A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

A replication-competent late liver stage-attenuated human malaria parasite.

Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage-arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage-arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2-. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2- liver stages in human liver-chimeric mice. However, P. falciparum mei2- liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2- is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.

Intracerebral tuberculomas: A rare cause of seizure in an immunocompetent young male.

Central nervous system (CNS) involvement occurs in about 1% of all tuberculosis (TB) cases, classically presenting as a meningitis. Intracerebral tuberculomas are a much rarer manifestation. We describe the case of a young black male who presented with new-onset seizure. Cerebral computerized tomography from an outside hospital reportedly showed findings concerning for septic emboli. Brain magnetic resonance imaging at our institution confirmed the presence of multiple, peripherally enhancing lesions in the right frontal and temporal lobes, cerebellum, and pons. Thoracentesis was performed for a concomitant pleural effusion, which contained elevated levels of adenosine deaminase and ultimately grew Mycobacterium tuberculosis. After ruling out other causes, we reached a diagnosis of CNS TB manifesting as cerebral tuberculomas. The patient was initiated on a course of rifampin, isoniazid, pyrazinamide, and ethambutol for two months, followed by rifampin and isoniazid to complete at least twelve months of antimicrobial therapy. We present this case to highlight this unusual manifestation of CNS TB and review the challenges in diagnosis.

The color of lactotroph secretory granules stained with FM1-43 depends on dye concentration.

When pituitary lactotroph granules undergo exocytosis in the presence of FM1-43, their cores absorb dye and fluoresce brightly. We report that different granules fluoresce with different colors, despite being stained with a single fluorescent dye; emission spectra from individual granules show up to a 25 nm difference between the greenest and reddest granules. We found a correlation between granule color and average fluorescence intensity, suggesting that granule color depends upon dye concentration. We confirmed this in two ways: by increasing FM dye concentration in granules, which red shifted granule color, and by partially photobleaching the FM dye in granules, which green shifted granule color. Increasing stimulation intensity (by increasing KCl concentration) increased the proportion of red granules, indicating that granules exocytosing during intense stimulation bound more dye. This, perhaps, reflects differences in granule core maturation and condensation in which mature granules with condensed cores bind more FM dye but require more intense stimulation to be released. Concentration-dependent color shifts of FM dyes may be useful for monitoring aggregation processes occurring on a size scale smaller than the optical limit.

Slow spontaneous secretion from single large dense-core vesicles monitored in neuroendocrine cells.

Hormones are released from cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. In stimulated exocytosis vesicle content is discharged swiftly. Although rapid vesicle discharge has also been proposed to mediate basal secretion, this has not been studied directly. We investigated basal hormone release by preloading fluorescent peptides into single vesicles. The hormone discharge, monitored with confocal microscopy, was compared with the simultaneous loading of vesicle by FM styryl dye. In stimulated vesicles FM 4-64 (4 microM), loading and hormone discharge occurs within seconds. In contrast, in approximately 50% of spontaneously releasing vesicles, the vesicle content discharge and the FM 4-64 loading were slow (approximately 3 min). These results show that in peptide secreting neuroendocrine cells the elementary vesicle content discharge differs in basal and in stimulated exocytosis. It is proposed that the view dating back for some decades, which is that, at rest, the vesicle discharge of hormones and neurotransmitters is similar to that occurring after stimulation, needs to be extended. In addition to the classical paradigm that secretory capacity of a cell is determined by controlling the probability of occurrence of elementary exocytotic events, one will have to consider activity modulation of elementary exocytotic events as well.

Effects of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one on synaptic vesicle cycling at the frog neuromuscular junction.

Inositol phospholipids are thought to play an important regulatory role in synaptic membrane traffic. We investigated the effects of perturbing 3-phosphoinositide metabolism on neurotransmission at the frog neuromuscular junction. We used the reversible phosphoinositide-3 kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one [LY294002 (LY)] and we examined its effects by intracellular recording, fluorescence imaging with styryl dyes (FM 1-43 and FM 2-10), calcium imaging, and electron microscopy. LY treatment reversibly inhibited vesicle cycling; electron micrographs indicated a dramatic reduction in the number of vesicles, balanced by the appearance of numerous cisternas. LY wash-off reverted the phenotype; terminals were refilled with vesicles, and they resumed normal FM 1-43 uptake and release. Surprisingly, LY treatment also enhanced the frequency of spontaneous release up to 100-fold in a calcium-independent manner. LY evoked similar effects in normal frog Ringer's solution, Ca-free Ringer's solution, and BAPTA AM-pretreated preparations; imaging of nerve terminals loaded with the calcium-sensitive fluorescent dye fluo-3 showed no significant change in fluorescence intensity during LY treatment. FM 1-43 imaging data suggested that LY evoked the cycling of 70-90% of all vesicles. The LY-induced effect on spontaneous release was reproduced by the casein kinase 2 inhibitor 5,6-dichlorobenzimidazole riboside but not, however, by the PI3K inhibitor wortmannin. Because LY has been shown recently to potently inhibit casein kinase 2 as well as PI3K, we hypothesize that casein kinase 2 inhibition is responsible for the enhancement of spontaneous release, whereas PI3K inhibition induces the block of vesicle cycling.