Loraine Campanati

Congenital Zika Virus Infection: Beyond Neonatal Microcephaly

Importance Recent studies have reported an increase in the number of fetuses and neonates with microcephaly whose mothers were infected with the Zika virus (ZIKV) during pregnancy. To our knowledge, most reports to date have focused on select aspects of the maternal or fetal infection and fetal effects. Objective To describe the prenatal evolution and perinatal outcomes of 11 neonates who had developmental abnormalities and neurological damage associated with ZIKV infection in Brazil. Design, Setting, and Participants We observed 11 infants with congenital ZIKV infection from gestation to 6 months in the state of Paraíba, Brazil. Ten of 11 women included in this study presented with symptoms of ZIKV infection during the first half of pregnancy, and all 11 had laboratory evidence of the infection in several tissues by serology or polymerase chain reaction. Brain damage was confirmed through intrauterine ultrasonography and was complemented by magnetic resonance imaging. Histopathological analysis was performed on the placenta and brain tissue from infants who died. The ZIKV genome was investigated in several tissues and sequenced for further phylogenetic analysis. Main Outcomes and Measures Description of the major lesions caused by ZIKV congenital infection. Results Of the 11 infants, 7 (63.6%) were female, and the median (SD) maternal age at delivery was 25 (6) years. Three of 11 neonates died, giving a perinatal mortality rate of 27.3%. The median (SD) cephalic perimeter at birth was 31 (3) cm, a value lower than the limit to consider a microcephaly case. In all patients, neurological impairments were identified, including microcephaly, a reduction in cerebral volume, ventriculomegaly, cerebellar hypoplasia, lissencephaly with hydrocephalus, and fetal akinesia deformation sequence (ie, arthrogryposis). Results of limited testing for other causes of microcephaly, such as genetic disorders and viral and bacterial infections, were negative, and the ZIKV genome was found in both maternal and neonatal tissues (eg, amniotic fluid, cord blood, placenta, and brain). Phylogenetic analyses showed an intrahost virus variation with some polymorphisms in envelope genes associated with different tissues. Conclusions and Relevance Combined findings from clinical, laboratory, imaging, and pathological examinations provided a more complete picture of the severe damage and developmental abnormalities caused by ZIKV infection than has been previously reported. The term congenital Zika syndrome is preferable to refer to these cases, as microcephaly is just one of the clinical signs of this congenital malformation disorder.

Gliomas and the vascular fragility of the blood brain barrier

Astrocytes, members of the glial family, interact through the exchange of soluble factors or by directly contacting neurons and other brain cells, such as microglia and endothelial cells. Astrocytic projections interact with vessels and act as additional elements of the Blood Brain Barrier (BBB). By mechanisms not fully understood, astrocytes can undergo oncogenic transformation and give rise to gliomas. The tumors take advantage of the BBB to ensure survival and continuous growth. A glioma can develop into a very aggressive tumor, the glioblastoma (GBM), characterized by a highly heterogeneous cell population (including tumor stem cells), extensive proliferation and migration. Nevertheless, gliomas can also give rise to slow growing tumors and in both cases, the afflux of blood, via BBB is crucial. Glioma cells migrate to different regions of the brain guided by the extension of blood vessels, colonizing the healthy adjacent tissue. In the clinical context, GBM can lead to tumor-derived seizures, which represent a challenge to patients and clinicians, since drugs used for its treatment must be able to cross the BBB. Uncontrolled and fast growth also leads to the disruption of the chimeric and fragile vessels in the tumor mass resulting in peritumoral edema. Although hormonal therapy is currently used to control the edema, it is not always efficient. In this review we comment the points cited above, considering the importance of the BBB and the concerns that arise when this barrier is affected.

Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM)

The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen’s topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.

Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.

Structure and elastic properties of tunneling nanotubes

We investigate properties of a reported new mechanism for cell–cell interactions, tunneling nanotubes (TNT’s). TNT’s mediate actin-based transfer of vesicles and organelles and they allow signal transmission between cells. The effects of lateral pulling with polystyrene beads trapped by optical tweezers on TNT’s linking separate U-87 MG human glioblastoma cells in culture are described. This cell line was chosen for handling ease and possible pathology implications of TNT persistence in communication between cancerous cells. Observed nanotubes are shown to have the characteristic features of TNT’s. We find that pulling induces two different types of TNT bifurcations. In one of them, termed V-Y bifurcation, the TNT is first distorted into a V-shaped form, following which a new branch emerges from the apex. In the other one, termed I-D bifurcation, the pulled TNT is bent into a curved arc of increasingly broader span. Curves showing the variation of pulling force with displacement are obtained. Results yield information on TNT structure and elastic properties.

