Pauline van Eeden

Timed Ablation of Regulatory CD4+ T Cells Can Prevent Murine AIDS Progression

We describe successful immunotherapy of murine AIDS (MAIDS) in C57BL/6J mice based on the elimination of replicating CD4+ regulator T cells. We demonstrate that a single injection of the antimitotic drug vinblastine (Vb) given 14 days postinfection (p.i.) with LP-BM5 can prevent MAIDS progression. Treatment with anti-CD4 mAb at 14 days p.i. is similarly able to prevent MAIDS. Treatment at other time points with Vb or anti-CD4 mAb is ineffective. The effect is based on ablation of a replicating dominantly suppressive CD4+ T cell population, as indicated by adoptive transfer and in vivo depletion experiments using mAbs against CD4 as well as combinations of mAbs against the known regulatory cell surface markers CD25, GITR, and CTLA-4. Cell surface marker analysis shows a population of CD4+CD25+ cells arising shortly before day 14 p.i. Cytokine analyses show a peak in IL-10 production from day 12 to day 16 p.i. MAIDS-infected mice also have CD4+ T cells with significantly higher expression levels of CD38 and particularly CD69, which have been demonstrated to be regulator T cell markers in the Friend retroviral model. The immunotherapy appears to prevent disease progression, although no protection against reinfection with LP-BM5 is generated. These data define a new therapy for murine retroviral infection, which has potential for use in other diseases where T regulator cell-mediated immunosuppression plays a role in the disease process.

Ultrarush versus semirush initiation of insect venom immunotherapy: A randomized controlled trial

BACKGROUND Venom immunotherapy can be initiated by different schedules, but randomized comparisons have not been performed. OBJECTIVE We aimed to compare the safety of 2 initiation schedules. METHODS Patients of any age with prior immediate generalized reactions to jack jumper ant (Myrmecia pilosula) stings were randomized to venom immunotherapy initiation by a semirush schedule over 10 visits (9 weeks) or an ultrarush schedule over 3 visits (2 weeks). In a concurrent treatment efficacy study, the target maintenance dose was randomized to either 50 μg or 100 μg. The primary outcome was the occurrence of 1 or more objective systemic reactions during venom immunotherapy initiation. Analyses were by intention to treat. We also assessed outcomes in patients who declined randomization. RESULTS Of 213 eligible patients, 93 were randomized to semirush (44 patients) or ultrarush (49 patients) initiation. Objective systemic reactions were more likely during ultrarush initiation (65% vs 29%; P < .001), as were severe reactions (12% vs 0%; P= .029). Times to maximal increases in venom-specific IgG(4) were no different between treatments, whereas the maximal increase in venom-specific IgE occurred earlier with ultrarush treatment. Similar differences between methods were observed in patients who declined randomization. One hundred seventy-eight patients were randomized to maintenance doses of either 50 μg (90 patients) or 100 μg (88 patients). The target maintenance dose had no effect on the primary outcome, but multiple-failure-per-subject analysis found that the 50 μg dose reduced the likelihood of reactions. CONCLUSION Ultrarush initiation increases the risk of systemic reactions. A lower maintenance dose reduces the risk of repeated reactions, but the effect on treatment efficacy is unknown.

Sustained elevation of resistin, NGAL and IL-8 are associated with severe sepsis/septic shock in the emergency department.

Objective To identify biomarkers which distinguish severe sepsis/septic shock from uncomplicated sepsis in the Emergency Department (ED). Methods Patients with sepsis underwent serial blood sampling, including arrival in the ED and up to three subsequent time points over the first 24 hours. Messenger RNA (mRNA) levels of 13 genes representing arms of the innate immune response, organ dysfunction or shock were measured in peripheral blood leucocytes using quantitative PCR, and compared with healthy controls. Serum protein concentrations of targets differentially expressed between uncomplicated sepsis and severe sepsis/septic shock were then measured at each time point and compared between the two patient groups. Results Of 27 participants (median age 66 years, (IQR 35, 78)), 10 had uncomplicated sepsis and 17 had sepsis with organ failure (14 septic shock; 3 had other sepsis-related organ failures). At the time of first sample collection in the ED, gene expression of Interleukin (IL)-10 and Neutrophil Gelatinase Associated Lipocalin (NGAL) were significantly higher in severe sepsis than uncomplicated sepsis. Expression did not significantly change over time for any target gene. Serum concentrations of IL-6, IL-8, IL-10, NGAL and Resistin were significantly higher in severe sepsis than uncomplicated sepsis at the time of first sample collection in the ED, but only IL-8, NGAL and Resistin were consistently higher in severe sepsis compared to uncomplicated sepsis at all time points up to 24 h after presentation. Conclusions These mediators, produced by both damaged tissues and circulating leukocytes, may have important roles in the development of severe sepsis. Further work will determine whether they have any value, in addition to clinical risk parameters, for the early identification of patients that will subsequently deteriorate and/or have a higher risk of death.

Critical illness in the emergency department: lessons learnt from the first 12 months of enrolments in the Critical Illness and Shock Study.

