In this study we show that intravenous administration of a synthetic tetravalent peptide (TVP) designed to counteract Stx2 activity within susceptible cells is capable of rescuing baboons from an otherwise lethal dose of toxin. Even when TVP administration was delayed by 24 hours, the survival rate and protection of organ function was substantial and significant. In other STEC animal models, delayed treatment with anti-toxins, such as antibodies [29
] or Gb3
receptor mimetics [31
] are beneficial and improve survival. However, unlike most other anti-toxin compounds, TVP is cell-permeable even in the absence of toxin ( and [23
]) and can complex with Stx2 within living cells [24
], so it has the potential to be effective long after toxin delivery.
The kidneys were primary beneficiaries of TVP treatment as judged by complete absence of clinical signs of acute kidney injury in Prevention Protocol animals and significantly reduced injury in Rescue Protocol animals. Urine output remained normal and BUN, creatinine, and urinalysis measures remained normal or considerably reduced despite challenge with a lethal Stx2 dose. Even years after hospitalization with diarrhea + HUS, 25–36% of children have one or more renal sequelae which may be predicted by duration of oliguria (>10 days) or anuria (>5 days) [32
]. Although we did not evaluate TVP efficacy beyond 24 hours post-toxin challenge or monitor animals beyond 28 days, the observation that TVP effectively protected urine output suggests that this treatment may have long-term benefits.
Most patients have a fall in platelet counts during the first illness week and monitoring platelet counts is recommended to follow the clinical course [1
]. Thrombocytopenia developed rapidly in the baboons after Stx2 whereas platelet counts in Prevention Protocol animals remained within normal ranges. Platelet loss was greater when TVP treatment was delayed, but recovered after the first week post-challenge. When compared to historical dose-response data in this baboon model, platelet counts in Rescue Protocol animals who received 50ng/kg Stx2 essentially overlay those from baboons who received 10ng/kg Stx2, which is a mild, non-lethal dose [25
]. The thrombocytopenia is not due to a disseminated consumptive coagulopathy, because prolongation of clotting times was modest, indicating sufficient levels of coagulation factors, and fibrinogen levels did not decrease. There is a pro-coagulant environment, and patients with diarrhea + HUS may have elevated D-dimer and F1+2 levels [33
]. Stx induces activation of platelet sub-populations [35
], and expression of adhesion [36
] and coagulant molecules [37
] on glomerular endothelial cells. However, although pro-coagulant activity occurs early in HUS patients [34
], there is little evidence to suggest anti-thrombotics are helpful [33
] and there is concern of increased bleeding risk.
We observed a moderate and slowly progressive anemia in all the baboons, which persisted even in rescued survivors through day 28. HUS patients can become profoundly anemic and ~80% need erythrocyte transfusions [39
]. We do not know the mechanism for prolonged anemia in the baboons, but RBC losses may be due to renal thrombotic microangiopathy which is characteristic of typical HUS. Consistent with this is the observation that the urine dipstick test for blood invariably showed a homogenous pattern, indicating hemoglobinuria. However, schistocyte increases are modest in our Stx2 model, only 2.8–3.5% by 72–96 hours. These observations and the lack of TVP effect suggest an early kidney injury at the cellular level that is not related to survival, but compromises red cell counts. If renal cortical interstitial cells are a target, then low erythropoietin production is possible. Argentina has a high incidence of STEC infection and pediatric HUS, and one study found erythropoietin levels in children with acute diarrhea + HUS to be similar to low levels observed in patients with chronic renal diseases [40
]. One clinical study described fewer RBC transfusions in five diarrhea + HUS pediatric patients treated early with erythropoietin [41
]. Low erythropoietin levels may explain the anemia in our baboons, but we cannot yet quantify baboon erythropoietin and are pursuing other approaches to evaluate potential Stx2 impact on epo
transcription. In the current study, we set the Stx2 dose to be lethal and this probably does not represent a typical toxin load in children with HUS (3–5% mortality), but the baboon toxemia model may be useful for testing efficacy and side effects of therapeutics intended to normalize the hematologic abnormalities caused by Stx.
TVP treatment moderated the systemic inflammation induced by Stx2, but the overall host response was relatively mild. Lipopolysaccharide (endotoxin) is a well known pro-inflammatory mediator from Gram negative bacteria, inducing systemic production of TNFα and other cytokines in humans [42
] and baboons [43
] through activation of Toll-like receptor 4. The lack of plasma or urinary TNFα in the current study is consistent with essentially undetectable levels of endotoxin in the Stx2 preparations used. It also argues that TNFα is not a required component for the kidney damage inflicted by Stx2. However, most STEC patients do have a robust systemic cytokine response [16
], even though the disease is considered to be a toxemia and patients are not bacteremic. It is very possible that intestinal colonization with bacteria that inflicts tissue injury produces damage associated molecular pattern molecules (DAMPs) that contribute to patient inflammation as occurs in other inflammatory bowel conditions [45
Notably, VEGF levels were markedly different between plasma and urine. Plasma levels remained undetectable, whereas urine VEGF increased to 400pg/ml after toxin, which were reduced to baseline by TVP. These observations suggest local influences contribute to urinary VEGF. Human glomerular epithelia and collecting duct cells have VEGF protein [46
] and glomerular capillaries express VEGF receptors [47
]. Hypoxia is a well-established VEGF stimulant and local ischemia due to microthrombi may contribute to elevated urinary VEGF. Conceivably, increased urine VEGF could be an early marker of kidney injury, but levels did not increase until two days after challenge, well after platelet counts started to decline. Alternatively, bedside measurement of urinary VEGF levels may provide a non-invasive way to monitor treatment efficacy and clinical course.
While informative, this baboon model is a model of Stx toxemia and will not completely replicate the consequences of intestinal infection with STEC bacteria. Although bacteremia is rare and the toxins drive organ injury, we would expect differences in the magnitude and timing of toxin delivery as bacteria colonize the intestine. In addition, neurologic abnormalities are a frequent complication of STEC infection in patients [48
]. We occasionally observe seizures or ataxia in the Stx2-challenged baboons, but they are not observed in all animals. Because of the infrequency and lack of quantifiable neural tissue injury marker(s), we do not know if TVP had any effect on the central nervous system. Finally, while Stx1 and Stx2 are primary virulence factors, other bacterial products likely contribute to host responses [49
]. Thus, while intravenous TVP administration was effective, it was effective with respect to the Stx2 challenge and not necessarily a bacterial infection. In ongoing studies, we are developing enteric infection models designed to recapitulate responses observed in patients and test therapeutics for dose, timing and route of administration.
In summary, we show that a small peptide delivered intravenously can protect and rescue nonhuman primates from a lethal dose of Stx2. Even with delayed therapeutic delivery, urine output is preserved, kidney function is protected, and platelet loss is minimized, all of which contributed to a better clinical outcome and improved survival.