Figure Legends

Figure Legends

Figure 1: Monocyte HLA-DR expression and ex vivo LPS-induced TNFa response in 27 children with MODS. These children underwent measurement of both biomarkers on day 7 of MODS, Top: Children who went on to develop nosocomial infection (gray squares) had lower ex vivo LPS-induced TNFα production and monocyte HLA-DR expression compared to children who recovered without developing infection (open circles). Bottom: Children who went on to die (gray squares) similarly demonstrated lower immune function compared to survivors (open circles). The dotted lines show the biomarker threshold levels which define Immunoparalysis. Patients below these thresholds had a greater risk of developing nosocomial infection and death (p < 0.01).

Figure 2: Persistence of nosocomial infection was associated with less ex vivo TNFα production and more systemic inflammation.

Ex vivo TNFα response was measured at the onset of infection and one week later in children with nosocomial infection. Top: Children with persistent nosocomial infection (■, n = 14) demonstrated lower ex vivo TNFα production over time compared to children whose nosocomial infection resolved (Δ, n = 12) (p < 0.0001; 2-way ANOVA). Bottom: Plasma IL-6 levels were higher in children with persistent infection (p = 0.01; 2-way ANOVA). One subject with immunoparalysis and persistent infection did not undergo testing at the second time point and was not included in this figure. *p 0.02; Mann-Whitney test. Data represent mean ± SEM.

Figure 3: Immune suppression therapy, GM-CSF therapy, ,ex vivo TNFa response and monocyte per cent HLA-DR expression biomarkers, and MRSA nosocomial infection in a transplant patient with immunoparalysis.

A patient with a history of lung transplantation who developed MODS was treated on two occasions with GM-CSF. Top: On both occasions her ex vivo LPS-induced TNFα response and monocyte HLA-DR expression increased and she demonstrated clearance of what had been refractory MRSA septicemia. Middle: Her ex vivo LPS-induced TNFα response remained above the immunoparalysis threshold following her second course of GM-CSF despite a high tacrolimus level. Bottom: The patient’s plasma IL-6 levels dropped during both GM-CSF treatment periods.

Figure 4: GM-CSF therapy in non-transplant, non-neutropenic patients resulted in increased LPS sensitivity and prevention of the development of nosocomial infection without increasing systemic inflammation.

Children with ex vivo LPS-induced TNFα production < 160 pg/mL on day 3 of MODS were randomized to receive GM-CSF or standard therapy. Top: Children receiving GM-CSF (○, n = 7) demonstrated a more rapid recovery of ex vivo LPS-induced TNFα production > 200 pg/mL compared to children receiving standard therapy alone (■, n = 7) (p = 0.001; 2-way RM-ANOVA). Middle: GM-CSF therapy did not result in increased systemic IL-6 production in treated children (p = 0.20; 2-way RM-ANOVA). Data represent mean ± SEM. Bottom: The seven patients who received Standard care all developed nosocomial infections with one patient developing two for a total of 8 nosocomial infections (3 gram negative, 5 fungal). At day 7 and 14, six patients had active infections and at day 2, three patients had active infections. No nosocomial infections were observed in the GM-CSF treated group (p < 0.05).

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