The ED is the most common setting for initial resuscitation of the undifferentiated critically ill, yet most research in this patient population occurs in the post-ED period, in intensive care unit (ICU) or other critical care wards. Thus, our knowledge of the pathophysiology of critical illness and the biological, particularly immunological, response to resuscitation in humans is limited. Early accurate diagnosis and resuscitation in the ED improves outcome in a variety of critical illnesses. However, most mechanistic studies commence many hours after ED resuscitation and are of limited value for understanding the early mechanisms of critical illness. There appears to be significant individual variability in the expression of key biological markers of inflammatory response during the early hours of illnesses, such as sepsis and anaphylaxis. Whether further research into the early inflammatory response to critical illness might identify novel therapeutic targets is unknown, but the possible translational benefits from a better understanding of disease processes in this population are substantial. Complex mechanistic research in the critically ill population in the ED is hampered by the overwhelming clinical needs of the patient. It is recognized that a lack of research infrastructure and personnel, and increasingly stringent ethical requirements, have also impeded such research. In 2010, we commenced the Critical Illness and Shock Study (CISS) to collect clinical data and sequential biological samples from critically ill patients in the initial hours of ED resuscitation. The wealth of information from this ongoing study will allow us to explore specific hypotheses relevant to the pathophysiology and outcomes of this heterogeneous population. Over time we aim to: (i) provide a repository of clinical and laboratory data and a DNA bank with which to explore hypotheses relating to a variety of life-threatening conditions; (ii) identify any common themes in the nature of immunological response to critical illness in general; and (iii) provide data to plan prospective interventional studies to improve patient outcomes. Here we describe the CISS protocol and basic findings from the initial 12 months of the study. In particular, we describe the feasibility and completeness of

Using Time-Resolved Fluorescence to Measure Serum Venom-Specific IgE and IgG

We adapted DELFIA™ (dissociation-enhanced lanthanide fluoroimmunoassay), a time resolved fluorescence method, to quantitate whole venom specific and allergenic peptide-specific IgE (sIgE), sIgG1 and sIgG4 in serum from people clinically allergic to Australian native ant venoms, of which the predominant cause of allergy is jack jumper ant venom (JJAV). Intra-assay CV was 6.3% and inter-assay CV was 13.7% for JJAV sIgE. DELFIA and Phadia CAP JJAV sIgE results correlated well and had similar sensitivity and specificity for the detection of JJAV sIgE against intradermal skin testing as the gold standard. DELFIA was easily adapted for detecting sIgE to a panel of other native ant venoms.

Genomic Responses during Acute Human Anaphylaxis Are Characterized by Upregulation of Innate Inflammatory Gene Networks

Background Systemic spread of immune activation and mediator release is required for the development of anaphylaxis in humans. We hypothesized that peripheral blood leukocyte (PBL) activation plays a key role. Objective To characterize PBL genomic responses during acute anaphylaxis. Methods PBL samples were collected at three timepoints from six patients presenting to the Emergency Department (ED) with acute anaphylaxis and six healthy controls. Gene expression patterns were profiled on microarrays, differentially expressed genes were identified, and network analysis was employed to explore underlying mechanisms. Results Patients presented with moderately severe anaphylaxis after oral aspirin (2), peanut (2), bee sting (1) and unknown cause (1). Two genes were differentially expressed in patients compared to controls at ED arrival, 67 genes at 1 hour post-arrival and 2,801 genes at 3 hours post-arrival. Network analysis demonstrated that three inflammatory modules were upregulated during anaphylaxis. Notably, these modules contained multiple hub genes, which are known to play a central role in the regulation of innate inflammatory responses. Bioinformatics analyses showed that the data were enriched for LPS-like and TNF activation signatures. Conclusion PBL genomic responses during human anaphylaxis are characterized by dynamic expression of innate inflammatory modules. Upregulation of these modules was observed in patients with different reaction triggers. Our findings indicate a role for innate immune pathways in the pathogenesis of human anaphylaxis, and the hub genes identified in this study represent logical candidates for follow-up studies.

Characterisation of a model for retinal neovascularisation. VEGF model Characterisation

Retinal neovascularisation is a major clinical complication of diabetic retinopathy that takes place late in the disease process and constitutes the most damaging phase resulting in loss of vision (Klein et al., 1984). Neovascularisation is defined as the growth of new blood vessels which, in a disease process such as diabetic retinopathy, occurs in abnormal retinal locations. Long term consequences of retinal neovascularisation include the formation of epiretinal membranes and retinal detachment (Smith et al., 1999). In addition, new blood vessels lack a patent blood retinal barrier and exhibit leukostasis presumably resulting in cytotoxic damage (Ishida et al., 2003; Qaum et al., 2001).

Changes in differential gene expression during a fatal stroke

We present a young woman (with an identical twin sister) who arrived at the Emergency Department (ED) within 1hour of her initial stroke symptoms. Previous microarray studies have demonstrated differential expression of multiple genes between stroke patients and healthy controls. However, for many of these studies there is a significant delay between the initial symptoms and collection of blood samples, potentially leaving the important early activators/regulators of the inflammatory response unrecognised. Blood samples were collected from the patient for an analysis of differential gene expression over time during the evolution of a fatal stroke. The time points for blood collection were ED arrival (T0) and 1, 3 and 24hours post ED arrival (T1, T3 and T24). This was compared to her identical twin and an additional two age and sex-matched healthy controls. When compared to the controls, the patient had 12 mRNA that were significantly upregulated at T0, and no downregulated mRNA (with a cut off fold change value ±1.5). Of the 12 upregulated mRNA at T0, granzyme B demonstrated the most marked upregulation on arrival, with expression steadily declining over time, whereas S100 calcium-binding protein A12 (S100A12) gene expression increased from T0 to T24, remaining >two-fold above that in the healthy controls at T24. Other genes, such as matrix metalloproteinase 9, high mobility group box 2 and interleukin-18 receptor I were not upregulated at T0, but they demonstrated clear upregulation from T1-T3, with gene expression declining by T24. A greater understanding of the underlying immunopathological mechanisms that are involved during the evolution of ischaemic stroke may help to distinguish between patients with stroke and stroke mimics.