Search

This case report describes a 68-year-old male who presented to the emergency department (ED) with nausea, vomiting, abdominal pain, diarrhea, and fatigue after starting adjuvant combination chemotherapy with capecitabine and oxaliplatin two weeks prior. Further evaluation of this patient in the ED revealed an incidentally discovered aortic thrombosis, of which this patient did not exhibit any specific symptoms. This case, among a few others, has described the development of arterial thrombosis in patients with cancer undergoing combination chemotherapy with capecitabine and oxaliplatin., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Burton et al.)

The protocols described in this unit provide detailed information on how to isolate and expand, in culture, ciliary epithelial cells (CECs), previously identified as retinal stem cells, from the adult mouse eye, and embryonic retinal progenitor cells (RPCs) from the developing retina. CECs are initially cultured in floating conditions as neurospheres and then expanded in monolayer cultures. RPCs are cultured in floating conditions. Detailed protocols for retinal differentiation, as well as exogenous gene expression using lentivirus are also described.

Photoreceptors have recently been generated from mouse and human embryonic stem cells (ESCs), although ethics concerns impede their utilization for cell replacement therapy for retinal disease. Extra-embryonic tissues have received attention as alternative therapeutic sources of stem cells. Human and mouse amniotic fluid-derived stem cells (AFCs) have been reported to be multipotent and express embryonic and adult stem cell markers. Here, in vitro conditions that generate retinal cells from ESCs were used to analyze and compare the retinal potential of murine AFCs and ESCs. We show that AFCs express pluripotency markers (Nanog, Sox2, and Oct3/4) as well as retinal transcription factor genes (Et, Lhx2, Tll1, Six6, Otx2, Pax6, and Fgf15). AFCs from amniotic fluid of Fgf15.gfp, Nrl.gfp, and Crx.gfp embryos cultured in retinal proliferation and differentiation conditions failed to switch on these retinal transgenes. AFCs cultured in retinal-promoting conditions, effective on ESCs, showed reduced expression of retinal markers. Retinal co-cultures activated retinal genes in ESCs but not in AFCs, and migration assays in retinal explants showed limited migration of AFCs compared with ESCs. Unlike ESCs, AFCs do not express the early embryonic ectodermal gene Utf1 and Western analysis of AFCs identified only the B isoform of Oct3/4, rather than the isoform A present in ESCs. We conclude that AFCs have restricted potential and differ considerably from ESCs and retinal progenitor cells. Reprogramming to induce pluripotency or new differentiation protocols will be required to confer retinal potential to AFCs as expression of a subset of pluripotency and retinal markers is not sufficient.

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus.

The ciliary margin in lower vertebrates is a site of continual retinal neurogenesis and a stem cell niche. By contrast, the human eye ceases retinal neuron production before birth and loss of photoreceptors during life is permanent and a major cause of blindness. The discovery of a proliferative cell population in the ciliary epithelium (CE) of the adult mammalian eye, designated retinal stem cells, raised the possibility that these cells could help to restore sight by replacing lost photoreceptors. We previously demonstrated the feasibility of photoreceptor transplantation using cells from the developing retina. CE cells could provide a renewable source of photoreceptors for transplantation. Several laboratories reported that these cells generate new photoreceptors, whereas a recent report questioned the existence of retinal stem cells. We used Nrl.gfp transgenic mice that express green fluorescent protein in rod photoreceptors to assess definitively the ability of CE cells to generate new photoreceptors. We report that CE cells expanded in monolayer cultures, lose pigmentation, and express a subset of eye field and retinal progenitor cell markers. Simultaneously, they continue to express some markers characteristic of differentiated CE and typically lack a neuronal morphology. Previously reported photoreceptor differentiation conditions used for CE cells, as well as conditions used to differentiate embryonic retinal progenitor cells (RPCs) and embryonic stem cell-derived RPCs, do not effectively activate the Nrl-regulated photoreceptor differentiation program. Therefore, we conclude that CE cells lack potential for photoreceptor differentiation and would require reprogramming to be useful as a source of new photoreceptors.

