Experimental Reptarenavirus Infection of Boa constrictor and Python regius.

Boid inclusion body disease (BIBD) causes losses in captive snake populations globally. BIBD is associated with the formation of cytoplasmic inclusion bodies (IBs), which mainly comprise reptarenavirus nucleoprotein (NP). In 2017, BIBD was reproduced by cardiac injection of boas and pythons with reptarenaviruses, thus demonstrating a causative link between reptarenavirus infection and the disease. Here, we report experimental infections of Python regius (n = 16) and Boa constrictor (n = 16) with three reptarenavirus isolates. First, we used pythons (n = 8) to test two virus delivery routes: intraperitoneal injection and tracheal instillation. Viral RNAs but no IBs were detected in brains and lungs at 2 weeks postinoculation. Next, we inoculated pythons (n = 8) via the trachea. During the 4 months following infection, snakes showed transient central nervous system (CNS) signs but lacked detectable IBs at the time of euthanasia. One of the snakes developed severe CNS signs; we succeeded in reisolating the virus from the brain of this individual and could demonstrate viral antigen in neurons. In a third attempt, we tested cohousing, vaccination, and sequential infection with multiple reptarenavirus isolates on boas (n = 16). At 10 months postinoculation, all but one snake tested positive for viral RNA in lung, brain, and/or blood, but none exhibited the characteristic IBs. Three of the four vaccinated snakes seemed to sustain challenge with the same reptarenavirus; however, neither of the two snakes rechallenged with different reptarenaviruses remained uninfected. Comparison of the antibody responses in experimentally versus naturally reptarenavirus-infected animals indicated differences in the responses.IMPORTANCE In the present study, we experimentally infected pythons and boas with reptarenavirus via either intraperitoneal injection or tracheal instillation. The aims were to experimentally induce boid inclusion body disease (BIBD) and to develop an animal model for studying disease transmission and pathogenesis. Both virus delivery routes resulted in infection, and infection via the trachea could reflect the natural route of infection. In the experimentally infected snakes, we did not find evidence of inclusion body (IB) formation, characteristic of BIBD, in pythons or boas. Most of the boas (11/12) remained reptarenavirus infected after 10 months, which suggests that they developed a persistent infection that could eventually have led to BIBD. We demonstrated that vaccination using recombinant protein or an inactivated virus preparation prevented infection by a homologous virus in three of four snakes. Comparison of the antibody responses of experimentally and naturally reptarenavirus-infected snakes revealed differences that merit further studies.

Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

BACKGROUND AND AIMS: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. METHODS: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. RESULTS: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. CONCLUSIONS: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

Chlamydiae in febrile children with respiratory tract symptoms and age-matched controls, Ghana.

Members of the Chlamydiales order are obligate intracellular pathogens causing acute and chronic infectious diseases. Chlamydiaceae are established agents of community- and zoonotically acquired respiratory tract infections, and emerging pathogens among the Chlamydia-related bacteria have been implicated in airway infections. The role of both in airway infections in Africa is underexplored. We performed a case -control study on the prevalence of Chlamydiaceae and Chlamydia-related emerging pathogens in children with febrile respiratory tract infections in West Africa, Ghana. Using a pan-Chlamydiales broad-range real-time PCR, we detected chlamydial DNA in 11 (1.9%) of 572 hospitalized febrile children with respiratory tract symptoms and in 24 (4.3%) of 560 asymptomatic age-matched controls (p 0.03). Chlamydiaceae were found to be common among both symptomatic and healthy Ghanaian children, with Chlamydia pneumoniae being the most prevalent species. Parachlamydiaceae were detected in two children without symptoms but not in the symptomatic group. We identified neither Chlamydia psittaci nor Simkania negevensis but a member of a new chlamydial family that shared 90.2% sequence identity with the 16S rRNA gene of the zoonotic pathogen Chlamydia pecorum. In addition, we found a new Chlamydia-related species that belonged to a novel family sharing 91.3% 16S rRNA sequence identity with Candidatus Syngnamydia venezia. The prevalence and spectrum of chlamydial species differed from previous results obtained from children of other geographic regions and our study indicates that both, Chlamydiaceae and Chlamydia-related bacteria, are not clearly linked to clinical symptoms in Ghanaian children.

