Human liver is the primary and obligatory site for malaria infection where sporozoites invade host hepatocytes. Malaria hepatic stages are asymptomatic and represent an attractive target for development of anti-malarial interventions and vaccines. However, owing to lack of robust and reproducible in vitro culture system, it is difficult to target and study this imperative malaria liver stage. Here, we describe a procedure that allow cultivation and visualization of malaria hepatic stages including dormant hypnozoites using primary simian hepatocytes. This method enables sensitive and quantitative assessment of different hepatic stages in vitro.

Plasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches.

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long‐term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host–parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR‐BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.

Relapses of Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.

Artemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13 propeller mutations.

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

DNA of malaria parasites, Plasmodium falciparum, is subjected to extraordinary high levels of genotoxic insults during its complex life cycle within both the mosquito and human host. Accordingly, most of the components of DNA repair machinery are conserved in the parasite genome. Here, we investigated the genome-wide responses of P. falciparum to DNA damaging agents and provided transcriptional evidence of the existence of the double strand break and excision repair system. We also showed that acetylation at H3K9, H4K8, and H3K56 play a role in the direct and indirect response to DNA damage induced by an alkylating agent, methyl methanesulphonate (MMS). Artemisinin, the first line antimalarial chemotherapeutics elicits a similar response compared to MMS which suggests its activity as a DNA damaging agent. Moreover, in contrast to the wild-type P. falciparum, two strains (Dd2 and W2) previously shown to exhibit a mutator phenotype, fail to induce their DNA repair upon MMS-induced DNA damage. Genome sequencing of the two mutator strains identified point mutations in 18 DNA repair genes which may contribute to this phenomenon.

Background: The vitiligo of the penis and vulva is a difficult to treat region of the body where the existing surgical repigmentation methods usually do not give satisfactory results.Objective: This study was conducted to evaluate the efficacy of microskin grafting in stable genital vitiligo.Methods: Four male patients and 1 female patient were included in this study, and microskin grafting was performed in the stable vitiliginous areas of genitals under regional anaesthesia.Results: The microskin grafting technique has shown desired results at these sites with near total pigmentation and negligible donor area deformity.Conclusion: The microskin grafting for vitiligo is simple, reliable, and the most cost-effective technique of tissue grafting that has all the benefits of the latest cellular grafting techniques without its high cost and infrastructure.

Parotid mass causing facial nerve palsy is rare, and is associated with malignant tumours. Acute infection or abscess leading to facial nerve palsy is an extremely rare complication. A literature review revealed only 16 cases of facial nerve palsy associated with suppurative parotitis or parotid abscess. We present a case of deep parotid abscess which is complicated by facial nerve dysfunction.

© 2014 by the American Society for Dermatologic Surgery, Inc. Published by Lippincott Williams & Wilkins

A single-stage technique for reconstruction of the medial nasal ala with a nasolabial flap and an inferiorly based remnant alar flap is presented in this article. The technique has been used in four cases. All the flaps healed uneventfully with aesthetically pleasing results using the one-stage technique. The subcutaneous nasolabial island flap and alar remnant flap have become the method of choice in the author's clinic for partial medial nasal ala reconstruction. It allows one-stage reconstruction with very similar tissue and a concealed scar in the natural groove. The remnant ala as an inferiorly based flap has been used by the author to cover the subcutaneous pedicle of the nasolabial flap to provide better shape to the alar base without its lateral drift during healing.Level of Evidence: Level V, therapeutic study.

Various methods have been described in the literature for earlobe reconstruction in one or many stages. Authors describe here a simple, single-stage technique to reconstruct the loss of the earlobe. The technique is clearly illustrated in a step-by-step manner and produces a naturallooking earlobe without an unpleasant secondary deformity with desirable texture and color match.Level of Evidence: Level IV, therapeutic study.

Reconstruction of the nasolabial columella complex is a technical challenge for which many solutions have been suggested. The peri-alar hatchet pedicle flap, herein described, allows the transposition of a well-matched tissue to reconstruct columellar and nasal tip defects while preserving the alar and nasolabial crease. This procedure did not require a columellar strut or a finishing touch-up procedure for sufficient columellar projection and achieved excellent cosmetic results.

Various methods of nasal alar reconstruction has been described in the medical literature but very few for defects involving the alar rim. These are single- or multistage procedures and have their pros and cons. The authors have designed a novel technique for alar rim defects by advancing a flap alongside the alar crease. This flap is simple, easy to execute and provides desirable results for full-thickness defects in a single-stage procedure.

We are conducting investigational dose escalation trials in patients with High Grade Glioma (HGG, NCT01156584 and NCT01470794), using a retroviral replicating vector (Toca 511).Toca 511 (vocimagene amiretrorepvec) encodes an optimized yeast cytosine deaminase that converts 5-fluorocytosine (5-FC) to the anti-cancer drug 5-FU in infected tumors.We report here results of a Phase 1/2 trial of direct intratumoral Toca 511 administration to recurrent HGG patients, followed by repeat courses of oral 5-FC.Six Toca 511 dose levels, escalated by half logs, the mode of administration and the use of extended release 5-FC (Toca FC) were investigated in 25 HGG patients to date.Treatment at all dose levels has been well tolerated.Post-treatment resection in two patients showed viral protein and DNA and RNA sequences including the CD gene, suggesting viral spread and persistence.MRI and clinical improvements were also occasionally observed.Virus was initially delivered via a brain biopsy needle and placement in the tumor was predicted using conventional neuro-navigation.Immediate MRI after injection of Toca 511 spiked with gadolinium showed inconsistent delivery of Toca 511 to tumors.As a result, real-time MRI-guided delivery was introduced using Toca 511 infusion with a stepped-tip cannula.Using this approach, delivery into as many as 4 locations of up to 3 mL of Toca 511 at flow rates of up to 30 mL/min has been achieved without reflux.In a tumor biopsy from a patient who received 1 mL of Toca 511 and subsequent Toca FC, we observed tumor selective vector transduction and expression by PCR and RT-PCR with concomitant tumor necrosis.At higher Toca 511 doses, significant MRI changes consistent with tumor regression were observed post-Toca FC dosing.Completion of this study is planned, including dose escalation of Toca 511 and increasing the dose and duration of Toca FC.