Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs.

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 min after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b.i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.

Recurrent epileptiform discharges in the medial entorhinal cortex of kainate-treated rats are differentially sensitive to antiseizure drugs.

OBJECTIVE: Approximately 30% of patients with epilepsy are refractory to existing antiseizure drugs (ASDs). Given that the properties of the central nervous systems of these patients are likely to be altered due to their epilepsy, tissues from rodents that have undergone epileptogenesis might provide a therapeutically relevant disease substrate for identifying compounds capable of attenuating pharmacoresistant seizures. To facilitate the development of such a model, this study describes the effects of classical glutamate receptor antagonists and 20 ASDs on recurrent epileptiform discharges (REDs) in brain slices derived from the kainate-induced status epilepticus model of temporal lobe epilepsy (KA-rats). METHODS: Horizontal brain slices containing the medial entorhinal cortex (mEC) were prepared from KA-rats, and REDs were recorded from the superficial layers. 6-cyano-7-nitroquinoxaline-2,3-dione, (2R)-amino-5-phosphonovaleric acid, tetrodotoxin, or ASDs were bath applied for 20 minutes. Concentration-dependent effects and half maximal effective concentration values were determined for RED duration, frequency, and amplitude. RESULTS: ASDs targeting sodium and potassium channels (carbamazepine, eslicarbazepine, ezogabine, lamotrigine, lacosamide, phenytoin, and rufinamide) attenuated REDs at concentrations near their average therapeutic plasma concentrations. γ-aminobutyric acid (GABA)ergic synaptic transmission-modulating ASDs (clobazam, midazolam, phenobarbital, stiripentol, tiagabine, and vigabatrin) attenuated REDs only at higher concentrations and, in some cases, prolonged RED durations. ASDs with other/mixed mechanisms of action (bumetanide, ethosuximide, felbamate, gabapentin, levetiracetam, topiramate, and valproate) and glutamate receptor antagonists weakly or incompletely inhibited RED frequency, increased RED duration, or had no significant effects. SIGNIFICANCE: Taken together, these data suggest that epileptiform activity recorded from the superficial layers of the mEC in slices obtained from KA-rats is differentially sensitive to existing ASDs. The different sensitivities of REDs to these ASDs may reflect persistent molecular, cellular, and/or network-level changes resulting from disease. These data are expected to serve as a foundation upon which future therapeutics may be differentiated and assessed for potentially translatable efficacy in patients with refractory epilepsy.

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

Frequency and Distribution of Rickettsiae, Borreliae, and Ehrlichiae Detected in Human-Parasitizing Ticks, Texas, USA.

To describe the presence and distribution of tickborne bacteria and their vectors in Texas, USA, we screened ticks collected from humans during 2008-2014 for Rickettsia, Borrelia, and Ehrlichia spp. Thirteen tick species were identified, and 23% of ticks carried bacterial DNA from at least 1 of the 3 genera tested.

Survey of Borreliae in ticks, canines, and white-tailed deer from Arkansas, U.S.A.

BACKGROUND: In the Eastern and Upper Midwestern regions of North America, Ixodes scapularis (L.) is the most abundant tick species encountered by humans and the primary vector of B. burgdorferi, whereas in the southeastern region Amblyomma americanum (Say) is the most abundant tick species encountered by humans but cannot transmit B. burgdorferi. Surveys of Borreliae in ticks have been conducted in the southeastern United States and often these surveys identify B. lonestari as the primary Borrelia species, surveys have not included Arkansas ticks, canines, or white-tailed deer and B. lonestari is not considered pathogenic. The objective of this study was to identify Borrelia species within Arkansas by screening ticks (n=2123), canines (n=173), and white-tailed deer (n=228) to determine the identity and locations of Borreliae endemic to Arkansas using PCR amplification of the flagellin (flaB) gene. METHODS: Field collected ticks from canines and from hunter-killed white-tailed were identified to species and life stage. After which, ticks and their hosts were screened for the presence of Borrelia using PCR to amplify the flaB gene. A subset of the positive samples was confirmed with bidirectional sequencing. RESULTS: In total 53 (21.2%) white-tailed deer, ten (6%) canines, and 583 (27.5%) Ixodid ticks (252 Ixodes scapularis, 161 A. americanum, 88 Rhipicephalus sanguineus, 50 Amblyomma maculatum, 19 Dermacentor variabilis, and 13 unidentified Amblyomma species) produced a Borrelia flaB amplicon. Of the positive ticks, 324 (22.7%) were collected from canines (151 A. americanum, 78 R. sanguineus, 43 I. scapularis, 26 A. maculatum, 18 D. variabilis, and 8 Amblyomma species) and 259 (37.2%) were collected from white-tailed deer (209 I. scapularis, 24 A. maculatum, 10 A. americanum, 10 R. sanguineus, 1 D. variabilis, and 5 Amblyomma species). None of the larvae were PCR positive. A majority of the flaB amplicons were homologous with B. lonestari sequences: 281 of the 296 sequenced ticks, 3 canines, and 27 deer. Only 22 deer, 7 canines, and 15 tick flaB amplicons (12 I. scapularis, 2 A. maculatum, and 1 Amblyomma species) were homologous with B. burgdorferi sequences. CONCLUSIONS: Data from this study identified multiple Borreliae genotypes in Arkansas ticks, canines and deer including B. burgdorferi and B. lonestari; however, B. lonestari was significantly more prevalent in the tick population than B. burgdorferi. Results from this study suggest that the majority of tick-borne diseases in Arkansas are not B. burgdorferi.