Bilateral form deprivation in monkeys. Electrophysiologic and anatomic consequences.

The response characteristics of neurons in the striate cortex are described for rhesus monkeys that underwent bilateral form deprivation by surgical closure of the eyelids starting within the first month of life and lasting for 2, 6, 7, 13, or 16 weeks. The monkeys had been tested for visual deficits resulting from these experimental deprivations. Single-unit recordings from the striate cortices of these animals showed a single significant abnormality; the absence of excitatory binocular input. Whereas, 76% of the neurons in the foveal striate cortex of the normal animals were binocular, fewer than 20% of the neurons in the experimental monkeys were binocular. However, each eye was well represented by monocular cells. As demonstrated in oblique microelectrode penetrations, the cortical eye-dominance zones for each eye appeared to be of equal width with sharp transitions at the monocular boundaries. The sizes of the cells of the lateral geniculate nuclei were smaller (-15%) than those in controls. Binocular form deprivation early in life has its most obvious effect on the physiology and function of cortical binocular neurons and secondarily on the size of neurons of the lateral geniculate nucleus.

Excitatory binocular neurons are lost following prismatic binocular dissociation in infant monkeys.

Four infant rhesus monkeys had prismatic dissociation of binocular vision by viewing the world through prisms. Those monkeys tested previously for ability to utilize horizontal disparity cues in detection of dynamic random dot stereograms, were found here to have few excitatory binocular neurons in visual cortex (V1). Each eye was well represented in the monocular ability to drive cortical neurons, whilst stimulus orientation tuning appeared normal in the monocular neurons, but somewhat less sensitive in the remaining binocular neurons. Binocular dissociation early in life constitutes conditions unfavorable for the maintenance of neural connections delivering binocular excitation to the visual cortex.

[Objective evaluation of visual strain in computer screen and office work by recording visually evoked cortical potentials (VECP) and determining changes in contrast sensitivity and refraction]. / Untersuchungen zum objektiven Nachweis der Beanspruchung des visuellen Systems bei Bildschirm- und Büroarbeit durch Ableitung visuell evozierter kortikaler Potentiale (VECP) und Erfassung der Anderungen von Kontrastsensitivität und Refraktion.

Visual evoked cortical potentials (VECP) elicited with checkerboard pattern reversal stimuli of high spatial frequency and low contrast were recorded. The changes in the VECP depending on the duration of task-oriented activity at video display units (VDU) and in the office were determined. The testing procedure can be used to decide on adequate recovery periods (breaks) for any visual workload and requires only a short time. We investigated 30 probands: 10 subjects tested before and after 4 h of VDU work under controlled standard conditions had statistically significant increases of latency (P < 0.001) and decreases of amplitude in VECP recordings (P < 0.05). Another 10 subjects tested at the same intervals but exposed to standard office working conditions also showed statistically significant increases of latency (P < 0.0001) and decrease of amplitude in VECP recordings (P < 0.01). The 10 control subjects tested at the same intervals spent the time between recording sessions on leisure activity (walking) and showed no significant VECP changes. Statistical evaluation was performed with Student's t-test for paired samples. Contrast sensitivity (Ginsburg test) decreased in both VDU and office groups after completion of their workload, but remained within normal limits. The maximal changes in refraction amounted to +/- 0.25 D after 4 h and +/- 0.5 D after 6 h and showed no bias. Extended periods of working at VDUs and conventional office work increase the latency and decrease the amplitude of the pattern-evoked VECP. This method can be used to determine optimal scheduling of breaks in for people working at VDUs and doing conventional office work.

Mast cells and the cyclooxygenase pathway mediate colonic afferent nerve sensitization in a murine colitis model.