Contribution of microscopy to a better knowledge of the biology of Giardia lamblia.

Giardia lamblia is a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown that G. lamblia does not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum-Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequelae

Zika virus (ZIKV) causes significant public health concerns because of its association with congenital malformations, neurological disorders in adults, and, more recently, death. Considering the necessity to mitigate ZIKV-associated diseases, antiviral interventions are an urgent necessity. Sofosbuvir, a drug in clinical use against hepatitis C virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 × 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90% in different anatomical compartments, such as the blood plasma, spleen, kidney, and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals. Sofosbuvir also prevented the acute neuromotor impairment triggered by ZIKV. In the long-term behavioural analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results indicate that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.

Chloroquine inhibits Zika Virus infection in different cellular models

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. In adults, cases of Guillain-Barré syndrome and meningoencephalitis associated with ZIKV infection have been reported, and no specific therapy is available so far. There is urgency for the discovery of antiviral agents capable of inhibiting viral replication and its deleterious effects. Chloroquine is widely administered as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in VERO, human brain microvascular endothelial, and neural stem cells. We demonstrated in vitro that chloroquine reduces the number of ZIKV-infected cells, virus production and cell death promoted by ZIKV infection without cytotoxic effects. Our results suggest that chloroquine is a promising candidate for ZIKV clinical trials, since it is already approved for clinical use and can be safely administered to pregnant woman.

Gold finder: a computer method for fast automatic double gold labeling detection, counting, and color overlay in electron microscopic images.

This work presents a computerized method to identify, detect, evaluate, and, by colored overlay, present gold particle pairs in electron microscopy (EM), even in wide-field views. Double gold immunolabeled specimens were analyzed in a LEO 912 electron microscope equipped with a 2k x 2k-pixel slow-scan cooled CCD camera connected to a computer with analySIS 3.1 PRO image processing software. The acquisition of a high-resolution and high-dynamic-range image by the camera allowed correct segmentation of the gold particles, separating them from other cell structures and from the substrate. Particle identification was performed by a classification module designed by us. Based on shape and size, the computer recognized the group of small particles and classified them as either singular or clustered and differentiated these from the single bigger type. The final image shows the particle types separated and colored, and indicates the total number of objects encountered in the specific region of interest. Moreover, a montage tool allowed us to obtain final representative images of large microscopic fields, which on analysis by the Gold Finder module provided information on the distribution and localization of antigens comparable to that provided by the wide-field light microscope images.

Giardia lamblia: a report of drug effects under cell differentiation.

The Giardia lamblia life cycle is characterized by two phases during which two major cell differentiation processes take place: encystation and excystation. During encystation, the trophozoites transform into cysts, the resistance form. Once ingested by a susceptible host, the cysts are stimulated to excyst in the stomach, and the excysted trophozoites adhere to the epithelium of the upper small intestine. Our work analyses the effects of four benzimidazole derivatives during Giardia differentiation into cysts and evaluates the excystation efficiency of water resistant cysts. Albendazole (AB) showed the most significant results by inhibiting encystation about 30% and a decreasing rate of excystation efficiency. The ultrastructural organization of the cyst adhesive disk was notably affected by AB treatment. Although other benzimidazoles showed some effect on encystation, they were not able to inhibit the excystation process. It is known that the benzimidazoles affect the cytoskeleton of many organisms but how it interferes in Giardia differentiation processes is our main focus. The importance of studying Giardias differentiation under drug action is reinforced by the following arguments: (1) Cysts eliminated by hosts undergoing treatment could still be potentially infective; (2) once the host has been treated, it would be desirable that the shedding of cysts into the environment is avoided; (3) the prevention of Giardia dissemination is a question of extreme importance mainly in underdeveloped countries, where poor sanitary conditions are related to high rates of giardiasis. This report concerns the importance of keeping the environment free from infective cysts and on Giardia’s drug resistance and differentiating abilities.