responder profiles of anti-D donors and could potentially be applied to new donors and transfusion-dependent patients in a clinical setting. Additionally, target polymorphisms identified in immunological genes could help to elucidate the immunomodulatory pathways regulating the immune response to the RhD antigen, and to other RBC antigens.

Genome-wide analysis of primary peripheral blood mononuclear cells from HIV + patients-pre-and post- HAART show immune activation and inflammation the main drivers of host gene expression.

BACKGROUND: Although the host gene expression in the context of HIV has been explored by several studies, it remains unclear how HIV is able to manipulate and subvert host gene machinery before and after highly active antiretroviral therapy (HAART) in the same individual. In order to define the underlying pharmaco-genomic basis of HIV control during HAART and genomic basis of immune deterioration prior to HAART initiation, we performed a genome-wide expression analysis using primary peripheral blood mononuclear cells (PBMC) derived from 14 HIV + subjects pre-highly active antiretroviral therapy (HAART) (time point-1 or TP1) with detectable plasma viremia and post-HAART (time point-2 or TP2) with effective control of plasma viremia (<40 HIV RNA copies/mL of plasma). METHODS: Genomic RNA extracted from the PBMCs was used in microarray analysis using HT-12V3 Illumina chips. Illumina®BeadStudio Software was used to obtain differentially expressed (DE) genes. Only the genes with p value <0.01 and FDR of <5% were considered for analysis. Pathway analysis was performed in MetaCore™ to derive functional annotations. Functionally significant genes were validated by qRT-PCR. RESULTS: Between TP1 and TP2, 234 genes were differentially expressed (DE). During viremic phase (TP1), there was an orchestrated and coordinated up-regulation of immune, inflammation and antiviral genes, consistent with HIV infection and immune activation, which comprised of genes mainly involved in antiviral action of interferons and their signalling. In contrast, the therapy-mediated control phase (TP2) showed systematic down-regulation of these pathways, suggesting that the reduction in plasma viremia with HAART has a considerable influence on reducing the immune activation, thereby implying a definitive role of HIV in subverting the human gene machinery. CONCLUSIONS: This is the first study to show the evidence for the differential regulation of gene expression between the untreated and treated time points, suggesting that gene expression is a consequence of cellular activation during plasma viremia. Affirmation to these observations comes from down-modulation of genes involved in cellular activation and inflammation upon initiation of HAART coinciding with below detectable levels of plasma viremia.

The Sydney Blood Bank Cohort: implications for viral fitness as a cause of elite control.

PURPOSE OF REVIEW: The Sydney Blood Bank Cohort comprised eight individuals who were infected with an attenuated, nef/LTR-deleted strain of HIV-1 from a single donor. All six recipients with sufficient follow-up, as well as the donor, were long-term nonprogressors. Only three recipients have maintained undetectable plasma viral loads, allowing investigation of factors that determined elite control of attenuated HIV-1 infection. RECENT FINDINGS: Follow-up of recipients showed that infection with this attenuated HIV-1 strain resulted in either low or absent viral replication in vivo for up to 29 years. The three patients without detectable viraemia have been studied for virological, genetic and immunological correlates of elite control. CD4 proliferation in vitro in response to p24 provided the clearest distinction of elite controllers from the slow progressors. Host factors are believed to differentiate the three elite controllers; only one, C135, has identifiable genetic polymorphisms that probably contributed to nonprogression: Δ32 CCR5 heterozygosity, HLA-B57 and HLA-DR13 alleles, in addition to infection with nef-defective HIV-1. SUMMARY: Even nef-defective HIV-1 can lead to sufficient replication in vivo to enable viral evolution and eventual progression to immunodeficiency. Host factors modified the outcome of infection with attenuated HIV-1, as exemplified by the unique patient C135.

Genome-wide analysis of primary CD4+ and CD8+ T cell transcriptomes shows evidence for a network of enriched pathways associated with HIV disease.

BACKGROUND: HIV preferentially infects CD4+ T cells, and the functional impairment and numerical decline of CD4+ and CD8+ T cells characterize HIV disease. The numerical decline of CD4+ and CD8+ T cells affects the optimal ratio between the two cell types necessary for immune regulation. Therefore, this work aimed to define the genomic basis of HIV interactions with the cellular transcriptome of both CD4+ and CD8+ T cells. RESULTS: Genome-wide transcriptomes of primary CD4+ and CD8+ T cells from HIV+ patients were analyzed at different stages of HIV disease using Illumina microarray. For each cell subset, pairwise comparisons were performed and differentially expressed (DE) genes were identified (fold change >2 and B-statistic >0) followed by quantitative PCR validation. Gene ontology (GO) analysis of DE genes revealed enriched categories of complement activation, actin filament, proteasome core and proton-transporting ATPase complex. By gene set enrichment analysis (GSEA), a network of enriched pathways functionally connected by mitochondria was identified in both T cell subsets as a transcriptional signature of HIV disease progression. These pathways ranged from metabolism and energy production (TCA cycle and OXPHOS) to mitochondria meditated cell apoptosis and cell cycle dysregulation. The most unique and significant feature of our work was that the non-progressing status in HIV+ long-term non-progressors was associated with MAPK, WNT, and AKT pathways contributing to cell survival and anti-viral responses. CONCLUSIONS: These data offer new comparative insights into HIV disease progression from the aspect of HIV-host interactions at the transcriptomic level, which will facilitate the understanding of the genetic basis of transcriptomic interaction of HIV in vivo and how HIV subverts the human gene machinery at the individual cell type level.