INTRODUCTION: Intestinal inflammation alters colonic afferent nerve sensitivity which may contribute to patients' perception of abdominal discomfort. We aimed to explore whether mast cells and the cyclooxygenase pathway are involved in altered afferent nerve sensitivity during colitis. METHODS: C57Bl6 mice received 3% dextran-sulfate sodium (DSS) in drinking water for 7 days to induce colitis. Control animals received regular water. On day 8 inflammation was assessed in the proximal colon by morphology and histology. Extracellular afferent nerve discharge was recorded from the mesenteric nerve of a 2 cm colonic segment. Subgroups were treated in vitro with the mast cell stabilizer doxantrazole (10⁻4M) or the cyclooxygenase inhibitor naproxen (10⁻5M). RESULTS: DSS colitis resulted in morphological and histological signs of inflammation. At baseline, peak firing was 11±2 imp s⁻¹ in colitis segments and 5±1 imp s⁻¹ in uninflamed control segments (p<0.05; mean ± SEM; each n=6). In colitis segments, afferent nerve discharge to bradykinin (0.5 µM) was increased to 47±7 compared to 23±6 imp s⁻¹ in recordings from non-inflamed control tissue (p<0.05). Mechanosensitivity during luminal ramp distension (0-80 cm H2O) was increased reaching 24±5 imp s⁻¹ at 80 cm H2O during colitis compared to 14±2 in non-inflamed controls (p<0.05). Doxantrazole or naproxen reduced afferent discharge to bradykinin and luminal ramp distension in colitis segments to control levels. CONCLUSION: Intestinal inflammation sensitizes mesenteric afferent nerve fibers to bradykinin and mechanical stimuli. The underlying mechanism responsible for this sensitization seems to involve mast cells and prostaglandins.

Afferent nerve sensitivity is decreased by an iNOS-dependent mechanism during indomethacin-induced inflammation in the murine jejunum in vitro.

Evidence exists that visceral afferent sensitivity is subject to regulatory mechanisms. We hypothesized that afferent sensitivity is decreased in the small intestine during intestinal inflammation by an inducible nitric oxide synthase (iNOS)-dependent mechanism. C57BL/6 mice were injected twice with vehicle or 60 mg kg(-1) indomethacin subcutaneously to induce intestinal inflammation. Afferent sensitivity was recorded on day 3 from a 2-cm segment of jejunum in vitro by extracellular multi-unit afferent recordings from the mesenteric nerve bundle. In subgroups (n = 6), iNOS was inhibited selectively by L-N6-(1-iminoethyl)-lysine (L-NIL) given either chronically from day 1-3 (3 mg kg(-1) twice daily i.p.) or acutely into the organ bath (30 micromol L(-1)). The indomethacin-induced increase of macroscopic and microscopic scores of intestinal inflammation (both P < 0.05) were unchanged after pretreatment with L-NIL. Peak afferent firing following bradykinin (0.5 micromol L(-1)) was 55 +/- 8 impulse s(-1) during inflammation vs 97 +/- 7 impulse s(-1) in controls (P < 0.05). Normal firing rate was preserved following L-NIL pretreatment (112 +/- 16 impulse s(-1)) or acute administration of L-NIL (108 +/- 14 impulse s(-1)). A similar L-NIL dependent reduction was observed for 5-HT (250 micromol L(-1)) and mechanical ramp distension from 20 to 60 cmH(2)O (both P < 0.05). Intraluminal pressure peaks were decreased to 0.66 +/- 0.1 cmH(2)O during inflammation compared to 2.51 +/- 0.3 in controls (P < 0.01). Afferent sensitivity is decreased by an iNOS-dependent mechanism during intestinal inflammation which appears to be independent of the inflammatory response. This suggests that iNOS-dependent nitric oxide production alters afferent sensitivity during inflammation by interfering with signal transduction to afferent nerves rather than by attenuating intestinal inflammation.