Rac GTPases are members of the Rho family regulating the actin cytoskeleton and implicated in neuronal development. Ubiquitous Rac1 and neuron-specific Rac3 GTPases are coexpressed in the developing mammalian brain. We used Cre-mediated conditional deletion of Rac1 in neurons combined with knockout of neuron-specific Rac3 to study the role of these GTPases in neural development. We found that lack of both genes causes motor behavioral defects, epilepsy, and premature death of mice. Deletion of either GTPase does not produce evident phenotypes. Double-knockout mice show specific defects in the development of the hippocampus. Selective impairment of the dorsal hilus of double-knockout animals is associated with alteration in the formation of the hippocampal circuitry. Axonal pathways to and from the dorsal hilus are affected because of the deficit of hilar mossy cells. Moreover, analysis of Rac function in hippocampal cultures shows that spine formation is strongly hampered only in neurons lacking both Rac proteins. These findings show for the first time that both Rac1 and Rac3 are important for the development of the nervous system, wherein they play complementary roles during late stages of neuronal and brain development.

Background Information: Rho family GTPases are required for cytoskeletal reorganization and are considered important for the maturation of neurons. Among these proteins, Rac1 is known to play a crucial role in the regulation of actin dynamics, and a number of studies indicate the involvement of this protein in different steps of vertebrate neuronal maturation. There are two distinct Rac proteins expressed in neurons, namely the ubiquitous Rac1 and the neuron-specific Rac3. The specific functions of each of these GTPases during early neuronal development are largely unknown., Results: The combination of the knockout of Rac3 with Rac1 down-regulation by siRNA (small interfering RNA) has been used to show that down-regulation of Rac1 affects dendritic development in mouse hippocampal neurons, without affecting axons. F-actin levels are strongly decreased in neuronal growth cones following down-regulation of Rac1, and time-lapse analysis indicated that the reduction of Rac1 levels decreases growth-cone dynamics., Conclusions: These results show that normal levels of endogenous Rac1 activity are critical for early dendritic development, whereas dendritic outgrowth is not affected in hippocampal neurons from Rac3-null mice. On the other hand, early axonal development appears normal after Rac1 down-regulation. Our findings also suggest that the initial establishment of neuronal polarity is not affected by Rac1 down-regulation.

Previous data from this laboratory suggest that Ca-induced reductions in blood pressure may result in part from Ca-induced natriuresis and a possible reduction in intravascular volume. Consequently, the present study was conducted to directly determine the relative effects of dietary Ca and Na on total body water (TBW) and extracellular water (ECW) as measured by tetrapolar bioelectrical impedance and to determine the potential relationship of these changes to diet-induced changes in blood pressure. Eleven hypertensive black adults were maintained for 14 days on each of four diets that contained 356 mg Ca or 1,000 mg Ca, each at 1,000 or 4,000 mg Na in a repeated measures design. Increasing dietary Na at the low Ca intake caused significant increases in supine systolic and diastolic pressure, reduced plasma renin activity (PRA), significantly increased TBW, and caused a significant reduction in total body reactance indicating an increase in ECW. Adding supplementary Ca to the low Na diet was without significant effect on blood pressure, PRA, TBW, or electrical reactance. In contrast, adding Ca to the low Ca-high Na diet caused a significant natriuretic effect that was accompanied by significant increases in PRA and electrical reactance and significant reductions in blood pressure and TBW, to baseline (low Ca-low Na levels). Thus, these data indicate that the antihypertensive effect of calcium in salt-sensitive blacks may be attributable, in part, to Ca-induced natriuresis and an associated volume contraction.

To examine changes in calcium metabolism related to aging in blacks and caucasians, we measured serum mid-molecule parathyroid hormone (PTH), total and ionised calcium in 83 black and 47 caucasian healthy males aged 31-77 yrs. Age and race were without effect on total or ionised calcium. PTH levels increased significantly (P less than 0.03) with age in black but not caucasian subjects. These results demonstrate an aging-associated increase in circulating PTH in blacks and are consistent with other data from our laboratory which suggest that PTH may be involved in the development of salt-sensitive hypertension in blacks.