Littermate cats rescued from a shelter succumbed to acute, primary toxoplasmosis associated with TOXO DB genotype #4, generally circulating in wildlife.

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts in the environment. Although exposure is common (approximately 30% of cats in the USA), clinical toxoplasmosis is relatively rare. Here, we report overwhelming disseminated toxoplasmosis in two litter mate 8-week-old kittens, thought to have acquired toxoplasmosis postnatally. Five domestic shorthair kittens, approximately 2-3 weeks of age, and the queen were found in upstate New York by a rescue group in spring of 2018. The kittens and queen were placed in a foster home for approximately 4-5 weeks and then transferred to a shelter. Two kittens died unexpectedly following a short illness. Postmortem examination of the two deceased kittens revealed overwhelming toxoplasmosis and the presence of entero-epithelial stages in small intestine, suggestive of recent ingestion of infected tissues. Antibodies to T. gondii were found in the deceased kittens and the queen but not in the three asymptomatic littermate kittens. No obvious cause of immunosuppression was demonstrated. Genetic typing of T. gondii from DNA extracted from liver and lungs of both kittens revealed Toxo DB #4 genotype, commonly found in wildlife. Owners and veterinarians should be aware of dangers of feeding raw meat to cats and contact with infected cat feces. Procedures to safely handle T. gondii infected feces in hospital setting are outlined.

Holoprosencephaly and Pure Red Cell Aplasia in a Feline Leukaemia Virus-Positive Kitten.

A 9-month-old, female, domestic longhair cat with severe anaemia tested positive for feline leukaemia virus (FeLV) and was humanely destroyed and submitted for necropsy examination. Gross findings included a non-divided rostral telencephalon, consistent with semilobar holoprosencephaly. Histological examination of the bone marrow revealed an almost complete absence of erythroid precursor cells, consistent with pure red cell aplasia, and mild to moderate myelofibrosis. This case demonstrates a very unusual central nervous system defect, as well as an atypical presentation of pure red cell aplasia, in a FeLV-positive kitten.

Stalled cerebral capillary blood flow in mouse models of essential thrombocythemia and polycythemia vera revealed by in vivo two-photon imaging.

BACKGROUND: Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) that share the JAK2(V617F) mutation in hematopoietic stem cells, leading to excessive production of predominantly platelets in ET, and predominantly red blood cells (RBCs) in PV. The major cause of morbidity and mortality in PV and ET is thrombosis, including cerebrovascular occlusive disease. OBJECTIVES: To identify the effect of excessive blood cells on cerebral microcirculation in ET and PV. METHODS: We used two-photon excited fluorescence microscopy to examine cerebral blood flow in transgenic mouse models that mimic MPNs. RESULTS AND CONCLUSIONS: We found that flow was 'stalled' in an elevated fraction of brain capillaries in ET (18%), PV (27%), mixed MPN (14%) and secondary (non-MPN) erythrocytosis (27%) mice, as compared with controls (3%). The fraction of capillaries with stalled flow increased when the hematocrit value exceeded 55% in PV mice, and the majority of stalled vessels contained only stationary RBCs. In contrast, the majority of stalls in ET mice were caused by platelet aggregates. Stalls had a median persistence time of 0.5 and 1 h in ET and PV mice, respectively. Our findings shed new light on potential mechanisms of neurological problems in patients with MPNs.

Role of DNA damage response pathways in preventing carcinogenesis caused by intrinsic replication stress.

Defective DNA replication can result in genomic instability, cancer and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4(Chaos3/Chaos3) ('Chaos3') mouse model that, by virtue of an amino-acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ataxia telangiectasia-mutated (ATM) double-strand break response pathway (Atm, p21/Cdkn1a and Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. Atm deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development.

Distal-less 3 haploinsufficiency results in elevated placental oxidative stress and altered fetal growth kinetics in the mouse.

Distal-less 3 (Dlx3)(-/-) mice die at E9.5 presumably due to an abnormal placental phenotype including reduced placental vasculature and secretion of placental growth factor. To examine the role of Dlx3 specifically within the epiblast, Dlx3 conditional knockout mice were generated using an epiblast-specific Meox2(CreSor) allele. Dlx3(-/fl), Meox2(CreSor) animals were born at expected frequencies and survived to weaning providing indirect evidence that loss of Dlx3 within the trophoectoderm plays a critical role in fetal survival in the Dlx3(-/-) mouse. We next examined the hypothesis that loss of a single Dlx3 allele would have a negative impact on placental and fetal fitness. Dlx3(+/-) mice displayed reduced fetal growth beginning at E12.5 compared with Dlx3(+/+) controls. Altered fetal growth trajectory occurred coincident with elevated oxidative stress and apoptosis within Dlx3(+/-) placentas. Oral supplementation with the superoxide dismutase mimetic, Tempol, rescued the fetal growth and placental cell death phenotypes in Dlx3(+/-) mice. To determine the potential mechanisms associated with elevated oxidative stress on the Dlx3(+/-) placentas, we next examined vascular characteristics within the feto-placental unit. Studies revealed reduced maternal spiral artery luminal area in the Dlx3(+/-) mice receiving water; Dlx3(+/-) mice receiving Tempol displayed maternal spiral artery luminal area similar to control Dlx3(+/+) mice. We conclude that reduced Dlx3 gene dose results in diminished fetal fitness associated with elevated placental cell oxidative stress and apoptosis coincident with altered vascular remodeling. Administration of antioxidant therapy ameliorated this feto-placental phenotype, suggesting that Dlx3 may be required for adaptation to oxidative stresses within the intrauterine environment.

Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts.

Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 microm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential-like activity, which had peak-to-peak amplitudes of < or = 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade; and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip < or = 3 microm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, wits averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording which typically lasted for 1-3 h. Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (<1 min) steady-state perforation at 22-25 degrees C. This adaptation of conventional perforated patch recording was termed "rapid perforated patch recording". The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].