Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3(+) structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival

Genital Herpes Has Played a More Important Role than Any Other Sexually Transmitted Infection in Driving HIV Prevalence in Africa

Extensive evidence from observational studies suggests a role for genital herpes in the HIV epidemic. A number of herpes vaccines are under development and several trials of the efficacy of HSV-2 treatment with acyclovir in reducing HIV acquisition, transmission, and disease progression have just reported their results or will report their results in the next year. The potential impact of these interventions requires a quantitative assessment of the magnitude of the synergy between HIV and HSV-2 at the population level.A deterministic compartmental model of HIV and HSV-2 dynamics and interactions was constructed. The nature of the epidemiologic synergy was explored qualitatively and quantitatively and compared to other sexually transmitted infections (STIs). The results suggest a more substantial role for HSV-2 in fueling HIV spread in sub-Saharan Africa than other STIs. We estimate that in settings of high HSV-2 prevalence, such as Kisumu, Kenya, more than a quarter of incident HIV infections may have been attributed directly to HSV-2. HSV-2 has also contributed considerably to the onward transmission of HIV by increasing the pool of HIV positive persons in the population and may explain one-third of the differential HIV prevalence among the cities of the Four City study. Conversely, we estimate that HIV had only a small net impact on HSV-2 prevalence.HSV-2 role as a biological cofactor in HIV acquisition and transmission may have contributed substantially to HIV particularly by facilitating HIV spread among the low-risk population with stable long-term sexual partnerships. This finding suggests that prevention of HSV-2 infection through a prophylactic vaccine may be an effective intervention both in nascent epidemics with high HIV incidence in the high risk groups, and in established epidemics where a large portion of HIV transmission occurs in stable partnerships

Pericyte-mediated regulation of cerebral and coronary blood flow in health and disease

The information processing power of the brain is constrained by its energy supply. Neuronal activity evokes local increases in blood flow to fuel signalling by active neurons. Recent evidence has overturned the dogma that brain blood flow is principally regulated by smooth muscle-covered arterioles. Instead, contractile pericytes on brain capillaries are the first responders to neuronal activity, dilating to supply nutrients in a process called functional hyperaemia. In this thesis, I have characterised the anatomical properties of brain capillary pericytes, their surface marker expression, and their interactions with other cells in the neurovascular unit. I probed the signaling pathways underlying capillary level neurovascular coupling in acute brain slices. Adrenergic compounds induced pericyte constriction. The neuron and astrocyte-derived vasoactive messengers nitric oxide and prostaglandin E2 dilated capillaries near pericytes, as did electrical stimulation of axons, mimicking neuronal activity. During ischaemia, I found that capillaries were rapidly constricted by pericytes, which subsequently died in a state of rigor, possibly underlying the no-reflow phenomenon encountered after stroke. Blocking voltage-gated calcium channels and inhibiting the synthesis of the vasoconstrictor 20-HETE significantly delayed the ischaemia-induced constriction. Capillaries also constricted following ischaemia in vivo. Given the similar lack of reperfusion seen in the heart after myocardial infarction, I investigated the anatomy and pathophysiology of cardiac pericytes. I discovered that pericytes are abundant on coronary capillaries and share many morphological properties with brain pericytes, suggesting they might play a role in coronary blood flow regulation as well as in coronary no-reflow after heart attack

Capillary pericytes mediate coronary no-reflow after myocardial ischaemia.

After cardiac ischaemia, a prolonged decrease of coronary microvascular perfusion often occurs even after flow is restored in an upstream artery. This 'no-reflow' phenomenon worsens patient prognosis. In the brain, after stroke, a similar post-ischaemic 'no-reflow' has been attributed to capillary constriction by contractile pericytes. We now show that occlusion of a rat coronary artery, followed by reperfusion, blocks 40% of cardiac capillaries and halves perfused blood volume within the affected region. Capillary blockages colocalised strongly with pericytes, where capillary diameter was reduced by 37%. The pericyte relaxant adenosine increased capillary diameter by 21% at pericyte somata, decreased capillary block by 25% and increased perfusion volume by 57%. Thus, cardiac pericytes constrict coronary capillaries and reduce microvascular blood flow after ischaemia, despite re-opening of the culprit artery. Cardiac pericytes are therefore a novel therapeutic target in ischaemic heart disease

Capillary pericytes regulate cerebral blood flow in health and disease.

Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke

Capillary pericytes regulate cerebral blood flow in health and disease

Brain blood flow increases, evoked by neuronal activity, power neural computation and are the basis of BOLD functional imaging. It is controversial whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease which damages neurons after stroke