Development of shuttle vectors for transformation of diverse Rickettsia species.

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.

Borrelia, Ehrlichia, and Rickettsia spp. in ticks removed from persons, Texas, USA.

Data regarding the type, frequency, and distribution of tick-borne pathogens and bacterial agents are not widely available for many tick species that parasitize persons in the southern United States. We therefore analyzed the frequency and identity of pathogens and bacterial agents in ticks removed from humans and subsequently submitted to the Texas Department of State Health Services, Zoonosis Control Program, from October 1, 2004, through September 30, 2008. The data showed associations of bacterial agents and potential vectors. Tick-related illnesses may pose unidentified health risks in areas such as Texas, where incidence of human disease related to tick bites is low but well above zero and where ticks are not routinely suspected as the cause of disease. Cause, treatment, and prevention strategies can be better addressed through collecting sufficient data to establish baseline assessments of risk.

Wide dispersal and possible multiple origins of low-copy-number plasmids in rickettsia species associated with blood-feeding arthropods.

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, "Candidatus Rickettsia amblyommii," R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two "Ca. Rickettsia amblyommii" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of "Ca. Rickettsia amblyommii," R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

Rickettsia amblyommii infecting Amblyomma americanum larvae.

Polymerase chain reaction analysis of Amblyomma americanum adults, nymphs, and larvae from Aberdeen Proving Ground, MD (APG), revealed a very high prevalence of a spotted fever group (SFG) rickettsia. Restriction fragment length polymorphism (RFLP) and sequence analysis identified "Rickettsia amblyommii." This organism is not yet described or well studied, and its pathogenicity is unknown; however, investigations of the organism are warranted because of its high prevalence in A. americanum. This tick is extremely abundant at military training facilities in the south, central, and Mid-Atlantic United States, and many soldiers experience multiple concurrent tick bites. Bites by R. amblyommii-infected A. americanum may account for rates of SFG rickettsia seropositivity that are higher than reported rates of Rocky Mountain spotted fever (RMSF) cases from the same location. Seroconversion to SFG rickettsia following bites of A. americanum may suggest that R. amblyommii is infectious in humans. Subclinical infection in the numerous A. americanum tick bite victims could contaminate donated blood and compromise immunodeficient recipients. Detection of R. amblyommii in questing A. americanum larvae suggests transovarial transmission. The absence of R. rickettsii, the agent of RMSF, in A. americanum may be due to transovarial interference by R. amblyommii. The likelihood of pathogen transmission by larvae is magnified by their habit of mass attack. The very small size of the larvae is also a risk factor for pathogen transmission. High R. amblyommii prevalence in populations of A. americanum presage co-infection with other A. americanum-borne pathogens. A. americanum nymphs and adults from APG were found to be co-infected with R. amblyommii and Borrelia lonestari, Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, and larval pools were infected with both R. amblyommii and B. lonestari. Co-infections can compound effects and complicate diagnosis of tick-borne disease.

Cubilin, a binding partner for galectin-3 in the murine utero-placental complex.

Galectin-3 is a lectin important in animal development and regulatory processes and is found selectively localized at the implantation site of the mouse embryo. To better understand the role of galectin-3 at the maternal-fetal interface, a binding partner was isolated and characterized. Homogenates of uteroplacental tissue were incubated with immobilized recombinant galectin-3, and specifically bound proteins were eluted using lactose. The principal protein, p400, had an M(r) of 400,000 in SDS-PAGE. Physical properties of p400 and amino acid sequences of seven tryptic peptides were similar to cubilin from rats, humans, and dogs, identifying p400 as the murine ortholog of cubilin. This was further supported by the tissue distribution observed only in yolk sac, kidney, and ileum with monospecific antiserum for p400. Cubilin occurred in yolk sac epithelium throughout pregnancy, but galectin-3 was there only during the last week. Unexpectedly, cubilin was found only in perforin-containing granules of uterine natural killer (uNK) cells, although galectin-3 occurred throughout the cell cytoplasm. In situ hybridization revealed cubilin mRNA in yolk sac epithelium but not uNK cells, implying that yolk sac-derived cubilin is endocytosed by uNK cells via galectin-3. This is consistent with cubilin being an endogenous partner of galectin-3 at the maternal-fetal interface and suggests an important role for cubilin in uNK cell function.